Matthew Bogyo

All Publications

• Polymer-Tethered Quenched Fluorescent Probes for Enhanced Imaging of Tumor-Associated Proteases. ACS sensors Hadzima, M., Faucher, F. F., Blažková, K., Yim, J. J., Guerra, M., Chen, S., Woods, E. C., Park, K. W., Šácha, P., Šubr, V., Kostka, L., Etrych, T., Majer, P., Konvalinka, J., Bogyo, M. 2024

  Abstract

  Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

  View details for DOI 10.1021/acssensors.4c00912

  View details for PubMedID 38941307

• DNA encoded peptide library for SARS-CoV-2 3CL protease covalent inhibitor discovery and profiling RSC CHEMICAL BIOLOGY Xing, Y., Zhang, H., Wang, Y., Zong, Z., Bogyo, M., Chen, S. 2024

  View details for DOI 10.1039/d4cb00097h

  View details for Web of Science ID 001243408700001

• Polymer-tethered quenched fluorescent probes for enhanced imaging of tumor associated proteases. bioRxiv : the preprint server for biology Hadzima, M., Faucher, F., Blažková, K., Yim, J. J., Guerra, M., Chen, S., Woods, E. C., Park, K. W., Šácha, P., Šubr, V., Kostka, L., Etrych, T., Majer, P., Konvalinka, J., Bogyo, M. 2024

  Abstract

  Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

  View details for DOI 10.1101/2024.05.06.592849

  View details for PubMedID 38766164

  View details for PubMedCentralID PMC11100723

• Molecularly targeted protease-activated probes for visualization of glioblastoma: a comparison with 5-ALA. Journal of neurosurgery Konečná, D., Výmola, P., Ternerová, N., Výmolová, B., Garcia-Borja, E., Mateu, R., Šroubek, F., Pankrác, J., Widen, J. C., Bogyo, M., Netuka, D., Bušek, P., Šedo, A. 2024: 1-12

  Abstract

  The highly infiltrative growth of glioblastoma (GBM) makes distinction between the tumor and normal brain tissue challenging. Therefore, fluorescence-guided surgery is often used to improve visual identification of radiological tumor margins. The aim of this study was to evaluate the ability of recently developed molecularly targeted near-infrared (NIR) protease-activated probes to visualize GBM tissue and to compare the most promising candidate with the gold standard, 5-aminolevulinic acid (5-ALA).Single-substrate probes 6QC-ICG and 6QC-Cy5 (cysteine cathepsin cleavable), double-substrate probes AG2-FNIR and AG2-Cy5 (cysteine cathepsin and caspase 3 cleavable), and 5-ALA were administered intravenously to mice with orthotopic tumors. Activation of the probes was also evaluated in cell cultures in vitro and in biopsy material from patients with GBM ex vivo. The tumor to normal brain tissue fluorescence ratio (TNR) was quantified in brain sections using preclinical and clinical visualization platforms, and in tissue homogenates and cell suspensions using spectrofluorimetry. Subcellular localization of the fluorophores was visualized by confocal microscopy.In vitro, the single-substrate probe 6QC-ICG was cleaved in glioma cells and macrophages, and the resulting fluorophore accumulated intracellularly. In experimental GBMs, both single- and double-substrate probes visualized tumor tissue, while in healthy brain tissue the signal was minimal. TNR was highest for 6QC-ICG and AG2-FNIR, but the signal intensity was higher for 6QC-ICG. Using xenograft and syngeneic mouse models, as well as human GBM biopsy material ex vivo, the authors confirmed the ability of 6QC-ICG to specifically visualize the glioma tissue using preclinical and clinical visualization platforms. Finally, a comparison with 5-ALA in animals coadministered with both compounds revealed a higher TNR for 6QC-ICG in experimental GBMs.The cysteine cathepsin-cleavable probe 6QC-ICG is activated by glioma cells and tumor-associated macrophages, leading to a high contrast between tumor and nontumorous brain tissue that is superior to that of the current standard, 5-ALA. In addition to a well-defined mechanism of action, protease-activated probes that use NIR fluorophores (e.g., indocyanine green) have the advantage of low absorption and scattering of the NIR light and lower tissue autofluorescence. These results suggest that 6QC-ICG has the potential to become the targeted agent in intraoperative detection of GBM tissue using fluorescence imaging.

  View details for DOI 10.3171/2024.1.JNS231137

  View details for PubMedID 38552239

• Recent advances in ratiometric fluorescence imaging of enzyme activity in vivo. Current opinion in chemical biology Cosco, E. D., Bogyo, M. 2024; 80: 102441

  Abstract

  Among molecular imaging modalities that can monitor enzyme activity in vivo, optical imaging provides sensitive, molecular-level information at low-cost using safe and non-ionizing wavelengths of light. Yet, obtaining quantifiable optical signals in vivo poses significant challenges. Benchmarking using ratiometric signals can overcome dependence on dosing, illumination variability, and pharmacokinetics to provide quantitative in vivo optical data. This review highlights recent advances using fluorescent probes that are processed by enzymes to induce photophysical changes that can be monitored by ratiometric imaging. These diverse strategies include caged fluorophores that change photophysical properties upon enzymatic cleavage, as well as multi-fluorophore systems that are triggered by enzymatic cleavage to alter optical outputs in one or more fluorescent channels. The strategies discussed here have great potential for further development as well as potential broad applications for targeting diverse enzymes important for a wide range of human diseases.

  View details for DOI 10.1016/j.cbpa.2024.102441

  View details for PubMedID 38457961

• Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections. Journal of the American Chemical Society Jo, J., Upadhyay, T., Woods, E. C., Park, K. W., Pedowitz, N. J., Jaworek-Korjakowska, J., Wang, S., Valdez, T. A., Fellner, M., Bogyo, M. 2024

  Abstract

  Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.

  View details for DOI 10.1021/jacs.3c13974

  View details for PubMedID 38411555

• Mixed Alkyl/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets. bioRxiv : the preprint server for biology Bennett, J. M., Narwal, S. K., Kabeche, S., Abegg, D., Hackett, F., Yeo, T., Li, V. L., Muir, R. K., Faucher, F. F., Lovell, S., Blackman, M. J., Adibekian, A., Yeh, E., Fidock, D. A., Bogyo, M. 2024

  Abstract

  Malaria, caused by Plasmodium falciparum, remains a significant health burden. A barrier for developing anti-malarial drugs is the ability of the parasite to rapidly generate resistance. We demonstrated that Salinipostin A (SalA), a natural product, kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism with a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent anti-parasitic potencies which enabled identification of therapeutically relevant targets. We also confirm that this compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor, Orlistat. Like SalA, our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are a promising, synthetically tractable anti-malarials with a low-propensity to induce resistance.

  View details for DOI 10.1101/2024.01.11.575224

  View details for PubMedID 38260474

• Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of S. aureus Infections. bioRxiv : the preprint server for biology Jo, J., Upadhyay, T., Woods, E. C., Park, K. W., Pedowitz, N. J., Jaworek-Korjakowska, J., Wang, S., Valdez, T. A., Fellner, M., Bogyo, M. 2023

  Abstract

  Staphylococcus aureus is a major human pathogen responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here we describe the development of oxadiazolonebased activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologs in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling and mouse models of infection we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

  View details for DOI 10.1101/2023.12.11.571116

  View details for PubMedID 38168396

  View details for PubMedCentralID PMC10760020

• Covalent Macrocyclic Proteasome Inhibitors Mitigate Resistance in Plasmodium falciparum. ACS infectious diseases Bennett, J. M., Ward, K. E., Muir, R. K., Kabeche, S., Yoo, E., Yeo, T., Lam, G., Zhang, H., Almaliti, J., Berger, G., Faucher, F. F., Lin, G., Gerwick, W. H., Yeh, E., Fidock, D. A., Bogyo, M. 2023

  Abstract

  The Plasmodium proteasome is a promising antimalarial drug target due to its essential role in all parasite lifecycle stages. Furthermore, proteasome inhibitors have synergistic effects when combined with current first-line artemisinin and related analogues. Linear peptides that covalently inhibit the proteasome are effective at killing parasites and have a low propensity for inducing resistance. However, these scaffolds generally suffer from poor pharmacokinetics and bioavailability. Here we describe the development of covalent, irreversible, macrocyclic inhibitors of the Plasmodium falciparum proteasome. We identified compounds with excellent potency and low cytotoxicity; however, the first generation suffered from poor microsomal stability. Further optimization of an existing macrocyclic scaffold resulted in an irreversible covalent inhibitor carrying a vinyl sulfone electrophile that retained high potency and low cytotoxicity and had acceptable metabolic stability. Importantly, unlike the parent reversible inhibitor that selected for multiple mutations in the proteasome, with one resulting in a 5,000-fold loss of potency, the irreversible analogue only showed a 5-fold loss in potency for any single point mutation. Furthermore, an epoxyketone analogue of the same scaffold retained potency against a panel of known proteasome mutants. These results confirm that macrocycles are optimal scaffolds to target the malarial proteasome and that the use of a covalent electrophile can greatly reduce the ability of the parasite to generate drug resistance mutations.

  View details for DOI 10.1021/acsinfecdis.3c00310

  View details for PubMedID 37712594

• Chemoproteomic identification of a DPP4 homolog in Bacteroides thetaiotaomicron. Nature chemical biology Keller, L. J., Nguyen, T. H., Liu, L. J., Hurysz, B. M., Lakemeyer, M., Guerra, M., Gelsinger, D. J., Chanin, R., Ngo, N., Lum, K. M., Faucher, F., Ipock, P., Niphakis, M. J., Bhatt, A. S., O'Donoghue, A. J., Huang, K. C., Bogyo, M. 2023

  Abstract

  Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

  View details for DOI 10.1038/s41589-023-01357-8

  View details for PubMedID 37349583

  View details for PubMedCentralID 6108420

• Protease Activated Probes for Real-Time Ratiometric Imaging of Solid Tumors ACS CENTRAL SCIENCE Faucher, F. F., Liu, K. J., Cosco, E. D., Widen, J. C., Sorger, J., Guerra, M., Bogyo, M. 2023: 1059-1069

  Abstract

  Surgery is the preferred treatment option for most solid tumors. However, inaccurate detection of cancer borders leads to either incomplete removal of malignant cells or excess excision of healthy tissue. While fluorescent contrast agents and imaging systems improve tumor visualization, they can suffer from low signal-to-background and are prone to technical artifacts. Ratiometric imaging has the potential to eliminate many of these issues such as uneven probe distribution, tissue autofluorescence, and changes in positioning of the light source. Here, we describe a strategy to convert quenched fluorescent probes into ratiometric contrast agents. Conversion of the cathepsin-activated probe, 6QC-Cy5, into a two-fluorophore probe, 6QC-RATIO, significantly improved signal-to-background in vitro and in a mouse subcutaneous breast tumor model. Tumor detection sensitivity was further enhanced using a dual-substrate AND-gate ratiometric probe, Death-Cat-RATIO, that fluoresces only after orthogonal processing by multiple tumor-specific proteases. We also designed and built a modular camera system that was coupled to the FDA-approved da Vinci Xi robot, to enable real-time imaging of ratiometric signals at video frame rates compatible with surgical workflows. Our results demonstrate that ratiometric camera systems and imaging probes have the potential to be clinically implemented to improve surgical resection of many types of cancer.

  View details for DOI 10.1021/acscentsci.3c00261

  View details for Web of Science ID 000985613600001

  View details for PubMedID 37252358

  View details for PubMedCentralID PMC10214504

• Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome. Cell chemical biology Deni, I., Stokes, B. H., Ward, K. E., Fairhurst, K. J., Pasaje, C. F., Yeo, T., Akbar, S., Park, H., Muir, R., Bick, D. S., Zhan, W., Zhang, H., Liu, Y. J., Ng, C. L., Kirkman, L. A., Almaliti, J., Gould, A. E., Duffey, M., O'Donoghue, A. J., Uhlemann, A. C., Niles, J. C., da Fonseca, P. C., Gerwick, W. H., Lin, G., Bogyo, M., Fidock, D. A. 2023

  Abstract

  The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic β2 and β5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the β5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.

  View details for DOI 10.1016/j.chembiol.2023.03.002

  View details for PubMedID 36963402

• Solid Phase Synthesis of Fluorosulfate Containing Macrocycles for Chemoproteomic Workflows. bioRxiv : the preprint server for biology Faucher, F. F., Abegg, D., Ipock, P., Adibekian, A., Lovell, S., Bogyo, M. 2023

  Abstract

  Macrocyclic peptides are attractive for chemoproteomic applications due to their modular synthesis and potential for high target selectivity. We describe a solid phase synthesis method for the efficient generation of libraries of small macrocycles that contain an electrophile and alkyne handle. The modular synthesis produces libraries that can be directly screened using simple SDS-PAGE readouts and then optimal lead molecules applied to proteomic analysis. We generated a library of 480 macrocyclic peptides containing the weakly reactive fluorosulfate (OSF) electrophile. Initial screening of a subset of the library containing each of the various diversity elements identified initial molecules of interest. The corresponding positional and confirmational isomers were then screened to select molecules that showed specific protein labeling patterns that were dependent on the probe structure. The most promising hits were applied to standard chemoproteomic workflows to identify protein targets. Our results demonstrate the feasibility of rapid, on-resin synthesis of diverse macrocyclic electrophiles to generate new classes of covalent ligands.

  View details for DOI 10.1101/2023.02.17.529022

  View details for PubMedID 36824748

  View details for PubMedCentralID PMC9949109

• Solid Phase Synthesis of Fluorosulfate Containing Macrocycles for Chemoproteomic Workflows Israel Journal of Chemistry Faucher, F., Abegg, D., Ipock, P., Adibekian, A., Lovell, S., Bogyo, M. 2023

  View details for DOI 10.1002/ijch.202300020

• Finding optimal drug target sites in parasite pathogens. Trends in parasitology Bogyo, M. 2022

  Abstract

  Benns et al. have recently combined a chemoproteomic profiling method with a CRISPR-based gene-editing method to identify chemically targetable residues essential for fitness in the parasite Toxoplasma gondii. The result is a strategy that enables rapid discovery of new drug targets to combat T. gondii and other related parasites.

  View details for DOI 10.1016/j.pt.2022.12.003

  View details for PubMedID 36564249

• Trypanosoma brucei Acyl-Protein Thioesterase-like (TbAPT-L) Is a Lipase with Esterase Activity for Short and Medium-Chain Fatty Acids but Has No Depalmitoylation Activity. Pathogens (Basel, Switzerland) Brown, R. W., Sharma, A. I., Villanueva, M. R., Li, X., Onguka, O., Zilbermintz, L., Nguyen, H., Falk, B. A., Olson, C. L., Taylor, J. M., Epting, C. L., Kathayat, R. S., Amara, N., Dickinson, B. C., Bogyo, M., Engman, D. M. 2022; 11 (11)

  Abstract

  Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. S-acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in Trypanosoma brucei parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in Trypanosoma brucei which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth in vitro, or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.

  View details for DOI 10.3390/pathogens11111245

  View details for PubMedID 36364996

• The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection. Science (New York, N.Y.) Richards, C. M., Jabs, S., Qiao, W., Varanese, L. D., Schweizer, M., Mosen, P. R., Riley, N. M., Klüssendorf, M., Zengel, J. R., Flynn, R. A., Rustagi, A., Widen, J. C., Peters, C. E., Ooi, Y. S., Xie, X., Shi, P. Y., Bartenschlager, R., Puschnik, A. S., Bogyo, M., Bertozzi, C. R., Blish, C. A., Winter, D., Nagamine, C. M., Braulke, T., Carette, J. E. 2022: eabn5648

  Abstract

  Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. Here, we used genome-scale CRISPR screens to identify Lysosomal Enzyme Trafficking factor (LYSET) as essential for infection by cathepsin-dependent viruses including SARS-CoV-2. LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery, and mutations in LYSET can explain the phenotype of the associated disorder.

  View details for DOI 10.1126/science.abn5648

  View details for PubMedID 36074821

• Formulation of a Thermosensitive Imaging Hydrogel for Topical Application and Rapid Visualization of Tumor Margins in the Surgical Cavity. Cancers Walker, E., Linders, D. G., Abenojar, E., Wang, X., Hazelbag, H. M., Straver, M. E., Bijlstra, O. D., March, T. L., Vahrmeijer, A. L., Exner, A., Bogyo, M., Basilion, J. P., Straight, B. 2022; 14 (14)

  Abstract

  BACKGROUND: Tumor-positive surgical margins during primary breast cancer (BCa) surgery are associated with a two-fold increase in the risk of local recurrence when compared with tumor-negative margins. Pathological microscopic evaluation of the samples only assesses about 1/10 of 1% of the entire volume of the removed BCa specimens, leading to margin under-sampling and potential local recurrence in patients with pathologically clean margins, i.e., false negative margins. In the case of tumor-positive margins, patients need to undergo re-excision and/or radiation therapy, resulting in increases in complications, morbidity, and healthcare costs. Development of a simple real-time imaging technique to identify residual BCa in the surgical cavity rapidly and precisely could significantly improve the quality of care.METHODS: A small-molecule, fluorescently quenched protease-substrate probe, AKRO-QC-ICG, was tested as part of a thermosensitive imaging gel formulated for topical application and imaging of the BCa surgical cavity.RESULTS: More than forty formulations of gel mixtures were investigated to enable easy fluid application and subsequent solidification once applied, preventing dripping and pooling in the surgical cavity. The final formulation was tested using human BCa orthotopic implants in nude and NSG patient-derived xenografts (PDX) mice. This formulation of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel was found to be a good solvent for the probe, with a desirable thermo-reversible solid-gel transition and mechanical strength for distribution of AKRO-QC-ICG on the surfaces of tissue. It demonstrated excellent ability to detect BCa tissue after 10 min exposure, with a high signal-to-noise ratio both in mouse xenografts and freshly excised human lumpectomy tissue. The in vivo efficacy of the AKRO-QC-ICG imaging gel to detect BCa revealed the levels of sensitivity/specificity = 0.92/1 in 12 nude mice, which was corroborated with the sensitivity/specificity = 0.94/1 in 10 PDX mice.CONCLUSIONS: Utilization of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel for topical application to detect BCa in the surgical cavity during surgery has the potential to reduce re-excisions, with consequent savings in healthcare costs and enhancement in patient quality of life.

  View details for DOI 10.3390/cancers14143459

  View details for PubMedID 35884520

• A cathepsin targeted quenched activity-based probe facilitates enhanced detection of human tumors during resection. Clinical cancer research : an official journal of the American Association for Cancer Research Kennedy, G. T., Holt, D. E., Azari, F. S., Bernstein, E., Nadeem, B., Chang, A., Sullivan, N. T., Segil, A., Deshpande, C., Bensen, E., Santini, J. T., Kucharczuk, J. C., Delikatny, E. J., Bogyo, M., Egan, A. J., Bradley, C. W., Eruslanov, E., Lickliter, J. D., Wright, G., Singhal, S. 2022

  Abstract

  PURPOSE: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity-based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity-based probe (VGT-309) for fluorescence-guided surgery.EXPERIMENTAL DESIGN: We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery.RESULTS: In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR range: 2.15-3.71). In the Phase 1 human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase 2 trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs= 2.83 & 7.18) in real-time to illustrate its successful clinical translation and potential to improve surgical management.CONCLUSIONS: This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors.

  View details for DOI 10.1158/1078-0432.CCR-22-1215

  View details for PubMedID 35792882

• Uncovering an overlooked consequence of phosphorylation: change in cysteine reactivity. Nature methods Lakemeyer, M., Bogyo, M. 2022

  View details for DOI 10.1038/s41592-022-01414-5

  View details for PubMedID 35228728

• Activity-Based Diagnostics: Recent Advances in the Development of Probes for Use with Diverse Detection Modalities. ACS chemical biology Muir, R. K., Guerra, M., Bogyo, M. M. 1800

  Abstract

  Abnormal enzyme expression and activity is a hallmark of many diseases. Activity-based diagnostics are a class of chemical probes that aim to leverage this dysregulated metabolic signature to produce a detectable signal specific to diseased tissue. In this Review, we highlight recent methodologies employed in activity-based diagnostics that provide exquisite signal sensitivity and specificity in complex biological systems for multiple disease states. We divide these examples based upon their unique signal readout modalities and highlight those that have advanced into clinical trials.

  View details for DOI 10.1021/acschembio.1c00753

  View details for PubMedID 35026106

• Integration of bioinformatic and chemoproteomic tools for the study of enzyme conservation in closely related bacterial species. Methods in enzymology Keller, L. J., Lakemeyer, M., Bogyo, M. 2022; 664: 1-22

  Abstract

  Activity-based protein profiling (ABPP) is a commonly utilized technique to globally characterize the endogenous activity of multiple enzymes within a related family. While it has been used extensively to identify enzymes that are differentially active across various mammalian tissues, recent efforts have expanded this technique to studying bacteria. As ABPP is applied to diverse sets of bacterial strains found in microbial communities, there is also an increasing need for robust tools for assessing the conservation of enzymes across closely related bacterial species and strains. In this chapter, we detail the integration of gel-based ABPP with basic bioinformatic tools to enable the analysis of enzyme activity, distribution, and homology. We use as an example the family of serine hydrolases identified in the skin commensal bacterium Staphylococcus epidermidis.

  View details for DOI 10.1016/bs.mie.2021.11.017

  View details for PubMedID 35331369

• A 'Swiss army knife' probe for metastatic cancers. Nature materials Bogyo, M. 2021; 20 (10): 1312-1314

  View details for DOI 10.1038/s41563-021-01105-0

  View details for PubMedID 34561628

• Chemiluminescent Protease Probe for Rapid, Sensitive, and Inexpensive Detection of Live Mycobacterium tuberculosis. ACS central science Babin, B. M., Fernandez-Cuervo, G., Sheng, J., Green, O., Ordonez, A. A., Turner, M. L., Keller, L. J., Jain, S. K., Shabat, D., Bogyo, M. 2021; 7 (5): 803-814

  Abstract

  Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, be cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a fast, luminescent, and affordable sensor of Hip1 (FLASH) for detecting and monitoring drug susceptibility of Mycobacterium tuberculosis (Mtb). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that, when processed, produces visible light that can be measured with a high signal-to-noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other nontuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings.

  View details for DOI 10.1021/acscentsci.0c01345

  View details for PubMedID 34079897

• Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility. mSphere Rompikuntal, P. K., Kent, R. S., Foe, I. T., Deng, B., Bogyo, M., Ward, G. E. 2021; 6 (3)

  Abstract

  Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of substrate-dependent gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin motor consisting of a class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. Therefore, the binding of TgMLC1 to TgGAP45 is considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation completely abrogate the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete.IMPORTANCE Gliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention or treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely held "linear motor" model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of, and between cells of the hosts they infect.

  View details for DOI 10.1128/mSphere.00823-20

  View details for PubMedID 34011689

• Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19. ACS infectious diseases Steuten, K., Kim, H., Widen, J. C., Babin, B. M., Onguka, O., Lovell, S., Bolgi, O., Cerikan, B., Neufeldt, C. J., Cortese, M., Muir, R. K., Bennett, J. M., Geiss-Friedlander, R., Peters, C., Bartenschlager, R., Bogyo, M. 2021

  Abstract

  Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsins L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

  View details for DOI 10.1021/acsinfecdis.0c00815

  View details for PubMedID 33570381

• A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions. Proceedings of the National Academy of Sciences of the United States of America Yim, J. J., Harmsen, S., Flisikowski, K., Flisikowska, T., Namkoong, H., Garland, M., van den Berg, N. S., Vilches-Moure, J. G., Schnieke, A., Saur, D., Glasl, S., Gorpas, D., Habtezion, A., Ntziachristos, V., Contag, C. H., Gambhir, S. S., Bogyo, M., Rogalla, S. 2021; 118 (1)

  Abstract

  Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 m, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

  View details for DOI 10.1073/pnas.2008072118

  View details for PubMedID 33443161

• The Thyroid Hormone Transporter Mct8 Restricts Cathepsin-Mediated Thyroglobulin Processing in Male Mice through Thyroid Auto-Regulatory Mechanisms That Encompass Autophagy. International journal of molecular sciences Venugopalan, V. n., Al-Hashimi, A. n., Rehders, M. n., Golchert, J. n., Reinecke, V. n., Homuth, G. n., Völker, U. n., Manirajah, M. n., Touzani, A. n., Weber, J. n., Bogyo, M. S., Verrey, F. n., Wirth, E. K., Schweizer, U. n., Heuer, H. n., Kirstein, J. n., Brix, K. n. 2021; 22 (1)

  Abstract

  The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.

  View details for DOI 10.3390/ijms22010462

  View details for PubMedID 33466458

• Identification of covalent inhibitors that disrupt M. tuberculosis growth by targeting multiple serine hydrolases involved in lipid metabolism. Cell chemical biology Babin, B. M., Keller, L. J., Pinto, Y., Li, V. L., Eneim, A. S., Vance, S. E., Terrell, S. M., Bhatt, A. S., Long, J. Z., Bogyo, M. 2021

  Abstract

  The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screen a library of small molecules containing serine-reactive electrophiles and identify narrow-spectrum inhibitors of M. tuberculosis growth. Using these lead molecules, we perform competitive activity-based protein profiling and identify multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures reveal an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis reveals a path to resistance via the synthesis of mycocerates, but not through mutations to SH targets. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections.

  View details for DOI 10.1016/j.chembiol.2021.08.013

  View details for PubMedID 34599874

• Toxoplasma gondii serine hydrolases regulate parasite lipid mobilization during growth and replication within the host. Cell chemical biology Onguka, O., Babin, B. M., Lakemeyer, M., Foe, I. T., Amara, N., Terrell, S. M., Lum, K. M., Cieplak, P., Niphakis, M. J., Long, J. Z., Bogyo, M. 2021

  Abstract

  The intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here, we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short-chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small-molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications.

  View details for DOI 10.1016/j.chembiol.2021.05.001

  View details for PubMedID 34043961

• Selective activation of PFKL suppresses the phagocytic oxidative burst. Cell Amara, N., Cooper, M. P., Voronkova, M. A., Webb, B. A., Lynch, E. M., Kollman, J. M., Ma, T., Yu, K., Lai, Z., Sangaraju, D., Kayagaki, N., Newton, K., Bogyo, M., Staben, S. T., Dixit, V. M. 2021

  Abstract

  In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.

  View details for DOI 10.1016/j.cell.2021.07.004

  View details for PubMedID 34320407

• Procathepsin V Is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe. International journal of molecular sciences Al-Hashimi, A., Venugopalan, V., Rehders, M., Sereesongsaeng, N., Hein, Z., Springer, S., Weber, E., Fuhrer, D., Bogyo, M. S., Scott, C. J., Burden, R. E., Brix, K. 2020; 21 (23)

  Abstract

  The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.

  View details for DOI 10.3390/ijms21239140

  View details for PubMedID 33266306

• Pre-Trained Deep Convolutional Neural Network for Clostridioides Difficile Bacteria Cytotoxicity Classification Based on Fluorescence Images. Sensors (Basel, Switzerland) Brodzicki, A., Jaworek-Korjakowska, J., Kleczek, P., Garland, M., Bogyo, M. 2020; 20 (23)

  Abstract

  Clostridioides difficile infection (CDI) is an enteric bacterial disease that is increasing in incidence worldwide. Symptoms of CDI range from mild diarrhea to severe life-threatening inflammation of the colon. While antibiotics are standard-of-care treatments for CDI, they are also the biggest risk factor for development of CDI and recurrence. Therefore, novel therapies that successfully treat CDI and protect against recurrence are an unmet clinical need. Screening for novel drug leads is often tested by manual image analysis. The process is slow, tedious and is subject to human error and bias. So far, little work has focused on computer-aided screening for drug leads based on fluorescence images. Here, we propose a novel method to identify characteristic morphological changes in human fibroblast cells exposed to C. difficile toxins based on computer vision algorithms supported by deep learning methods. Classical image processing algorithms for the pre-processing stage are used together with an adjusted pre-trained deep convolutional neural network responsible for cell classification. In this study, we take advantage of transfer learning methodology by examining pre-trained VGG-19, ResNet50, Xception, and DenseNet121 convolutional neural network (CNN) models with adjusted, densely connected classifiers. We compare the obtained results with those of other machine learning algorithms and also visualize and interpret them. The proposed models have been evaluated on a dataset containing 369 images with 6112 cases. DenseNet121 achieved the highest results with a 93.5% accuracy, 92% sensitivity, and 95% specificity, respectively.

  View details for DOI 10.3390/s20236713

  View details for PubMedID 33255305

• Identification of highly selective covalent inhibitors by phage display. Nature biotechnology Chen, S., Lovell, S., Lee, S., Fellner, M., Mace, P. D., Bogyo, M. 2020

  Abstract

  Molecules that covalently bind macromolecular targets have found widespread applications as activity-based probes and as irreversibly binding drugs. However, the general reactivity of the electrophiles needed for covalent bond formation makes control of selectivity difficult. There is currently no rapid, unbiased screening method to identify new classes of covalent inhibitors from highly diverse pools of candidate molecules. Here we describe a phage display method to directly screen for ligands that bind to protein targets through covalent bond formation. This approach makes use of a reactive linker to form cyclic peptides on the phage surface while simultaneously introducing an electrophilic 'warhead' to covalently react with a nucleophile on the target. Using this approach, we identified cyclic peptides that irreversibly inhibited a cysteine protease and a serine hydrolase with nanomolar potency and exceptional specificity. This approach should enable rapid, unbiased screening to identify new classes of highly selective covalent inhibitors for diverse molecular targets.

  View details for DOI 10.1038/s41587-020-0733-7

  View details for PubMedID 33199876

• Short-Wave Infrared Fluorescence Chemical Sensor for Detection of Otitis Media. ACS sensors Yim, J. J., Singh, S. P., Xia, A., Kashfi-Sadabad, R., Tholen, M., Huland, D. M., Zarabanda, D., Cao, Z., Solis-Pazmino, P., Bogyo, M., Valdez, T. A. 2020

  Abstract

  Otitis media (OM) or middle ear infection is one of the most common diseases in young children around the world. The diagnosis of OM is currently performed using an otoscope to detect middle ear fluid and inflammatory changes manifested in the tympanic membrane. However, conventional otoscopy cannot visualize across the tympanic membrane or sample middle ear fluid. This can lead to low diagnostic certainty and overdiagnoses of OM. To improve the diagnosis of OM, we have developed a short-wave infrared (SWIR) otoscope in combination with a protease-cleavable biosensor, 6QC-ICG, which can facilitate the detection of inflammatory proteases in the middle ear with an increase in contrast. 6QC-ICG is a fluorescently quenched probe, which is activated in the presence of cysteine cathepsin proteases that are up-regulated in inflammatory immune cells. Using a preclinical model and custom-built SWIR otomicroscope in this proof-of-concept study, we successfully demonstrated the feasibility of robustly distinguishing inflamed ears from controls (p = 0.0006). The inflamed ears showed an overall signal-to-background ratio of 2.0 with a mean fluorescence of 81 ± 17 AU, while the control ear exhibited a mean fluorescence of 41 ± 11 AU. We envision that these fluorescently quenched probes in conjunction with SWIR imaging tools have the potential to be used as an alternate/adjunct tool for objective diagnosis of OM.

  View details for DOI 10.1021/acssensors.0c01272

  View details for PubMedID 33175516

• Fluorescent image-guided surgery in breast cancer by intravenous application of a quenched fluorescence activity-based probe for cysteine cathepsins in a syngeneic mouse model. EJNMMI research Suurs, F. V., Qiu, S., Yim, J. J., Schroder, C. P., Timmer-Bosscha, H., Bensen, E. S., Santini, J. T., de Vries, E. G., Bogyo, M., van Dam, G. M. 2020; 10 (1): 111

  Abstract

  PURPOSE: The reoperation rate for breast-conserving surgery is as high as 15-30% due to residual tumor in the surgical cavity after surgery. In vivo tumor-targeted optical molecular imaging may serve as a red-flag technique to improve intraoperative surgical margin assessment and to reduce reoperation rates. Cysteine cathepsins are overexpressed in most solid tumor types, including breast cancer. We developed a cathepsin-targeted, quenched fluorescent activity-based probe, VGT-309, and evaluated whether it could be used for tumor detection and image-guided surgery in syngeneic tumor-bearing mice.METHODS: Binding specificity of the developed probe was evaluated in vitro. Next, fluorescent imaging in BALB/c mice bearing a murine breast tumor was performed at different time points after VGT-309 administration. Biodistribution of VGT-309 after 24h in tumor-bearing mice was compared to control mice. Image-guided surgery was performed at multiple time points tumors with different clinical fluorescent camera systems and followed by ex vivo analysis.RESULTS: The probe was specifically activated by cathepsins X, B/L, and S. Fluorescent imaging revealed an increased tumor-to-background contrast over time up to 15.1 24h post probe injection. In addition, VGT-309 delineated tumor tissue during image-guided surgery with different optical fluorescent imaging camera systems.CONCLUSION: These results indicate that optical fluorescent molecular imaging using the cathepsin-targeted probe, VGT-309, may improve intraoperative tumor detection, which could translate to more complete tumor resection when coupled with commercially available surgical tools and techniques.

  View details for DOI 10.1186/s13550-020-00688-0

  View details for PubMedID 32990883

• The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis. PLoS pathogens Bilverstone, T. W., Garland, M., Cave, R. J., Kelly, M. L., Tholen, M., Bouley, D. M., Kaye, P., Minton, N. P., Bogyo, M., Kuehne, S. A., Melnyk, R. A. 2020; 16 (9): e1008852

  Abstract

  Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Deltaerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630DeltaermDeltatcdA (DeltatcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or DeltatcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.

  View details for DOI 10.1371/journal.ppat.1008852

  View details for PubMedID 32960931

• Structural Basis for the Inhibitor and Substrate Specificity of the Unique Fph Serine Hydrolases of Staphylococcus aureus. ACS infectious diseases Fellner, M., Lentz, C. S., Jamieson, S. A., Brewster, J. L., Chen, L., Bogyo, M., Mace, P. D. 2020

  Abstract

  Staphylococcus aureus is a prevalent bacterial pathogen in both community and hospital settings, and its treatment is made particularly difficult by resilience within biofilms. Within this niche, serine hydrolase enzymes play a key role in generating and maintaining the biofilm matrix. Activity-based profiling has previously identified a family of serine hydrolases, designated fluorophosphonate-binding hydrolases (Fph's), some of which contribute to the virulence of S. aureus in vivo. These 10 Fph proteins have limited annotation and have few, if any, characterized bacterial or mammalian homologues. This suggests unique hydrolase functions even within bacterial species. Here we report structures of one of the most abundant Fph family members, FphF. Our structures capture FphF alone, covalently bound to a substrate analogue and bound to small molecule inhibitors that occupy the hydrophobic substrate-binding pocket. In line with these findings, we show that FphF has promiscuous esterase activity toward hydrophobic lipid substrates. We present docking studies that characterize interactions of inhibitors and substrates within the active site environment, which can be extended to other Fph family members. Comparison of FphF to other esterases and the wider Fph protein family suggest that FphF forms a new esterase subfamily. Our data suggest that other Fph enzymes, including the virulence factor FphB, are likely to have more restricted substrate profiles than FphF. This work demonstrates a clear molecular rationale for the specificity of fluorophosphonate probes that target FphF and provides a structural template for the design of enhanced probes and inhibitors of the Fph family of serine hydrolases.

  View details for DOI 10.1021/acsinfecdis.0c00503

  View details for PubMedID 32865965

• Strategies for Tuning the Selectivity of Chemical Probes that Target Serine Hydrolases. Cell chemical biology Faucher, F., Bennett, J. M., Bogyo, M., Lovell, S. 2020

  Abstract

  Serine hydrolases comprise a large family of enzymes that have diverse roles in key cellular processes, such as lipid metabolism, cell signaling, and regulation of post-translation modifications of proteins. They are also therapeutic targets for multiple human pathologies, including viral infection, diabetes, hypertension, and Alzheimer disease; however, few have well-defined substrates and biological functions. Activity-based probes (ABPs) have been used as effective tools to both profile activity and screen for selective inhibitors of serine hydrolases. One broad-spectrum ABP containing a fluorophosphonate electrophile has been used extensively to advance our understanding of diverse serine hydrolases. Due to the success of this single reagent, several robust chemistries have been developed to further diversify and tune the selectivity of ABPs used to target serine hydrolases. In this review, we highlight approaches to identify selective serine hydrolase ABPs and suggest new synthetic methodologies that could be applied to further advance probe development.

  View details for DOI 10.1016/j.chembiol.2020.07.008

  View details for PubMedID 32726586

• The Clinical Drug Ebselen Attenuates Inflammation and Promotes Microbiome Recovery in Mice after Antibiotic Treatment for CDI. Cell reports medicine Garland, M., Hryckowian, A. J., Tholen, M., Bender, K. O., Van Treuren, W. W., Loscher, S., Sonnenburg, J. L., Bogyo, M. 2020; 1 (1)

  Abstract

  Clostridium difficile infection (CDI) is an enteric bacterial disease that is increasing in prevalence worldwide. C. difficile capitalizes on gut inflammation and microbiome dysbiosis to establish infection, with symptoms ranging from watery diarrhea to toxic megacolon. We reported that the safe-in-human clinical drug ebselen (ClinicalTrials.gov: NCT03013400, NCT01452607, NCT00762671, and NCT02603081) has biochemical, cell-based, and in vivo efficacy against the toxins of C. difficile. Here, we show that ebselen treatment reduces recurrence rates and decreases colitis in a hamster model of relapsing CDI. Furthermore, ebselen treatment does not alter microbiome diversity and promotes recovery back to that of healthy controls after antibiotic-induced dysbiosis in healthy and C. difficile-infected mice. This increased microbiome recovery upon ebselen treatment correlates with a decrease in host-derived inflammatory markers, suggesting that the anti-inflammatory properties of ebselen, combined with its anti-toxin function, help to mitigate the major clinical challenges of CDI, including recurrence, microbial dysbiosis, and colitis.

  View details for DOI 10.1016/j.xcrm.2020.100005

  View details for PubMedID 32483557

• A Protease-Activated Fluorescent Probe Allows Rapid Visualization of Keratinocyte Carcinoma During Excision. Cancer research Walker, E., Liu, Y., Kim, I., Biro, M., Iyer, S. R., Ezaldein, H., Scott, J., Merati, M., Mistur, R., Zhou, B., Straight, B., Yim, J. J., Bogyo, M., Mann, M., Wilson, D. L., Basilion, J. P., Popkin, D. L. 2020

  Abstract

  Keratinocyte carcinomas (KC), including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all KC (4 million annual cases in the US) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathological assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to ninety specimens ex vivo immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence resulting in sensitivities of 62-78% and specificities of 92-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day re-excision when needed, reducing patient anxiety and improving compliance by expediting post-surgical specimen assessment.

  View details for DOI 10.1158/0008-5472.CAN-19-3067

  View details for PubMedID 32132111

• The Antimalarial Natural Product Salinipostin A Identifies Essential α/β Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites. Cell chemical biology Yoo, E. n., Schulze, C. J., Stokes, B. H., Onguka, O. n., Yeo, T. n., Mok, S. n., Gnädig, N. F., Zhou, Y. n., Kurita, K. n., Foe, I. T., Terrell, S. M., Boucher, M. J., Cieplak, P. n., Kumpornsin, K. n., Lee, M. C., Linington, R. G., Long, J. Z., Uhlemann, A. C., Weerapana, E. n., Fidock, D. A., Bogyo, M. n. 2020

  Abstract

  Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/β serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

  View details for DOI 10.1016/j.chembiol.2020.01.001

  View details for PubMedID 31978322

• Characterization of Serine Hydrolases Across Clinical Isolates of Commensal Skin Bacteria Staphylococcus epidermidis Using Activity-Based Protein Profiling. ACS infectious diseases Keller, L. J., Lentz, C. S., Chen, Y. E., Metivier, R. J., Weerapana, E. n., Fischbach, M. A., Bogyo, M. n. 2020

  Abstract

  The bacterial genus Staphylococcus comprises diverse species that colonize the skin as commensals but can also cause infection. Previous work identified a family of serine hydrolases termed fluorophoshonate-binding hydrolases (Fphs) in the pathogenic bacteria Staphylococcus aureus, one of which, FphB, functions as a virulence factor. Using a combination of bioinformatics and activity-based protein profiling (ABPP), we identify homologues of these enzymes in the related commensal bacteria Staphylococcus epidermidis. Two of the S. aureus Fph enzymes were not identified in S. epidermidis. Using ABPP, we identified several candidate hydrolases that were not previously identified in S. aureus that may be functionally related to the Fphs. Interestingly, the activity of the Fphs vary across clinical isolates of S. epidermidis. Biochemical characterization of the FphB homologue in S. epidermidis (SeFphB) suggests it is a functional homologue of FphB in S. aureus, but our preliminary studies suggest it may not have a role in colonization in vivo. This potential difference in biological function between the Fphs of closely related staphylococcal species may provide mechanisms for specific inhibition of S. aureus infection without perturbing commensal communities of related bacteria.

  View details for DOI 10.1021/acsinfecdis.0c00095

  View details for PubMedID 32298574

• Discovery of small molecules that normalize the transcriptome and enhance cysteine cathepsin activity in progranulin-deficient microglia. Scientific reports Telpoukhovskaia, M. A., Liu, K. n., Sayed, F. A., Etchegaray, J. I., Xie, M. n., Zhan, L. n., Li, Y. n., Zhou, Y. n., Le, D. n., Bahr, B. A., Bogyo, M. n., Ding, S. n., Gan, L. n. 2020; 10 (1): 13688

  Abstract

  Patients with frontotemporal dementia (FTD) resulting from granulin (GRN) haploinsufficiency have reduced levels of progranulin and exhibit dysregulation in inflammatory and lysosomal networks. Microglia produce high levels of progranulin, and reduction of progranulin in microglia alone is sufficient to recapitulate inflammation, lysosomal dysfunction, and hyperproliferation in a cell-autonomous manner. Therefore, targeting microglial dysfunction caused by progranulin insufficiency represents a potential therapeutic strategy to manage neurodegeneration in FTD. Limitations of current progranulin-enhancing strategies necessitate the discovery of new targets. To identify compounds that can reverse microglial defects in Grn-deficient mouse microglia, we performed a compound screen coupled with high throughput sequencing to assess key transcriptional changes in inflammatory and lysosomal pathways. Positive hits from this initial screen were then further narrowed down based on their ability to rescue cathepsin activity, a critical biochemical readout of lysosomal capacity. The screen identified nor-binaltorphimine dihydrochloride (nor-BNI) and dibutyryl-cAMP, sodium salt (DB-cAMP) as two phenotypic modulators of progranulin deficiency. In addition, nor-BNI and DB-cAMP also rescued cell cycle abnormalities in progranulin-deficient cells. These data highlight the potential of a transcription-based platform for drug screening, and advance two novel lead compounds for FTD.

  View details for DOI 10.1038/s41598-020-70534-9

  View details for PubMedID 32792571

• Methods for analysis of near-infrared (NIR) quenched-fluorescent contrast agents in mouse models of cancer. Methods in enzymology Widen, J. C., Tholen, M., Yim, J. J., Bogyo, M. 2020; 639: 141–66

  Abstract

  Optical contrast agents containing near-infrared (NIR) fluorophores are useful for visualizing biological landmarks, enzyme activities and biological processes in live animals and humans. Activatable (smart) quenched-fluorescent probes are sensors that become fluorescent after processing by an enzyme or in response to a physiological change (i.e., pH, ROS, etc.). Recently, there has been increased interest in developing activatable probes for research and clinical applications. This requires evaluation using in vivo animal models to gain insights into the pharmacodynamic and pharmacokinetic properties of a given probe. Important parameters to measure when evaluating quenched-fluorescent probes are signal brightness and signal-to-background ratios, which define the sensitivity and specificity of a probe. In this chapter, we discuss methods to evaluate activatable quenched-fluorescent probes in mouse models of cancer. Quantification of fluorescent signal intensity, calculation of tumor-to-background ratios, comparison of fluorescent activation in specific organ compartments, and fluorescence scanning of sectioned tissue will be discussed.

  View details for DOI 10.1016/bs.mie.2020.04.012

  View details for PubMedID 32475399

• AND-gate contrast agents for enhanced fluorescence-guided surgery. Nature biomedical engineering Widen, J. C., Tholen, M. n., Yim, J. J., Antaris, A. n., Casey, K. M., Rogalla, S. n., Klaassen, A. n., Sorger, J. n., Bogyo, M. n. 2020

  Abstract

  Surgical resection of tumours requires precisely locating and defining the margins between lesions and normal tissue. However, this is made difficult by irregular margin borders. Although molecularly targeted optical contrast agents can be used to define tumour margins during surgery in real time, the selectivity of the contrast agents is often limited by the target being expressed in both healthy and tumour tissues. Here, we show that AND-gate optical imaging probes that require the processing of two substrates by multiple tumour-specific enzymes produce a fluorescent signal with significantly improved specificity and sensitivity to tumour tissue. We evaluated the performance of the probes in mouse models of mammary tumours and of metastatic lung cancer, as well as during fluorescence-guided robotic surgery. Imaging probes that rely on multivariate activation to selectively target complex patterns of enzymatic activity should be useful in disease detection, treatment and monitoring.

  View details for DOI 10.1038/s41551-020-00616-6

  View details for PubMedID 32989286

• Design of optical imaging probes by screening of diverse substrate libraries directly in disease tissue extracts. Angewandte Chemie (International ed. in English) Tholen, M. n., Yim, J. J., Groborz, K. n., Yoo, E. n., Martin, B. A., van den Berg, N. S., Drag, M. n., Bogyo, M. n. 2020

  Abstract

  Fluorescently-quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis, early detection and surgical guidance. These probes are often designed to target specific enzymes associated with disease by direct optimization using single purified enzymes. However, this can result in painstaking chemistry efforts to produce a probe with suboptimal performance when applied in vivo. We describe here an alternate, unbiased activity-profiling approach in which whole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of tumor extracts with a hybrid combinatorial substrate library (HyCoSuL) identified a combination of natural and non-natural amino acid residues that was used to generate highly efficient tumor-specific probes. This new strategy simplifies and enhances the process of probe optimization without any a priori knowledge of enzyme targets and has the potential to be applied to diverse disease states using clinical or animal model tissues samples.

  View details for DOI 10.1002/anie.202006719

  View details for PubMedID 32589815

• Activity-based protein profiling in bacteria: Applications for identification of therapeutic targets and characterization of microbial communities. Current opinion in chemical biology Keller, L. J., Babin, B. M., Lakemeyer, M., Bogyo, M. 2019; 54: 45–53

  Abstract

  Activity-based protein profiling (ABPP) is a robust chemoproteomic technique that uses activity-based probes to globally measure endogenous enzymatic activity in complex proteomes. It has been utilized extensively to characterize human disease states and identify druggable targets in diverse disease conditions. ABPP has also recently found applications in microbiology. This includes using activity-based probes (ABPs) for functional studies of pathogenic bacteria as well as complex communities within a microbiome. This review will focus on recent advances in the use of ABPs to profile enzyme activity in disease models, screen for selective inhibitors of key enzymes, and develop imaging tools to better understand the host-bacterial interface.

  View details for DOI 10.1016/j.cbpa.2019.10.007

  View details for PubMedID 31835131

• Leveraging Peptide Substrate Libraries to Design Inhibitors of Bacterial Lon Protease ACS CHEMICAL BIOLOGY Babin, B. M., Kasperkiewicz, P., Janiszewski, T., Yoo, E., Drag, M., Bogyo, M. 2019; 14 (11): 2453–62

  Abstract

  Lon is a widely conserved housekeeping protease found in all domains of life. Bacterial Lon is involved in recovery from various types of stress, including tolerance to fluoroquinolone antibiotics, and is linked to pathogenesis in a number of organisms. However, detailed functional studies of Lon have been limited by the lack of selective, cell-permeant inhibitors. Here, we describe the use of positional scanning libraries of hybrid peptide substrates to profile the primary sequence specificity of bacterial Lon. In addition to identifying optimal natural amino acid binding preferences, we identified several non-natural residues that were leveraged to develop optimal peptide substrates as well as a potent peptidic boronic acid inhibitor of Lon. Treatment of Escherichia coli with this inhibitor promotes UV-induced filamentation and reduces tolerance to ciprofloxacin, phenocopying established lon-deletion phenotypes. It is also nontoxic to mammalian cells due to its selectivity for Lon over the proteasome. Our results provide new insight into the primary substrate specificity of Lon and identify substrates and an inhibitor that will serve as useful tools for dissecting the diverse cellular functions of Lon.

  View details for DOI 10.1021/acschembio.9b00529

  View details for Web of Science ID 000497263200011

  View details for PubMedID 31464417

  View details for PubMedCentralID PMC6858493

• Synthetic and biological approaches to map substrate specificities of proteases. Biological chemistry Chen, S., Yim, J. J., Bogyo, M. 2019

  Abstract

  Proteases are regulators of diverse biological pathways including protein catabolism, antigen processing and inflammation, as well as various diseases conditions, such as malignant metastasis, viral infection, and parasite invasion. The identification of substrates of a given protease is essential to understand its function and this information can also aid in the design of specific inhibitors and active site probes. However, the diversity of putative protein and peptide substrates makes connecting a protease to its downstream substrates technically difficult and time-consuming. To address this challenge in protease research, a range of methods have been developed to identify natural protein substrates as well as map the overall substrate specificity patterns of proteases. In this review, we highlight recent examples of both synthetic and biological methods that are being used to define the substrate specificity of protease so that new protease-specific tools and therapeutic agents can be developed.

  View details for DOI 10.1515/hsz-2019-0332

  View details for PubMedID 31639098

• Treatment of rat thyrocytes in vitro with cathepsin B and L inhibitors results in disruption of primary cilia leading to redistribution of the trace amine associated receptor 1 to the endoplasmic reticulum. Biochimie Szumska, J., Batool, Z., Al-Hashimi, A., Venugopalan, V., Skripnik, V., Schaschke, N., Bogyo, M., Brix, K. 2019

  Abstract

  Taar1 is a G protein-coupled receptor (GPCR) confined to primary cilia of rodent thyroid epithelial cells. Taar1-deficient mouse thyroid follicles feature luminal accumulation of thyroglobulin suggesting that Taar1 acts as a regulator of extra- and pericellular thyroglobulin processing, which is mediated by cysteine cathepsin proteases present at the apical plasma membrane of rodent thyrocytes. Here, by immunostaining and confocal laser scanning microscopy, we demonstrated co-localization of cathepsin L, but only little cathepsin B, with Taar1 at primary cilia of rat thyrocytes, the FRT cells. Because proteases were shown to affect half-lives of certain receptors, we determined the effect of cathepsin activity inhibition on sub-cellular localization of Taar1 in FRT cells, whereupon Taar1 localization altered such that it was retained in compartments of the secretory pathway. Since the same effect on Taar1 localization was observed in both cathepsin B and L inhibitor-treated cells, the interaction of cathepsin activities and sub-cellular localization of Taar1 was thought to be indirect. Indeed, we observed that cathepsin inhibition resulted in a lack of primary cilia from FRT cells. Next, we proved that primary cilia are a necessity for Taar1 trafficking to reach the plasma membrane of FRT cells, since the disruption of primary cilia by treatment with beta-cyclodextrin resulted in Taar1 retention in compartments of the secretory pathway. Furthermore, in less well-polarized rat thyrocytes, namely in FRTL-5 cells lacking primary cilia, Taar1 was mainly confined to the compartments of the secretory pathway. We conclude that Taar1 localization in polarized thyroid epithelial cells requires the presence of primary cilia, which is dependent on the proteolytic activity of cysteine cathepsins B and L.

  View details for DOI 10.1016/j.biochi.2019.07.010

  View details for PubMedID 31302164

• Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis. Biochimie Veillard, F., Sztukowska, M., Nowakowska, Z., Mizgalska, D., Thogersen, I. B., Enghild, J. J., Bogyo, M., Potempa, B., Nguyen, K., Potempa, J. 2019

  Abstract

  Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the DeltaRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

  View details for DOI 10.1016/j.biochi.2019.06.010

  View details for PubMedID 31212040

• Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide-based substrates and covalent inhibitors. The FEBS journal de Vries, L. E., Sanchez, M. I., Groborz, K., Kuppens, L., Poreba, M., Lehmann, C., Nevins, N., Withers-Martinez, C., Hirst, D. J., Yuan, F., Arastu-Kapur, S., Horn, M., Mares, M., Bogyo, M., Drag, M., Deu, E. 2019

  Abstract

  Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of non-optimal peptide substrates. Finally, our homology modelling and docking studies provide potential structural explanations of the differences in specificity between DPAP1, DPAP3, and CatC, and between substrates and inhibitors in the case of DPAP3. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1111/febs.14953

  View details for PubMedID 31177613

• Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases CELL CHEMICAL BIOLOGY Amara, N., Foe, I. T., Onguka, O., Garland, M., Bogyo, M. 2019; 26 (1): 35-+

  View details for DOI 10.1016/j.chembiol.2018.10.005

  View details for Web of Science ID 000456042100007

• Identification of Plasmodiumdipeptidyl aminopeptidase allosteric inhibitors by high throughput screening. PloS one Sanchez, M. I., de Vries, L. E., Lehmann, C., Lee, J. T., Ang, K. K., Wilson, C., Chen, S., Arkin, M. R., Bogyo, M., Deu, E. 2019; 14 (12): e0226270

  Abstract

  Dipeptidyl aminopeptidases (DPAPs) are cysteine proteases that cleave dipeptides from the N-terminus of protein substrates and have been shown to play important roles in many pathologies including parasitic diseases such as malaria, toxoplasmosis and Chagas's disease. Inhibitors of the mammalian homologue cathepsin C have been used in clinical trials as potential drugs to treat chronic inflammatory disorders, thus proving that these enzymes are druggable. In Plasmodium species, DPAPs play important functions at different stages of parasite development, thus making them potential antimalarial targets. Most DPAP inhibitors developed to date are peptide-based or peptidomimetic competitive inhibitors. Here, we used a high throughput screening approach to identify novel inhibitor scaffolds that block the activity of Plasmodium falciparum DPAP1. Most of the hits identified in this screen also inhibit Plasmodium falciparum DPAP3, cathepsin C, and to a lesser extent other malarial clan CA proteases, indicating that these might be general DPAP inhibitors. Interestingly, our mechanism of inhibition studies indicate that most hits are allosteric inhibitors, which opens a completely new strategy to inhibit these enzymes, study their biological function, and potentially develop new inhibitors as starting points for drug development.

  View details for DOI 10.1371/journal.pone.0226270

  View details for PubMedID 31851699

• Catalytic linkage between caspase activity and proteostasis in Archaea ENVIRONMENTAL MICROBIOLOGY Seth-Pasricha, M., Senn, S., Sanman, L. E., Bogyo, M., Nanda, V., Bidle, K. A., Bidle, K. D. 2019; 21 (1): 286-298

  View details for DOI 10.1111/1462-2920.14456

  View details for Web of Science ID 000456278900020

• Molecular imaging and validation of margins in surgically excised nonmelanoma skin cancer specimens. Journal of medical imaging (Bellingham, Wash.) Liu, Y., Walker, E., Iyer, S. R., Biro, M., Kim, I., Zhou, B., Straight, B., Bogyo, M., Basilion, J. P., Popkin, D. L., Wilson, D. L. 2019; 6 (1): 016001

  Abstract

  In an effort to increase the efficiency and cure rate of nonmelanoma skin cancer (NMSC) excisions, we have developed a point-of-care method of imaging and evaluation of skin cancer margins. We evaluate the skin surgical specimens using a smart, near-infrared probe (6qcNIR) that fluoresces in the presence of cathepsin proteases overexpressed in NMSC. Imaging is done with an inverted, flying-spot fluorescence scanner that reduces scatter, giving a 70% improved step response as compared to a conventional imaging system. We develop a scheme for careful comparison of fluorescent signals to histological annotation, which involves image segmentation, fiducial-based registration, and nonrigid free-form deformation on fluorescence images, corresponding color images, "bread-loafed" tissue images, hematoxylin and eosin (H&E)-stained slides, and pathological annotations. From epidermal landmarks, spatial accuracy in the bulk of the sample is 500 mu m , which when extrapolated with a linear stretch model, suggests an error at the margin of 100 mu m , within clinical reporting standards. Cancer annotations on H&E slides are transformed and superimposed on the fluorescence images to generate the final results. Using this methodology, fluorescence cancer signals are generally found to correspond spatially with histological annotations. This method will allow us to accurately analyze molecular probes for imaging skin cancer margins.

  View details for PubMedID 30915384

• Molecular imaging and validation of margins in surgically excised nonmelanoma skin cancer specimens JOURNAL OF MEDICAL IMAGING Liu, Y., Walker, E., Iyer, S., Biro, M., Kim, I., Zhou, B., Straight, B., Bogyo, M., Basilion, J. P., Popkin, D. L., Wilson, D. L. 2019; 6 (1)

  View details for DOI 10.1117/1.JMI.6.1.016001

  View details for Web of Science ID 000463892000024

• Fluorescent Triazole Urea Activity-Based Probes for the Single-Cell Phenotypic Characterization of Staphylococcus aureus. Angewandte Chemie (International ed. in English) Chen, L. n., Keller, L. J., Cordasco, E. n., Bogyo, M. n., Lentz, C. S. 2019; 58 (17): 5643–47

  Abstract

  Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity-based probes as chemical tools for the single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3-triazole urea library to identify selective inhibitors of fluorophosphonate-binding serine hydrolases and lipases in S. aureus and synthesized target-selective activity-based probes. Molecular imaging and activity-based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single-cell level.

  View details for PubMedID 30768830

• Covalent Plasmodium falciparum-selective proteasome inhibitors exhibit a low propensity for generating resistance in vitro and synergize with multiple antimalarial agents. PLoS pathogens Stokes, B. H., Yoo, E. n., Murithi, J. M., Luth, M. R., Afanasyev, P. n., da Fonseca, P. C., Winzeler, E. A., Ng, C. L., Bogyo, M. n., Fidock, D. A. 2019; 15 (6): e1007722

  Abstract

  Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the β2, β5 or β6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria.

  View details for DOI 10.1371/journal.ppat.1007722

  View details for PubMedID 31170268

• Covalent Modifiers of Botulinum Neurotoxin Counteract Toxin Persistence ACS CHEMICAL BIOLOGY Garland, M., Babin, B. M., Miyashita, S., Loscher, S., Shen, Y., Dong, M., Bogyo, M. 2019; 14 (1): 76–87

  Abstract

  Botulinum neurotoxins (BoNTs) are the most potent toxin known to man and a significant threat as a weapon of bioterrorism. BoNTs contain a metalloprotease domain that blocks neurotransmitter release in nerve terminals, resulting in a descending, flaccid paralysis with a 5-10% mortality rate. Existing treatment options cannot access or neutralize toxin following its endocytosis, so there is a clear need to develop novel therapies. Numerous substrate-based and zinc-chelating small-molecule inhibitors have been reported, however none have progressed to the clinic. This is likely due to the difficulty of reversible inhibitors to achieve sustained inhibition of the toxin, which has a months-long half-life in vivo. An alternative strategy to mitigate BoNT persistence is through covalent, irreversible inhibition of toxin function. However, few examples of covalent BoNT inhibitors have been reported. Here, we describe a competition-based screen to identify covalent modifiers of the conserved active-site adjacent cysteine C165 in the BoNT/A serotype. We found that compounds containing cysteine-reactive electrophiles designed to target cysteine proteases failed to bind C165 while selenide compounds were efficient covalent binders of this cysteine. Importantly, covalent modification at C165 resulted in sustained, irreversible inhibition of BoNT/A protease activity. Covalent selenide inhibitors were non-toxic and protective in a neuronal assay of intoxication making them promising new scaffolds for the study of the BoNT/A toxin as well as for the design of novel therapy agents.

  View details for PubMedID 30571080

• Validation of near infrared fluorescence (NIRF) probes in vivo with dual laser NIRF endoscope PLOS ONE Shrivastav, M., Gounaris, E., Khan, M. W., Ko, J., Ryu, S. H., Bogyo, M., Larson, A., Barret, T. A., Bentrem, D. J. 2018; 13 (11)

  View details for DOI 10.1371/journal.pone.0206568

  View details for Web of Science ID 000449289800043

• Catalytic linkage between caspase activity and proteostasis in Archaea. Environmental microbiology Seth-Pasricha, M., Senn, S., Sanman, L. E., Bogyo, M., Nanda, V., Bidle, K. A., Bidle, K. D. 2018

  Abstract

  The model haloarchaeon, Haloferax volcanii possess an extremely high, and highly specific, basal caspase activity in exponentially growing cells that closely resembles caspase-4, is specifically inhibited by the pan-caspase inhibitor, z-VAD-FMK, has no cross-reactivity with other known protease families. Although it is one of the dominant cellular proteolytic activities in exponentially growing H. volcanii cells, the interactive cellular roles remain unknown and the protein(s) responsible for this activity remain elusive. Here, biochemical purification and in situ trapping with caspase targeted covalent inhibitors combined with genome-enabled proteomics, structural analysis, targeted gene knockouts, and treatment with canavanine demonstrated a catalytic linkage between caspase activity and thermosomes, proteasomes, and cdc48b, a cell division protein and proteasomal degradation facilitating ATPase, as part of an 'interactase' of stress-related protein complexes with an established link to the unfolded protein response (UPR). Our findings provide novel cellular and biochemical context for the observed caspase activity in Archaea and add new insight to understanding the role of this activity, implicating their possible role in the establishment of protein stress and ER associated degradation pathways in Eukarya. This article is protected by copyright. All rights reserved.

  View details for PubMedID 30370585

• Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases. Cell chemical biology Amara, N., Foe, I. T., Onguka, O., Garland, M., Bogyo, M. 2018

  Abstract

  Palmitoylation is a post-translational modification involving the thioesterification of cysteine residues with a 16-carbon-saturated fatty acid. Little is known about rates of depalmitoylation or the parameters that dictate these rates. Here we report a modular strategy to synthesize quenched fluorogenic substrates for the specific detection of depalmitoylase activity and for mapping the substrate specificity of individual depalmitoylases. We demonstrate that human depalmitoylases APT1 and APT2, and TgPPT1 from the parasite Toxoplasma gondii, have distinct specificities that depend on amino acid residues distal to the palmitoyl cysteine. This information informs the design of optimal and non-optimal substrates as well as isoform-selective substrates to detect the activity of a specific depalmitoylase in complex proteomes. In addition to providing tools for studying depalmitoylases, our findings identify a previously unrecognized mechanism for regulating steady-state levels of distinct palmitoylation sites by sequence-dependent control of depalmitoylation rates.

  View details for PubMedID 30393067

• Erratum for Foe et al., "The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture". mSphere Foe, I. T., Onguka, O., Amberg-Johnson, K., Garner, R. M., Amara, N., Beatty, W., Yeh, E., Bogyo, M. 2018; 3 (5)

  Abstract

  [This corrects the article DOI: 10.1128/mSphere.00393-18.].

  View details for DOI 10.1128/mSphere.00535-18

  View details for PubMedID 31329810

  View details for PubMedCentralID PMC6180226

• The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture. mSphere Foe, I. T., Onguka, O., Amberg-Johnson, K., Garner, R. M., Amara, N., Beatty, W., Yeh, E., Bogyo, M. 2018; 3 (5)

  Abstract

  Hydrolase are enzymes that regulate diverse biological processes, including posttranslational protein modifications. Recent work identified four active serine hydrolases (ASHs) in Toxoplasma gondii as candidate depalmitoylases. However, only TgPPT1 (ASH1) has been confirmed to remove palmitate from proteins. ASH4 (TgME49_264290) was reported to be refractory to genetic disruption. We demonstrate that recombinant ASH4 is an esterase that processes short acyl esters but not palmitoyl thioesters. Genetic disruption of ASH4 causes defects in cell division and premature scission of parasites from residual bodies. These defects lead to the presence of vacuoles with a disordered intravacuolar architecture, with parasites arranged in pairs around multiple residual bodies. Importantly, we found that the deletion of ASH4 correlates with a defect in radial dispersion from host cells after egress. This defect in dispersion of parasites is a general phenomenon that is observed for disordered vacuoles that occur at low frequency in wild-type parasites, suggesting a possible general link between intravacuolar organization and dispersion after egress.IMPORTANCE This work defines the function of an enzyme in the obligate intracellular parasite Toxoplasma gondii We show that this previously uncharacterized enzyme is critical for aspects of cellular division by the parasite and that loss of this enzyme leads to parasites with cell division defects and which also are disorganized inside their vacuoles. This leads to defects in the ability of the parasite to disseminate from the site of an infection and may have a significant impact on the parasite's overall infectivity of a host organism.

  View details for PubMedID 30232166

• Defining the Determinants of Specificity of Plasmodium Proteasome Inhibitors JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yoo, E., Stokes, B. H., de Jong, H., Vanaerscho, M., Kumar, T. S., Lawrence, N., Njoroge, M., Garcia, A., Van der Westhuyzen, R., Momper, J. D., Ng, C. L., Fidock, D. A., Bogyo, M. 2018; 140 (36): 11424–37

  Abstract

  The Plasmodium proteasome is an emerging antimalarial target due to its essential role in all the major life cycle stages of the parasite and its contribution to the establishment of resistance to artemisinin (ART)-based therapies. However, because of a similarly essential role for the host proteasome, the key property of any antiproteasome therapeutic is selectivity. Several parasite-specific proteasome inhibitors have recently been reported, however, their selectivity must be improved to enable clinical development. Here we describe screening of diverse libraries of non-natural synthetic fluorogenic substrates to identify determinants at multiple positions on the substrate that produce enhanced selectivity. We find that selection of an optimal electrophilic "warhead" is essential to enable high selectivity that is driven by the peptide binding elements on the inhibitor. We also find that host cell toxicity is dictated by the extent of coinhibition of the human β2 and β5 subunits. Using this information, we identify compounds with over 3 orders of magnitude selectivity for the parasite enzyme. Optimization of the pharmacological properties resulted in molecules that retained high potency and selectivity, were soluble, sufficiently metabolically stable and orally bioavailable. These molecules are highly synergistic with ART and can clear parasites in a mouse model of infection, making them promising leads as antimalarial drugs.

  View details for DOI 10.1021/jacs.8b06655

  View details for Web of Science ID 000444793400042

  View details for PubMedID 30107725

• The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture MSPHERE Foe, I. T., Onguka, O., Amberg-Johnson, K., Garner, R. M., Amara, N., Beatty, W., Yeh, E., Bogyo, M. 2018; 3 (5)

  View details for DOI 10.1128/mSphere.00393-18

  View details for Web of Science ID 000449522900018

• The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture (vol 3, e00393-18, 2018) MSPHERE Foe, I. T., Onguka, O., Amberg-Johnson, K., Garner, R. M., Amara, N., Beatty, W., Yeh, E., Bogyo, M. 2018; 3 (5)

  View details for Web of Science ID 000449522900054

• The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture (vol 3, e00393-18, 2018) MSPHERE Foe, I. T., Onguka, O., Amberg-Johnson, K., Garner, R. M., Amara, N., Beatty, W., Yeh, E., Bogyo, M. 2018; 3 (5)

  View details for DOI 10.1128/mSphere.00535-18

  View details for Web of Science ID 000449522900019

• An Automatic Analysis System for High-Throughput Clostridium Difficile Toxin Activity Screening APPLIED SCIENCES-BASEL Garland, M., Jaworek-Korjakowska, J., Libal, U., Bogyo, M., Sienczyk, M. 2018; 8 (9)

  View details for DOI 10.3390/app8091512

  View details for Web of Science ID 000445760200101

• Chemical Tools for Selective Activity Profiling of Endogenously Expressed MMP-14 in Multicellular Models ACS CHEMICAL BIOLOGY Amara, N., Tholen, M., Bogyo, M. 2018; 13 (9): 2645–54

  Abstract

  Matrix metalloproteases (MMPs) are a large family of zinc-dependent endopeptidases involved in a diverse set of physiological and pathological processes, most notably in cancer. Current methods for imaging and quantifying MMP activity lack sufficient selectivity and spatiotemporal resolution to allow studies of specific MMP function in vivo. Previously, we reported a strategy for selective targeting of MMPs by engineering a functionally silent cysteine mutation that enables highly specific covalent modification by a designed activity-based probe. Here, we describe the translation of that technology into a mouse model of breast cancer and subsequent demonstration of the utility of the approach for studies of MMP-14 activation in the tumor microenvironment. Using this approach, we find that MMP-14 is active in late stage tumors and is predominantly associated with stromal cell populations that have been activated by specific signaling molecules (e.g., TGFβ) produced by tumor cells. Our data demonstrate the applicability of this approach for studies of MMP function in whole organisms and identify important regulatory mechanisms for MMP-14 activity in the tumor microenvironment.

  View details for DOI 10.1021/acschembio.8b00562

  View details for Web of Science ID 000445713100032

  View details for PubMedID 30160940

• PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells IMMUNITY Stathopoulou, C., Gangaplara, A., Mallett, G., Flomerfelt, F. A., Liniany, L. P., Knight, D., Samsel, L. A., Berlinguer-Palmini, R., Yim, J. J., Felizardo, T. C., Eckhaus, M. A., Edgington-Mitchell, L., Martinez-Fabregas, J., Zhu, J., Fowler, D. H., van Kasteren, S. I., Laurence, A., Bogyo, M., Watts, C., Shevach, E. M., Amarnath, S. 2018; 49 (2): 247-+

  Abstract

  CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.

  View details for PubMedID 30054205

  View details for PubMedCentralID PMC6105434

• Identifying compounds that restore normal cellular function in Frontotemporal dementia caused by progranulin haploinsufficiency Telpoukhovskaia, M., Liu, K., Sayed, F., Etchegaray, J., Zhou, Y., Le, D., Xie, M., Bogyo, M., Ding, S., Gan, L. AMER CHEMICAL SOC. 2018

  View details for Web of Science ID 000447609102353

• Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP) NATURE CHEMICAL BIOLOGY Lentz, C. S., Sheldon, J. R., Crawford, L. A., Cooper, R., Garland, M., Amieva, M. R., Weerapana, E., Skaar, E. P., Bogyo, M. 2018; 14 (6): 609-+

  View details for DOI 10.1038/s41589-018-0060-1

  View details for Web of Science ID 000435445100018

• Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP). Nature chemical biology Lentz, C. S., Sheldon, J. R., Crawford, L. A., Cooper, R., Garland, M., Amieva, M. R., Weerapana, E., Skaar, E. P., Bogyo, M. 2018

  Abstract

  Serine hydrolases play diverse roles in regulating host-pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here we describe a chemical proteomic screen that identified ten previously uncharacterized S. aureus serine hydrolases that mostly lack human homologs. We termed these enzymes fluorophosphonate-binding hydrolases (FphA-J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of fphB confirmed that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduced infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.

  View details for PubMedID 29769740

• Multiplexed in vivo small molecule screening for immediate drug repositioning reveals novel therapeutic targets in metastatic pancreatic cancer. Dorsch, M., Schulze, C., Yang, D., Bogyo, M., Winslow, M., Gruener, B. AMER ASSOC CANCER RESEARCH. 2018: 34–35

  View details for Web of Science ID 000432307300040

• Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes NATURE COMMUNICATIONS Hewings, D. S., Heideker, J., Ma, T. P., AhYoung, A. P., El Oualid, F., Amore, A., Costakes, G. T., Kirchhofer, D., Brasher, B., Pillow, T., Popovych, N., Maurer, T., Schwerdtfeger, C., Forrest, W. F., Yu, K., Flygare, J., Bogyo, M., Wertz, I. E. 2018; 9: 1162

  Abstract

  Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

  View details for PubMedID 29563501

• TGF-beta Regulates Cathepsin Activation during Normal and Pathogenic Development CELL REPORTS Flanagan-Steet, H., Christian, C., Lu, P., Aarnio-Peterson, M., Sanman, L., Archer-Hartmann, S., Azadi, P., Bogyo, M., Steet, R. A. 2018; 22 (11): 2964–77

  Abstract

  Cysteine cathepsins play roles during development and disease beyond their function in lysosomal protein turnover. Here, we leverage a fluorescent activity-based probe (ABP), BMV109, to track cysteine cathepsins in normal and diseased zebrafish embryos. Using this probe in a model of mucolipidosis II, we show that loss of carbohydrate-dependent lysosomal sorting alters the activity of several cathepsin proteases. The data support a pathogenic mechanism where TGF-ß signals enhance the proteolytic processing of pro-Ctsk by modulating the expression of chondroitin 4-sulfate (C4-S). In MLII, elevated C4-S corresponds with TGF-ß-mediated increases in chst11 expression. Inhibiting chst11 impairs the proteolytic activation of Ctsk and alleviates the MLII phenotypes. These findings uncover a regulatory loop between TGF-ß signaling and Ctsk activation that is altered in the context of lysosomal disease. This work highlights the power of ABPs to identify mechanisms underlying pathogenic development in living animals.

  View details for PubMedID 29539424

• Development of an activity-based probe for acyl-protein thioesterases PLOS ONE Garland, M., Schulze, C. J., Foe, I. T., van der Linden, W. A., Child, M. A., Bogyo, M. 2018; 13 (1): e0190255

  Abstract

  Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation-PATs, APTs and PPTs-will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe.

  View details for PubMedID 29364904

• Optimization of a Protease Activated Probe for Optical Surgical Navigation. Molecular pharmaceutics Yim, J. J., Tholen, M. n., Klaassen, A. n., Sorger, J. n., Bogyo, M. n. 2018; 15 (3): 750–58

  Abstract

  Molecularly targeted optical contrast agents have the potential to enable surgeons to visualize specific molecular markers that can help improve surgical precision and thus outcomes. Fluorescently quenched substrates can be used to highlight tumor lesions by targeting proteases that are highly abundant in the tumor microenvironment. However, the majority of these and other molecularly targeted optical contrast agents are labeled with reporter dyes that are not ideally matched to the properties of clinical camera systems, which are typically optimized for detection of indocyanine-green (ICG). While a wide range of near-infrared (NIR) dyes are suitable for use with highly sensitive and highly tunable research-focused small animal imaging systems, most have not been evaluated for use with commonly used clinical imaging systems. Here we report the optimization of a small molecule fluorescently quenched protease substrate probe 6QC-ICG, which uses the indocyanine green (ICG) dye as its optical reporter. We evaluated dosing and kinetic parameters of this molecule in tumor-bearing mice and observed optimal tumor over background signals in as little as 90 min with a dose of 2.3 mg/kg. Importantly, the fluorescence intensity of the probe signal in tumors did not linearly scale with dose, suggesting the importance of detailed dosing studies. Furthermore, when imaged using the FDA approved da Vinci Si surgical system with Firefly detection, signals were significantly higher for the ICG probe compared to a corresponding probe containing a dye with similar quantum yield but with a slightly shifted excitation and emission profile. The increased signal intensity generated by the optimal dye and dose of the ICG labeled probe enabled detection of small, flat lesions that were less than 5 mm in diameter. Therefore, 6QC-ICG is a highly sensitive probe that performs optimally with clinical imaging systems and has great potential for applications in optical surgical navigation.

  View details for DOI 10.1021/acs.molpharmaceut.7b00822

  View details for PubMedID 29172524

• Validation of near infrared fluorescence (NIRF) probes in vivo with dual laser NIRF endoscope. PloS one Shrivastav, M., Gounaris, E., Khan, M. W., Ko, J., Ryu, S. H., Bogyo, M., Larson, A., Barret, T. A., Bentrem, D. J. 2018; 13 (11): e0206568

  Abstract

  PURPOSE: The development of NIRF cathepsin activity probes offered the ability to visualize tumor associated tumor reaction and act as a surrogate marker to delineate the dysplastic lesions. One major type is a NIRF substrate of cathepsins (SBP), which is involved in catalytic way to produce high levels of fluorescence emission. The other major type (ABP) reacts with active cathepsins in stoichiometric manner since they bind covalently with their active center. Little is known about the sensitivity and the specificity of the NIRF probes to detect autochthonous developed dysplastic lesions. Dual laser NIRF endoscope provides a good tool to determine the efficiency of various NIRF probes in vivo in the same lesions.EXPERIMENTAL DESIGN: In the current study, we validated both types of NIRF probes by using the dual laser NIRF endoscope to detect lesions colon cancer mouse model (TS4Cre/cAPC +/lox).RESULTS: The dual laser NIRF endoscope is emitting equal power with both lasers. It can detect with the same efficiency in 680 mode, as well as, 750 mode when NIFR probes of the same scaffold in vivo. When SBP and ABP were used, our results showed both probes are efficient enough to detect large polyps but small dysplastic lesions could not efficiently imaged with the ABP.CONCLUSIONS: The dual laser NIRF endoscope is a powerful tool to validate probes. The probes that react catalytically with the active center of cathepsins are more efficient than the ones that react stoichiometrically in detecting small lesions.

  View details for PubMedID 30388158

• PROTEASOME INHIBITOR-BASED COMBINATION THERAPY POTENTLY INHIBITS ARTEMISININ-RESISTANT <it>PLASMODIUM FALCIPARUM</it> Stokes, B., Yoo, E., Muriungi, J., Ottilie, S., Luth, M., Winzeler, E., Bogyo, M., Fidock, D., Ng, C. AMER SOC TROP MED & HYGIENE. 2018: 231

  View details for Web of Science ID 000461386603071

• Molecular imaging and validation of non-melanoma skin cancer margins Liu, Y., Walker, E., Kim, I., Biro, M., Iyer, S., Zhou, B., Bogyo, M., Basilion, J. P., Popkin, D., Wilson, D., Zhang, J., Chen, P. H. SPIE-INT SOC OPTICAL ENGINEERING. 2018

  View details for DOI 10.1117/12.2293847

  View details for Web of Science ID 000442112200028

• New Blood Test SEEKs To Detect and Localize Cancer before It's Too Late. Biochemistry Bogyo, M. n., Yim, J. J., Rosenthal, E. L. 2018; 57 (10): 1561–62

  View details for DOI 10.1021/acs.biochem.8b00179

  View details for PubMedID 29489339

• Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation SCIENCE Ailland, C., Bose, C., Peris, L., Bosson, A., Heemeryck, P., Van Dijk, J., Le Friec, J., Boulan, B., Vossier, F., Sanman, L. E., Syed, S., Amara, N., Coute, Y., Lafanechere, L., Denarier, E., Delphin, C., Pelletier, L., Humbert, S., Bogyo, M., Andrieux, A., Rogowski, K., Moutin, M. 2017; 358 (6369): 1448–52

  Abstract

  Reversible detyrosination of α-tubulin is crucial to microtubule dynamics and functions, and defects have been implicated in cancer, brain disorganization, and cardiomyopathies. The identity of the tubulin tyrosine carboxypeptidase (TCP) responsible for detyrosination has remained unclear. We used chemical proteomics with a potent irreversible inhibitor to show that the major brain TCP is a complex of vasohibin-1 (VASH1) with the small vasohibin binding protein (SVBP). VASH1 and its homolog VASH2, when complexed with SVBP, exhibited robust and specific Tyr/Phe carboxypeptidase activity on microtubules. Knockdown of vasohibins or SVBP and/or inhibitor addition in cultured neurons reduced detyrosinated α-tubulin levels and caused severe differentiation defects. Furthermore, knockdown of vasohibins disrupted neuronal migration in developing mouse neocortex. Thus, vasohibin/SVBP complexes represent long-sought TCP enzymes.

  View details for PubMedID 29146868

• Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion JOURNAL OF PATHOLOGY Duivenvoorden, H. M., Rautela, J., Edgington-Mitchell, L. E., Spurling, A., Greening, D. W., Nowell, C. J., Molloy, T. J., Robbins, E., Brockwell, N. K., Lee-, C., Chen, M., Holliday, A., Selinger, C. I., Hu, M., Britt, K. L., Stroud, D. A., Bogyo, M., Moeller, A., Polyak, K., Sloane, B. F., O'Toole, S. A., Parker, B. S. 2017; 243 (4): 496–509

  View details for DOI 10.1002/path.4990

  View details for Web of Science ID 000415685900009

• Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion. The Journal of pathology Duivenvoorden, H. M., Rautela, J., Edgington-Mitchell, L. E., Spurling, A., Greening, D. W., Nowell, C. J., Molloy, T. J., Robbins, E., Brockwell, N. K., Lee, C. S., Chen, M., Holliday, A., Selinger, C. I., Hu, M., Britt, K. L., Stroud, D. A., Bogyo, M., Möller, A., Polyak, K., Sloane, B. F., O'Toole, S. A., Parker, B. S. 2017; 243 (4): 496-509

  Abstract

  Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  View details for DOI 10.1002/path.4990

  View details for PubMedID 29086922

• Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection NATURE MICROBIOLOGY Di Cristina, M., Dou, Z., Lunghi, M., Kannan, G., My-Hang Huynh, McGovern, O. L., Schultz, T. L., Schultz, A. J., Miller, A. J., Hayes, B. M., van der Linden, W., Emiliani, C., Bogyo, M., Besteiro, S., Coppens, I., Carruthers, V. B. 2017; 2 (8): 17096

  Abstract

  Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii1. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses2 and cognitive impairment3. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.

  View details for PubMedID 28628099

• The lysosomal protein cathepsin L is a progranulin protease MOLECULAR NEURODEGENERATION Lee, C. W., Stankowski, J. N., Chew, J., Cook, C. N., Lam, Y., Almeida, S., Carlomagno, Y., Lau, K., Prudencio, M., Gao, F., Bogyo, M., Dickson, D. W., Petrucelli, L. 2017; 12: 55

  Abstract

  Haploinsufficiency of GRN, the gene encoding progranulin (PGRN), causes frontotemporal lobar degeneration (FTLD), the second most common cause of early-onset dementia. Receptor-mediated lysosomal targeting has been shown to regulate brain PGRN levels, and complete deficiency of PGRN is a direct cause of neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Here we show that the lysosomal cysteine protease cathepsin L (Cat L) can mediate the proteolytic cleavage of intracellular PGRN into poly-granulin and granulin fragments. Further, PGRN and Cat L co-localize in lysosomes of HEK293 cells, iPSC-derived neurons and human cortical neurons from human postmortem tissue. These data identify Cat L as a key intracellular lysosomal PGRN protease, and provides an intriguing new link between lysosomal dysfunction and FTLD.

  View details for PubMedID 28743268

• Frontline Science: Multiple cathepsins promote inflammasome-independent, particle-induced cell death during NLRP3-dependent IL-1 beta activation JOURNAL OF LEUKOCYTE BIOLOGY Orlowski, G. M., Sharma, S., Colbert, J. D., Bogyo, M., Robertson, S. A., Kataoka, H., Chan, F. K., Rock, K. L. 2017; 102 (1): 7–17

  Abstract

  Sterile particles cause several chronic, inflammatory diseases, characterized by repeating cycles of particle phagocytosis and inflammatory cell death. Recent studies have proposed that these processes are driven by the NLRP3 inflammasome, a platform activated by phagocytosed particles, which controls both caspase-1-dependent cell death (pyroptosis) and mature IL-1β secretion. After phagocytosis, particles can disrupt lysosomes, and inhibitor studies have suggested that the resulting release of a lysosomal protease-cathepsin B-into the cytosol somehow activates NLRP3. However, using primary murine macrophages, we found that particle-induced cell death occurs independent of NLRP3/caspase-1 and depends instead on multiple, redundant cathepsins. In contrast, nigericin, a soluble activator of NLRP3 inflammasomes, induced cell death that was dependent on the NLRP3. Interestingly, nigericin-induced cell death depended partly on a single cathepsin, cathepsin X. By inhibiting or silencing multiple cathepsins in macrophages, several key proinflammatory events induced by sterile particles are blocked, including cell death, pro-IL-1β production, and IL-1β secretion. These data suggest that cathepsins might be potential therapeutic targets in particulate-mediated inflammatory disease. In support of this concept, we find that a broad-spectrum cathepsin inhibitor can suppress particle-induced IL-1-dependent peritonitis.

  View details for PubMedID 28087651

• Activity-based probes for the ubiquitin conjugation-deconjugation machinery: new chemistries, new tools, and new insights FEBS JOURNAL Hewings, D. S., Flygare, J. A., Bogyo, M., Wertz, I. E. 2017; 284 (10): 1555-1576

  Abstract

  The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology.

  View details for DOI 10.1111/febs.14039

  View details for Web of Science ID 000401328000011

  View details for PubMedID 28196299

• Activity-based probes for the multicatalytic proteasome FEBS JOURNAL Hewings, D. S., Flygare, J. A., Wertz, I. E., Bogyo, M. 2017; 284 (10): 1540-1554

  Abstract

  Proteasomes are multisubunit protease complexes responsible for degrading most intracellular proteins. In addition to removing damaged proteins, they regulate many important cellular processes through the controlled degradation of transcription factors, cell cycle regulators, and enzymes. Eukaryotic proteasomes have three catalytic subunits, β1, β2, and β5, that each has different substrate specificities. Additionally, although we know that diverse cell types express proteasome variants with distinct activity and specificity profiles, the functions of these different pools of proteasomes are not fully understood. Covalent inhibitors of the protease activity of the proteasome have been developed as drugs for hematological malignancies and are currently under investigation for other diseases. Therefore, there is a need for tools that allow direct monitoring of proteasome activity in live cells and tissues. Activity-based probes have proven valuable for biochemical and cell biological studies of the role of individual proteasome subunits, and for evaluating the efficacy and selectivity of proteasome inhibitors. These probes react covalently with the protease active sites, and contain a reporter tag to identify the probe-labeled proteasome subunits. This review will describe the development of broad-spectrum and subunit-specific proteasome activity-based probes, and discuss how these probes have contributed to our understanding of proteasome biology, and to the development of proteasome inhibitors.

  View details for DOI 10.1111/febs.14016

  View details for Web of Science ID 000401328000010

  View details for PubMedID 28107776

• Individuals with progranulin haploinsufficiency exhibit features of neuronal ceroid lipofuscinosis SCIENCE TRANSLATIONAL MEDICINE Ward, M. E., Chen, R., Huang, H., Ludwig, C., Telpoukhovskaia, M., Taubes, A., Boudin, H., Minami, S. S., Reichert, M., Albrecht, P., Gelfand, J. M., Cruz-Herranz, A., Cordano, C., Alavi, M. V., Leslie, S., Seeley, W. W., Miller, B. L., Bigio, E., Mesulam, M., Bogyo, M. S., Mackenzie, I. R., Staropoli, J. F., Cotman, S. L., Huang, E. J., Gan, L., Green, A. J. 2017; 9 (385)

  Abstract

  Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.

  View details for DOI 10.1126/scitranslmed.aah5642

  View details for Web of Science ID 000399009200002

  View details for PubMedID 28404863

• Chemical correction of cellular dysfunction caused by progranulin deficiency in frontotemporal dementia Telpoukhovskaia, M., Liu, K., Ludwig, C., Etchegaray, J., Sayed, F., Zhou, Y., Bogyo, M., Ding, S., Gan, L. AMER CHEMICAL SOC. 2017

  View details for Web of Science ID 000430569104382

• Chemical Strategies To Target Bacterial Virulence. Chemical reviews Garland, M., Loscher, S., Bogyo, M. 2017; 117 (5): 4422-4461

  Abstract

  Antibiotic resistance is a significant emerging health threat. Exacerbating this problem is the overprescription of antibiotics as well as a lack of development of new antibacterial agents. A paradigm shift toward the development of nonantibiotic agents that target the virulence factors of bacterial pathogens is one way to begin to address the issue of resistance. Of particular interest are compounds targeting bacterial AB toxins that have the potential to protect against toxin-induced pathology without harming healthy commensal microbial flora. Development of successful antitoxin agents would likely decrease the use of antibiotics, thereby reducing selective pressure that leads to antibiotic resistance mutations. In addition, antitoxin agents are not only promising for therapeutic applications, but also can be used as tools for the continued study of bacterial pathogenesis. In this review, we discuss the growing number of examples of chemical entities designed to target exotoxin virulence factors from important human bacterial pathogens.

  View details for DOI 10.1021/acs.chemrev.6b00676

  View details for PubMedID 28234447

• Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1. mBio Child, M. A., Garland, M., Foe, I., Madzelan, P., Treeck, M., van der Linden, W. A., Oresic Bender, K., Weerapana, E., Wilson, M. A., Boothroyd, J. C., Reese, M. L., Bogyo, M. 2017; 8 (1)

  Abstract

  Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

  View details for DOI 10.1128/mBio.02189-16

  View details for PubMedID 28246362

• Fluorescence Imaging for Cancer Screening and Surveillance. Molecular imaging and biology Tipirneni, K. E., Rosenthal, E. L., Moore, L. S., Haskins, A. D., Udayakumar, N., Jani, A. H., Carroll, W. R., Morlandt, A. B., Bogyo, M., Rao, J., Warram, J. M. 2017

  Abstract

  The advent of fluorescence imaging (FI) for cancer cell detection in the field of oncology is promising for both cancer screening and surgical resection. Particularly, FI in cancer screening and surveillance is actively being evaluated in many new clinical trials with over 30 listed on Clinical Trials.gov . While surgical resection forms the foundation of many oncologic treatments, early detection is the cornerstone for improving outcomes and reducing cancer-related morbidity and mortality. The applications of FI are twofold as it can be applied to high-risk patients in addition to those undergoing active surveillance. This technology has the promise of highlighting lesions not readily detected by conventional imaging or physical examination, allowing disease detection at an earlier stage of development. Additionally, there is a persistent need for innovative, cost-effective imaging modalities to ameliorate healthcare disparities and the global burden of cancer worldwide. In this review, we outline the current utility of FI for screening and detection in a range of cancer types.

  View details for DOI 10.1007/s11307-017-1050-5

  View details for PubMedID 28155079

• Membrane skeletal association and post-translational allosteric regulation of Toxoplasma gondii GAPDH1. Molecular microbiology Dubey, R., Staker, B. L., Foe, I. T., Bogyo, M., Myler, P. J., Ngô, H. M., Gubbels, M. 2017; 103 (4): 618-634

  Abstract

  When Toxoplasma gondii egresses from the host cell, glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1), which is primary a glycolysis enzyme but actually a quintessential multifunctional protein, translocates to the unique cortical membrane skeleton. Here, we report the 2.25 Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex providing the basis for the molecular dissection of GAPDH1 structure-function relationships Knockdown of GAPDH1 expression and catalytic site disruption validate the essentiality of GAPDH1 in intracellular replication but we confirmed that glycolysis is not strictly essential. We identify, for the first time, S-loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S-loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by the N-terminus, likely palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post-translational modifications.

  View details for DOI 10.1111/mmi.13577

  View details for PubMedID 27859784

  View details for PubMedCentralID PMC5296235

• Rapid visualization of nonmelanoma skin cancer. Journal of the American Academy of Dermatology Walker, E., Mann, M., Honda, K., Vidimos, A., Schluchter, M. D., Straight, B., Bogyo, M., Popkin, D., Basilion, J. P. 2017; 76 (2): 209-216 e9

  Abstract

  Mohs micrographic surgery examines all margins of the resected sample and has a 99% cure rate. However, many nonmelanoma skin cancers (NMSCs) are not readily amenable to Mohs micrographic surgery. This defines an unmet clinical need to assess the completeness of non-Mohs micrographic surgery resections during surgery to prevent re-excision/recurrence.We sought to examine the utility of quenched activity-based probe imaging to discriminate cancerous versus normal-appearing skin tissue.The quenched activity-based probe GB119 was applied to NMSC excised from 68 patients. We validated activation of the probe for hematoxylin-eosin-confirmed cancerous tissue versus normal-appearing skin tissue.Topical application of the probe differentiated basal cell carcinoma and squamous cell carcinoma from normal-appearing skin with overall estimated sensitivity and specificity of 0.989 (95% confidence interval 0.940-1.00) and 0.894 (95% confidence interval 0.769-0.965), respectively. Probe activation accurately defined peripheral margins of NMSC as compared with conventional hematoxylin-eosin-based pathology.This study only examined NMSC debulking excision specimens. The sensitivity and specificity for this approach using final NMSC excision margins will be clinically important.These findings merit further studies to determine whether quenched activity-based probe technology may enable cost-effective increased cure rates for patients with NMSC by reducing re-excision and recurrence rates with a rapid and easily interpretable technological advance.

  View details for DOI 10.1016/j.jaad.2016.09.008

  View details for PubMedID 27876303

  View details for PubMedCentralID PMC5341746

• Live Cell Imaging and Profiling of Cysteine Cathepsin Activity Using a Quenched Activity-Based Probe. Methods in molecular biology (Clifton, N.J.) Edgington-Mitchell, L. E., Bogyo, M., Verdoes, M. 2017; 1491: 145-159

  Abstract

  Since protease activity is highly regulated by structural and environmental influences, the abundance of a protease often does not directly correlate with its activity. Because in most of the cases it is the activity of a protease that gives rise to its biological relevance, tools to report on this activity are of great value to the research community. Activity-based probes (ABPs) are small molecule tools that allow for the monitoring and profiling of protease activities in complex biological systems. The class of fluorescent quenched ABPs (qABPs), being intrinsically "dark" and only emitting fluorescence after reaction with the target protease, are ideally suited for imaging techniques such as small animal noninvasive fluorescence imaging and live cell fluorescence microscopy. An additional powerful characteristic of qABPs is their covalent and irreversible modification of the labeled protease, enabling in-depth target characterization. Here we describe the synthesis of a pan-cysteine cathepsin qABP BMV109 and the application of this probe to live cell fluorescence imaging and fluorescent SDS-PAGE cysteine cathepsin activity profiling.

  View details for PubMedID 27778287

• New chemical probe technologies: applications to imaging and drug discovery (Conference Presentation) Bogyo, M., Pogue, B. W., Gioux, S. SPIE-INT SOC OPTICAL ENGINEERING. 2017

  View details for DOI 10.1117/12.2257636

  View details for Web of Science ID 000406427900014

• Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS central science Tomlin, F. M., Gerling-Driessen, U. I., Liu, Y. C., Flynn, R. A., Vangala, J. R., Lentz, C. S., Clauder-Muenster, S. n., Jakob, P. n., Mueller, W. F., Ordoñez-Rueda, D. n., Paulsen, M. n., Matsui, N. n., Foley, D. n., Rafalko, A. n., Suzuki, T. n., Bogyo, M. n., Steinmetz, L. M., Radhakrishnan, S. K., Bertozzi, C. R. 2017; 3 (11): 1143–55

  Abstract

  Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.

  View details for PubMedID 29202016

• Introduction to the Special Issue on Proteases and Proteolysis in Health and Disease. The FEBS journal Bogyo, M. n. 2017; 284 (10): 1392–93

  Abstract

  This Special Issue on Proteases and Proteolysis in Health and Disease comprises 11 reviews that cover a broad range of topics in this diverse field. We hope you find these pieces as engaging and informative as we have and we are grateful to their authors for taking the time to write for The FEBS Journal.

  View details for PubMedID 28503839

• Protein Degradation Systems as Antimalarial Therapeutic Targets. Trends in parasitology Ng, C. L., Fidock, D. A., Bogyo, M. n. 2017; 33 (9): 731–43

  Abstract

  Artemisinin (ART)-based combination therapies are the most efficacious treatment of uncomplicated Plasmodium falciparum malaria. Alarmingly, P. falciparum strains have acquired resistance to ART across much of Southeast Asia. ART creates widespread protein and lipid damage inside intraerythrocytic parasites, necessitating macromolecule degradation. The proteasome is the main engine of Plasmodium protein degradation. Indeed, proteasome inhibition and ART have shown synergy in ART-resistant parasites. Moreover, ubiquitin modification is associated with altered parasite susceptibility to multiple antimalarials. Targeting the ubiquitin-proteasome system (UPS), therefore, is an attractive avenue to combat drug resistance. Here, we review recent advances leading to specific targeting of the Plasmodium proteasome. We also highlight the potential for targeting other nonproteasomal protein degradation systems as an additional strategy to disrupt protein homeostasis.

  View details for PubMedID 28688800

  View details for PubMedCentralID PMC5656264

• Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites. PLoS pathogens Hopp, C. S., Bennett, B. L., Mishra, S. n., Lehmann, C. n., Hanson, K. K., Lin, J. W., Rousseau, K. n., Carvalho, F. A., van der Linden, W. A., Santos, N. C., Bogyo, M. n., Khan, S. M., Heussler, V. n., Sinnis, P. n. 2017; 13 (9): e1006586

  Abstract

  Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical.

  View details for PubMedID 28922424

  View details for PubMedCentralID PMC5602738

• Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1 MBIO Child, M. A., Garland, M., Foe, I., Madzelan, P., Treeck, M., van der Linden, W. A., Bender, K. O., Weerapana, E., Wilson, M. A., Boothroyd, J. C., Reese, M. L., Bogyo, M. 2017; 8 (1)

  Abstract

  Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

  View details for DOI 10.1128/mBio.02189-16

  View details for Web of Science ID 000395835000009

  View details for PubMedCentralID PMC5347346

• A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer. Molecular imaging and biology Sensarn, S., Zavaleta, C. L., Segal, E., Rogalla, S., Lee, W., Gambhir, S. S., Bogyo, M., Contag, C. H. 2016; 18 (6): 820-829

  Abstract

  Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes.We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice.This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model.The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection.

  View details for PubMedID 27154508

• A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer MOLECULAR IMAGING AND BIOLOGY Sensarn, S., Zavaleta, C. L., Segal, E., Rogalla, S., Lee, W., Gambhir, S. S., Bogyo, M., Contag, C. H. 2016; 18 (6): 820–29

  View details for DOI 10.1007/s11307-016-0956-7

  View details for Web of Science ID 000387367100004

• An in vivo multiplexed small-molecule screening platform. Nature methods Grüner, B. M., Schulze, C. J., Yang, D., Ogasawara, D., Dix, M. M., Rogers, Z. N., Chuang, C., McFarland, C. D., Chiou, S., Brown, J. M., Cravatt, B. F., Bogyo, M., Winslow, M. M. 2016; 13 (10): 883-889

  Abstract

  Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.

  View details for DOI 10.1038/nmeth.3992

  View details for PubMedID 27617390

  View details for PubMedCentralID PMC5088491

• Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation JOURNAL OF NUCLEAR MEDICINE Withana, N. P., Saito, T., Ma, X., Garland, M., Liu, C., Kosuge, H., Amsallem, M., Verdoes, M., Ofori, L. O., Fischbein, M., Arakawa, M., Cheng, Z., McConnell, M. V., Bogyo, M. 2016; 57 (10): 1583-1590

  Abstract

  Macrophages are cellular mediators of vascular inflammation and are involved in the formation of atherosclerotic plaques. These immune cells secrete proteases such as matrix metalloproteinases and cathepsins that contribute to disease formation and progression. Here, we demonstrate that activity-based probes (ABPs) targeting cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes can also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations.Macrophage-rich carotid lesions were induced in FVB mice fed on a high-fat diet by streptozotocin injection followed by ligation of the left common carotid artery. Mice with carotid atherosclerotic plaques were injected with the optical or dual-modality probes BMV109 and BMV101, respectively, via the tail vein and noninvasively imaged by optical and small-animal PET/CT at different time points. After noninvasive imaging, the murine carotid arteries were imaged in situ and ex vivo, followed by immunofluorescence staining to confirm target labeling. Additionally, human carotid plaques were topically labeled with the probe and analyzed by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunofluorescence staining to confirm the primary targets of the probe.Quantitative analysis of the signal intensity from both optical and PET/CT imaging showed significantly higher levels of accumulation of BMV109 and BMV101 (P < 0.005 and P < 0.05, respectively) in the ligated left carotid arteries than the right carotid or healthy arteries. Immunofluorescence staining for macrophages in cross-sectional slices of the murine artery demonstrated substantial infiltration of macrophages in the neointima and adventitia of the ligated left carotid arteries compared with the right. Analysis of the human plaque tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that the primary targets of the probe were cathepsins X, B, S, and L. Immunofluorescence labeling of the human tissue with the probe demonstrated colocalization of the probe with CD68, elastin, and cathepsin S, similar to that observed in the experimental carotid inflammation murine model.We demonstrate that ABPs targeting the cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes could also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Therefore, ABPs targeting the cysteine cathepsins are potentially valuable new reagents for rapid and noninvasive imaging of atherosclerotic disease progression and plaque vulnerability.

  View details for DOI 10.2967/jnumed.115.171553

  View details for PubMedID 27199363

• Legumain is activated in macrophages during pancreatitis. American journal of physiology. Gastrointestinal and liver physiology Edgington-Mitchell, L. E., Wartmann, T., Fleming, A. K., Gocheva, V., van der Linden, W. A., Withana, N. P., Verdoes, M., Aurelio, L., Edgington-Mitchell, D., Lieu, T., Parker, B. S., Graham, B., Reinheckel, T., Furness, J. B., Joyce, J. A., Storz, P., Halangk, W., Bogyo, M., Bunnett, N. W. 2016; 311 (3): G548-60

  Abstract

  Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68(+) macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

  View details for DOI 10.1152/ajpgi.00047.2016

  View details for PubMedID 27514475

• Bifunctional Probes of Cathepsin Protease Activity and pH Reveal Alterations in Endolysosomal pH during Bacterial Infection. Cell chemical biology Sanman, L. E., van der Linden, W. A., Verdoes, M., Bogyo, M. 2016; 23 (7): 793-804

  Abstract

  Cysteine cathepsins are lysosomal proteases involved in regulation of both normal cellular processes and disease. Biochemical studies with peptide substrates indicate that cathepsins have optimal activity at acidic pH and highly attenuated activity at neutral pH. In contrast, there is mounting evidence that cathepsins have biological roles in environments that have non-acidic pH. To further define the specific pH environments where cathepsins act, we designed bifunctional activity-based probes (ABPs) that allow simultaneous analysis of cathepsin protease activity and pH. We use these probes to analyze the steady-state environment of cathepsin activity in macrophages and to measure dynamic changes in activity and pH upon stimulation. We show that Salmonella typhimurium induces a change in lysosomal pH that ultimately impairs cathepsin activity in both infected cells and a fraction of bystander cells, highlighting a mechanism by which Salmonella can simultaneously flourish within host cells and alter the behavior of nearby uninfected cells.

  View details for DOI 10.1016/j.chembiol.2016.05.019

  View details for PubMedID 27427229

  View details for PubMedCentralID PMC4982764

• Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS infectious diseases Lentz, C. S., Ordonez, A. A., Kasperkiewicz, P., La Greca, F., O'Donoghue, A. J., Schulze, C. J., Powers, J. C., Craik, C. S., Drag, M., Jain, S. K., Bogyo, M. 2016: -?

  Abstract

  Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

  View details for PubMedID 27739665

  View details for PubMedCentralID PMC5109297

• The cryo-EM structure of the Plasmodium falciparum 20S proteasome and its use in the fight against malaria. FEBS journal Li, H., Bogyo, M., da Fonseca, P. C. 2016

  Abstract

  Plasmodium falciparum is the parasite responsible for the most severe form of malaria. Its increasing resistance to existing antimalarials represents a major threat to human health and urges the development of new therapeutic strategies to fight malaria. The proteasome is a protease complex essential in all eukaryotes. Accordingly, inhibition of the Plasmodium 20S proteasome is highly toxic for the parasite at all of its infective and developmental stages. Proteasome inhibitors have antimalarial potential both as curative and transmission blocking agents, but in order to have therapeutic application, they must specifically target the Plasmodium proteasome and not its human counterpart. X-ray crystallography has been widely used to determine structures of yeast and mammalian 20S proteasomes with ligands. However, crystallisation of the Plasmodium proteasome is challenging, as only small quantities of the complex can be directly purified from the parasite. Furthermore, most X-ray structures of proteasome-inhibitor complexes require soaking of crystals with high concentrations of ligand, thus preventing analysis of inhibitor subunit specificity. Instead we chose to determine the Plasmodium falciparum 20S proteasome structure, in the presence of a new rationally designed parasite-specific inhibitor, by high-resolution electron cryo-microscopy and single particle analysis. The resulting map, at a resolution of about 3.6 Å, allows a direct molecular analysis of inhibitor/enzyme interactions. Here we present an overview of this structure, and how it provides valuable information that can be used to assist in the design of improved proteasome inhibitors with the potential to be developed as next-generation antimalarial drugs.

  View details for DOI 10.1111/febs.13780

  View details for PubMedID 27286897

  View details for PubMedCentralID PMC5140733

• Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death ELIFE Sanman, L. E., Qian, Y., Eisle, N. A., Ng, T. M., van der Linden, W. A., Monack, D. M., Weerapana, E., Bogyo, M. 2016; 5

  Abstract

  When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1β production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production. This signal is also important for host-cell response to the intracellular pathogen Salmonella typhimurium, which can disrupt metabolism by uptake of host-cell glucose. These results reveal an important inflammatory signaling network used by immune cells to sense metabolic dysfunction or infection by intracellular pathogens.

  View details for DOI 10.7554/eLife.13663

  View details for Web of Science ID 000387452200001

  View details for PubMedCentralID PMC4846378

• Cysteine Cathepsin Inhibitors as Anti-Ebola Agents. ACS infectious diseases van der Linden, W. A., Schulze, C. J., Herbert, A. S., Krause, T. B., Wirchnianski, A. A., Dye, J. M., Chandran, K., Bogyo, M. 2016; 2 (3): 173-179

  Abstract

  The recent Ebola virus outbreak in western Africa highlights the need for novel therapeutics that target Ebola virus and other filoviruses. Filoviruses require processing by host cell-derived cysteine cathepsins for productive infection. Here we report the generation of a focused library of cysteine cathepsin inhibitors and subsequent screening to identify compounds with potent activity against viral entry and replication. Our top compounds show highly potent and broad-spectrum activity against cysteine cathepsins and were able to effectively block entry of Ebola and Marburg viruses. These agents are promising leads for development as antifilovirus therapeutics.

  View details for DOI 10.1021/acsinfecdis.5b00130

  View details for PubMedID 27347558

  View details for PubMedCentralID PMC4918641

• Cysteine Cathepsin Inhibitors as Anti-Ebola Agents ACS INFECTIOUS DISEASES van der Linden, W. A., Schulze, C. J., Herbert, A. S., Krause, T. B., Wirchnianski, A. A., Dye, J. M., Chandran, K., Bogyo, M. 2016; 2 (3): 173-179

  Abstract

  The recent Ebola virus outbreak in western Africa highlights the need for novel therapeutics that target Ebola virus and other filoviruses. Filoviruses require processing by host cell-derived cysteine cathepsins for productive infection. Here we report the generation of a focused library of cysteine cathepsin inhibitors and subsequent screening to identify compounds with potent activity against viral entry and replication. Our top compounds show highly potent and broad-spectrum activity against cysteine cathepsins and were able to effectively block entry of Ebola and Marburg viruses. These agents are promising leads for development as antifilovirus therapeutics.

  View details for DOI 10.1021/acsinfecdis.5b00130

  View details for Web of Science ID 000372213100002

  View details for PubMedCentralID PMC4918641

• Structure- and function-based design of Plasmodium-selective proteasome inhibitors NATURE Li, H., O'Donoghue, A. J., van der Linden, W. A., Xie, S. C., Yoo, E., Foe, I. T., Tilley, L., Craik, C. S., da Fonseca, P. C., Bogyo, M. 2016; 530 (7589): 233-?

  Abstract

  The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

  View details for DOI 10.1038/nature16936

  View details for Web of Science ID 000369916700042

  View details for PubMedID 26863983

  View details for PubMedCentralID PMC4755332

• Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes SCIENTIFIC REPORTS Withana, N. P., Ma, X., McGuire, H. M., Verdoes, M., van der Linden, W. A., Ofori, L. O., Zhang, R., Li, H., Sanman, L. E., Wei, K., Yao, S., Wu, P., Li, F., Huang, H., Xu, Z., Wolters, P. J., Rosen, G. D., Collard, H. R., Zhu, Z., Cheng, Z., Bogyo, M. 2016; 6

  Abstract

  Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF. Therefore, imaging probes that specifically recognize these pools of activated immune cells could provide valuable information about how these cells contribute to the pathobiology of the disease. Here we demonstrate that cysteine cathepsin-targeted imaging probes can be used to monitor the contribution of macrophages to fibrotic disease progression in the bleomycin-induced murine model of pulmonary fibrosis. Furthermore, we show that the probes highlight regions of macrophage involvement in fibrosis in human biopsy tissues from IPF patients. Finally, we present first-in-human results demonstrating non-invasive imaging of active cathepsins in fibrotic lesions of patients with IPF. Together, our findings validate small molecule cysteine cathepsin probes for clinical PET imaging and suggest that they have the potential to be used to generate mechanistically-informative molecular information regarding cellular drivers of IPF disease severity and progression.

  View details for DOI 10.1038/srep19755

  View details for Web of Science ID 000368667700001

  View details for PubMedCentralID PMC4726431

• A Bright Future for Precision Medicine: Advances in Fluorescent Chemical Probe Design and Their Clinical Application. Cell chemical biology Garland, M., Yim, J. J., Bogyo, M. 2016; 23 (1): 122-136

  Abstract

  The Precision Medicine Initiative aims to use advances in basic and clinical research to develop therapeutics that selectively target and kill cancer cells. Under the same doctrine of precision medicine, there is an equally important need to visualize these diseased cells to enable diagnosis, facilitate surgical resection, and monitor therapeutic response. Therefore, there is a great opportunity for chemists to develop chemically tractable probes that can image cancer in vivo. This review focuses on recent advances in the development of optical probes, as well as their current and future applications in the clinical management of cancer. The progress in probe development described here suggests that optical imaging is an important and rapidly developing field of study that encourages continued collaboration among chemists, biologists, and clinicians to further refine these tools for interventional surgical imaging, as well as for diagnostic and therapeutic applications.

  View details for DOI 10.1016/j.chembiol.2015.12.003

  View details for PubMedID 26933740

  View details for PubMedCentralID PMC4779185

• Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes NATURE PROTOCOLS Withana, N. P., Garland, M., Verdoes, M., Ofori, L. O., Segal, E., Bogyo, M. 2016; 11 (1): 184-191

  Abstract

  Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

  View details for DOI 10.1038/nprot.2016.004

  View details for Web of Science ID 000367462100012

• Response to Comment on "A small-molecule antivirulence agent for treating Clostridium difficile infection". Science translational medicine Bender, K. O., Garland, M. n., Bogyo, M. n. 2016; 8 (370): 370tr2

  Abstract

  Ebselen's antivirulence activity in Clostridium difficile infection is likely due to multiple modes of action, but the contribution of each to its efficacy remains unclear.

  View details for PubMedID 28003551

• Successful Translation of Fluorescence Navigation During Oncologic Surgery: A Consensus Report. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Rosenthal, E. L., Warram, J. M., de Boer, E., Basilion, J. P., Biel, M. A., Bogyo, M., Bouvet, M., Brigman, B. E., Colson, Y. L., DeMeester, S. R., Gurtner, G. C., Ishizawa, T., Jacobs, P. M., Keereweer, S., Liao, J. C., Nguyen, Q. T., Olson, J. M., Paulsen, K. D., Rieves, D., Sumer, B. D., Tweedle, M. F., Vahrmeijer, A. L., Weichert, J. P., Wilson, B. C., Zenn, M. R., Zinn, K. R., van Dam, G. M. 2016; 57 (1): 144-150

  Abstract

  Navigation with fluorescence guidance has emerged in the last decade as a promising strategy to improve the efficacy of oncologic surgery. To achieve routine clinical use, the onus is on the surgical community to objectively assess the value of this technique. This assessment may facilitate both the Food and Drug Administration (FDA) approval of new optical imaging agents and reimbursement for the imaging procedures. It is critical to characterize fluorescence-guided procedural benefits over existing practices and to elucidate both the costs and safety risks. This report is the result of a meeting of the International Society of Image Guided Surgery (ISIGS, www.isigs.org) on February 6th, 2015 in Miami, Florida and reflects a consensus of the participants' opinions. Our objective is to critically evaluate the imaging platform technology and optical imaging agents, and to make recommendations for successful clinical trial development of this highly promising approach in oncologic surgery.

  View details for DOI 10.2967/jnumed.115.158915

  View details for PubMedID 26449839

• Non-invasive Imaging of Idiopathic Pulmonary Fibrosis Using Cathepsin Protease Probes. Scientific reports Withana, N. P., Ma, X., McGuire, H. M., Verdoes, M., van der Linden, W. A., Ofori, L. O., Zhang, R., Li, H., Sanman, L. E., Wei, K., Yao, S., Wu, P., Li, F., Huang, H., Xu, Z., Wolters, P. J., Rosen, G. D., Collard, H. R., Zhu, Z., Cheng, Z., Bogyo, M. 2016; 6: 19755-?

  Abstract

  Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF. Therefore, imaging probes that specifically recognize these pools of activated immune cells could provide valuable information about how these cells contribute to the pathobiology of the disease. Here we demonstrate that cysteine cathepsin-targeted imaging probes can be used to monitor the contribution of macrophages to fibrotic disease progression in the bleomycin-induced murine model of pulmonary fibrosis. Furthermore, we show that the probes highlight regions of macrophage involvement in fibrosis in human biopsy tissues from IPF patients. Finally, we present first-in-human results demonstrating non-invasive imaging of active cathepsins in fibrotic lesions of patients with IPF. Together, our findings validate small molecule cysteine cathepsin probes for clinical PET imaging and suggest that they have the potential to be used to generate mechanistically-informative molecular information regarding cellular drivers of IPF disease severity and progression.

  View details for DOI 10.1038/srep19755

  View details for PubMedID 26797565

• Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes. Nature protocols Withana, N. P., Garland, M., Verdoes, M., Ofori, L. O., Segal, E., Bogyo, M. 2016; 11 (1): 184-191

  Abstract

  Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

  View details for DOI 10.1038/nprot.2016.004

  View details for PubMedID 26716706

• Detection of Active Caspases During Apoptosis Using Fluorescent Activity-Based Probes. Methods in molecular biology (Clifton, N.J.) Edgington-Mitchell, L. E., Bogyo, M. 2016; 1419: 27-39

  Abstract

  Activity-based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this chapter, we provide detailed methods for using fluorescent ABPs to detect the activity of caspases during the onset of apoptosis in vitro. We describe how these probes can be used to biochemically profile caspase activity in vitro using fluorescent SDS-PAGE as well as their application to imaging protease activity in live animals and tissues.

  View details for DOI 10.1007/978-1-4939-3581-9_3

  View details for PubMedID 27108429

• Cathepsin Activity-Based Probes and Inhibitor for Preclinical Atherosclerosis Imaging and Macrophage Depletion. PloS one Abd-Elrahman, I., Kosuge, H., Wises Sadan, T., Ben-Nun, Y., Meir, K., Rubinstein, C., Bogyo, M., McConnell, M. V., Blum, G. 2016; 11 (8)

  Abstract

  Cardiovascular disease is the leading cause of death worldwide, mainly due to an increasing prevalence of atherosclerosis characterized by inflammatory plaques. Plaques with high levels of macrophage infiltration are considered "vulnerable" while those that do not have significant inflammation are considered stable; cathepsin protease activity is highly elevated in macrophages of vulnerable plaques and contributes to plaque instability. Establishing novel tools for non-invasive molecular imaging of macrophages in plaques could aid in preclinical studies and evaluation of therapeutics. Furthermore, compounds that reduce the macrophage content within plaques should ultimately impact care for this disease.We have applied quenched fluorescent cathepsin activity-based probes (ABPs) to a murine atherosclerosis model and evaluated their use for in vivo imaging using fluorescent molecular tomography (FMT), as well as ex vivo fluorescence imaging and fluorescent microscopy. Additionally, freshly dissected human carotid plaques were treated with our potent cathepsin inhibitor and macrophage apoptosis was evaluated by fluorescent microscopy.We demonstrate that our ABPs accurately detect murine atherosclerotic plaques non-invasively, identifying cathepsin activity within plaque macrophages. In addition, our cathepsin inhibitor selectively induced cell apoptosis of 55%±10% of the macrophage within excised human atherosclerotic plaques.Cathepsin ABPs present a rapid diagnostic tool for macrophage detection in atherosclerotic plaque. Our inhibitor confirms cathepsin-targeting as a promising approach to treat atherosclerotic plaque inflammation.

  View details for DOI 10.1371/journal.pone.0160522

  View details for PubMedID 27532109

• Disruption of glycolytic flux is a signal for inflammasome signaling and pyroptotic cell death. eLife Sanman, L. E., Qian, Y., Eisele, N. A., Ng, T. M., van der Linden, W. A., Monack, D. M., Weerapana, E., Bogyo, M. 2016; 5

  Abstract

  When innate immune cells such as macrophages are challenged with environmental stresses or infection by pathogens, they trigger the rapid assembly of multi-protein complexes called inflammasomes that are responsible for initiating pro-inflammatory responses and a form of cell death termed pyroptosis. We describe here the identification of an intracellular trigger of NLRP3-mediated inflammatory signaling, IL-1β production and pyroptosis in primed murine bone marrow-derived macrophages that is mediated by the disruption of glycolytic flux. This signal results from a drop of NADH levels and induction of mitochondrial ROS production and can be rescued by addition of products that restore NADH production. This signal is also important for host-cell response to the intracellular pathogen Salmonella typhimurium, which can disrupt metabolism by uptake of host-cell glucose. These results reveal an important inflammatory signaling network used by immune cells to sense metabolic dysfunction or infection by intracellular pathogens.

  View details for DOI 10.7554/eLife.13663

  View details for PubMedID 27011353

  View details for PubMedCentralID PMC4846378

• Successful Translation of Fluorescence Navigation During Oncologic Surgery: A Consensus Report JOURNAL OF NUCLEAR MEDICINE Rosenthal, E. L., Warram, J. M., de Boer, E., Basilion, J. P., Biel, M. A., Bogyo, M., Bouvet, M., Brigman, B. E., Colson, Y. L., DeMeester, S. R., Gurtner, G. C., Ishizawa, T., Jacobs, P. M., Keereweer, S., Liao, J. C., Nguyen, Q. T., Olson, J. M., Paulsen, K. D., Rieves, D., Sumer, B. D., Tweedle, M. F., Vahrmeijer, A. L., Weichert, J. P., Wilson, B. C., Zenn, M. R., Zinn, K. R., van Dam, G. M. 2016; 57 (1): 144-150

  Abstract

  Navigation with fluorescence guidance has emerged in the last decade as a promising strategy to improve the efficacy of oncologic surgery. To achieve routine clinical use, the onus is on the surgical community to objectively assess the value of this technique. This assessment may facilitate both the Food and Drug Administration (FDA) approval of new optical imaging agents and reimbursement for the imaging procedures. It is critical to characterize fluorescence-guided procedural benefits over existing practices and to elucidate both the costs and safety risks. This report is the result of a meeting of the International Society of Image Guided Surgery (ISIGS, www.isigs.org) on February 6th, 2015 in Miami, Florida and reflects a consensus of the participants' opinions. Our objective is to critically evaluate the imaging platform technology and optical imaging agents, and to make recommendations for successful clinical trial development of this highly promising approach in oncologic surgery.

  View details for DOI 10.2967/jnumed.115.158915

  View details for Web of Science ID 000367862700025

• The protease cathepsin L regulates Th17 cell differentiation JOURNAL OF AUTOIMMUNITY Hou, L., Cooley, J., Swanson, R., Poh Chee Ong, P. C., Pike, R. N., Bogyo, M., Olson, S. T., Remold-O'Donnell, E. 2015; 65: 56-63

  Abstract

  Previously we reported that IL-17(+) T cells, primarily IL-17(+) γδ cells, are increased in mice lacking the protease inhibitor serpinB1 (serpinb1(-/-) mice). Here we show that serpinB1-deficient CD4 cells exhibit a cell-autonomous and selective deficiency in suppressing T helper 17 (Th17) cell differentiation. This suggested an opposing role for one or more protease in promoting Th17 differentiation. We found that several SerpinB1-inhibitable cysteine cathepsins are induced in Th17 cells, most prominently cathepsin L (catL); this was verified by peptidase assays, active site labeling and Western blots. Moreover, Th17 differentiation was suppressed by both broad cathepsin inhibitors and catL selective inhibitors. CatL is present in Th17 cells as single chain (SC)- and two-chain (TC)-forms. Inhibiting asparagine endopeptidase (AEP) blocked conversion of SC-catL to TC-catL and increased generation of serpinb1(-/-) Th17 cells, but not wild-type Th17 cells. These findings suggest that SC-catL is biologically active in promoting Th17 generation and is counter-regulated by serpinB1 and secondarily by AEP. Thus, in addition to regulation by cytokines and transcription factors, differentiation of CD4 cells to Th17 cells is actively regulated by a catL-serpinB1-AEP module. Targeting this protease regulatory module could be an approach to treating Th17 cell-driven autoimmune disorders.

  View details for DOI 10.1016/j.jaut.2015.08.006

  View details for Web of Science ID 000366779100006

  View details for PubMedCentralID PMC4679515

• The protease cathepsin L regulates Th17 cell differentiation. Journal of autoimmunity Hou, L., Cooley, J., Swanson, R., Ong, P. C., Pike, R. N., Bogyo, M., Olson, S. T., Remold-O'Donnell, E. 2015; 65: 56-63

  Abstract

  Previously we reported that IL-17(+) T cells, primarily IL-17(+) γδ cells, are increased in mice lacking the protease inhibitor serpinB1 (serpinb1(-/-) mice). Here we show that serpinB1-deficient CD4 cells exhibit a cell-autonomous and selective deficiency in suppressing T helper 17 (Th17) cell differentiation. This suggested an opposing role for one or more protease in promoting Th17 differentiation. We found that several SerpinB1-inhibitable cysteine cathepsins are induced in Th17 cells, most prominently cathepsin L (catL); this was verified by peptidase assays, active site labeling and Western blots. Moreover, Th17 differentiation was suppressed by both broad cathepsin inhibitors and catL selective inhibitors. CatL is present in Th17 cells as single chain (SC)- and two-chain (TC)-forms. Inhibiting asparagine endopeptidase (AEP) blocked conversion of SC-catL to TC-catL and increased generation of serpinb1(-/-) Th17 cells, but not wild-type Th17 cells. These findings suggest that SC-catL is biologically active in promoting Th17 generation and is counter-regulated by serpinB1 and secondarily by AEP. Thus, in addition to regulation by cytokines and transcription factors, differentiation of CD4 cells to Th17 cells is actively regulated by a catL-serpinB1-AEP module. Targeting this protease regulatory module could be an approach to treating Th17 cell-driven autoimmune disorders.

  View details for DOI 10.1016/j.jaut.2015.08.006

  View details for PubMedID 26343333

  View details for PubMedCentralID PMC4679515

• Trioxolane-Mediated Delivery of Mefloquine Limits Brain Exposure in a Mouse Model of Malaria. ACS medicinal chemistry letters Lauterwasser, E. M., Fontaine, S. D., Li, H., Gut, J., Katneni, K., Charman, S. A., Rosenthal, P. J., Bogyo, M., Renslo, A. R. 2015; 6 (11): 1145-1149

  Abstract

  Peroxidic antimalarial agents including the sequiterpene artemisinins and the synthetic 1,2,4-trioxolanes function via initial intraparasitic reduction of an endoperoxide bond. By chemically coupling this reduction to release of a tethered drug species it is possible to confer two distinct pharmacological effects in a parasite-selective fashion, both in vitro and in vivo. Here we demonstrate the trioxolane-mediated delivery of the antimalarial agent mefloquine in a mouse malaria model. Selective partitioning of the trioxolane-mefloquine conjugate in parasitized erythrocytes, combined with effective exclusion of the conjugate from brain significantly reduced brain exposure as compared to mice directly administered mefloquine. These studies suggest the potential of trioxolane-mediated drug delivery to mitigate off-target effects of existing drugs, including the adverse neuropsychiatric effects of mefloquine use in therapeutic and chemoprophylactic settings.

  View details for DOI 10.1021/acsmedchemlett.5b00296

  View details for PubMedID 26617969

  View details for PubMedCentralID PMC4645246

• Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase JOURNAL OF BIOLOGICAL CHEMISTRY Staniec, D., Ksiazek, M., Thogersen, I. B., Enghild, J. J., Sroka, A., Bryzek, D., Bogyo, M., Abrahamson, M., Potempa, J. 2015; 290 (45): 27248-27260

  Abstract

  Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease) that has sequence similarity to cysteine peptidases of the papain and calpain families. In this study we biochemically characterize Tpr. We found that the 55-kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48, 37, and 33 kDa via sequential truncations at the N terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations >1 mm, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4, and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.

  View details for DOI 10.1074/jbc.M115.648782

  View details for Web of Science ID 000364794000033

  View details for PubMedID 26385924

  View details for PubMedCentralID PMC4646396

• Trioxolane-Mediated Delivery of Mefloquine Limits Brain Exposure in a Mouse Model of Malaria ACS MEDICINAL CHEMISTRY LETTERS Lauterwasser, E. M., Fontaine, S. D., Li, H., Gut, J., Katneni, K., Charman, S. A., Rosenthal, P. J., Bogyo, M., Renslo, A. R. 2015; 6 (11): 1145-1149

  Abstract

  Peroxidic antimalarial agents including the sequiterpene artemisinins and the synthetic 1,2,4-trioxolanes function via initial intraparasitic reduction of an endoperoxide bond. By chemically coupling this reduction to release of a tethered drug species it is possible to confer two distinct pharmacological effects in a parasite-selective fashion, both in vitro and in vivo. Here we demonstrate the trioxolane-mediated delivery of the antimalarial agent mefloquine in a mouse malaria model. Selective partitioning of the trioxolane-mefloquine conjugate in parasitized erythrocytes, combined with effective exclusion of the conjugate from brain significantly reduced brain exposure as compared to mice directly administered mefloquine. These studies suggest the potential of trioxolane-mediated drug delivery to mitigate off-target effects of existing drugs, including the adverse neuropsychiatric effects of mefloquine use in therapeutic and chemoprophylactic settings.

  View details for DOI 10.1021/acsmedchemlett.5b00296

  View details for Web of Science ID 000364803200012

  View details for PubMedID 26617969

  View details for PubMedCentralID PMC4645246

• Global Analysis of Palmitoylated Proteins in Toxoplasma gondii CELL HOST & MICROBE Foe, I. T., Child, M. A., Majmudar, J. D., Krishnamurthy, S., van der Linden, W. A., Ward, G. E., Martin, B. R., Bogyo, M. 2015; 18 (4): 501-511

  Abstract

  Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.

  View details for DOI 10.1016/j.chom.2015.09.006

  View details for Web of Science ID 000365111600018

  View details for PubMedID 26468752

  View details for PubMedCentralID PMC4694575

• Cysteine cathepsin activity suppresses osteoclastogenesis of myeloid-derived suppressor cells in breast cancer ONCOTARGET Edgington-Mitchell, L. E., Rautela, J., Duivenvoorden, H. M., Jayatilleke, K. M., van der Linden, W. A., Verdoes, M., Bogyo, M., Parker, B. S. 2015; 6 (29): 27008-27022

  Abstract

  Cysteine cathepsin proteases contribute to many normal cellular functions, and their aberrant activity within various cell types can contribute to many diseases, including breast cancer. It is now well accepted that cathepsin proteases have numerous cell-specific functions within the tumor microenvironment that function to promote tumor growth and invasion, such that they may be valid targets for anti-metastatic therapeutic approaches. Using activity-based probes, we have examined the activity and expression of cysteine cathepsins in a mouse model of breast cancer metastasis to bone. In mice bearing highly metastatic tumors, we detected abundant cysteine cathepsin expression and activity in myeloid-derived suppressor cells (MDSCs). These immature immune cells have known metastasis-promoting roles, including immunosuppression and osteoclastogenesis, and we assessed the contribution of cysteine cathepsins to these functions. Blocking cysteine cathepsin activity with multiple small-molecule inhibitors resulted in enhanced differentiation of multinucleated osteoclasts. This highlights a potential role for cysteine cathepsin activity in suppressing the fusion of osteoclast precursor cells. In support of this hypothesis, we found that expression and activity of key cysteine cathepsins were downregulated during MDSC-osteoclast differentiation. Another cysteine protease, legumain, also inhibits osteoclastogenesis, in part through modulation of cathepsin L activity. Together, these data suggest that cysteine protease inhibition is associated with enhanced osteoclastogenesis, a process that has been implicated in bone metastasis.

  View details for DOI 10.18632/oncotarget.4714

  View details for PubMedID 26308073

  View details for PubMedCentralID PMC4694970

• A small-molecule antivirulence agent for treating Clostridium difficile infection. Science translational medicine Bender, K. O., Garland, M., Ferreyra, J. A., Hryckowian, A. J., Child, M. A., Puri, A. W., Solow-Cordero, D. E., Higginbottom, S. K., Segal, E., Banaei, N., Shen, A., Sonnenburg, J. L., Bogyo, M. 2015; 7 (306): 306ra148-?

  Abstract

  Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.

  View details for DOI 10.1126/scitranslmed.aac9103

  View details for PubMedID 26400909

• A small-molecule antivirulence agent for treating Clostridium difficile infection SCIENCE TRANSLATIONAL MEDICINE Bender, K. O., Garland, M., Ferreyra, J. A., Hryckowian, A. J., Child, M. A., Puri, A. W., Solow-Cordero, D. E., Higginbottom, S. K., Segal, E., Banaei, N., Shen, A., Sonnenburg, J. L., Bogyo, M. 2015; 7 (306)

  Abstract

  Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.

  View details for DOI 10.1126/scitranslmed.aac9103

  View details for Web of Science ID 000365232600002

  View details for PubMedID 26400909

• Design of Protease Activated Optical Contrast Agents That Exploit a Latent Lysosomotropic Effect for Use in Fluorescence-Guided Surgery. ACS chemical biology Ofori, L. O., Withana, N. P., Prestwood, T. R., Verdoes, M., Brady, J. J., Winslow, M. M., Sorger, J., Bogyo, M. 2015; 10 (9): 1977-1988

  Abstract

  There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivo could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.

  View details for DOI 10.1021/acschembio.5b00205

  View details for PubMedID 26039341

  View details for PubMedCentralID PMC4577961

• Design and Synthesis of Activity-Based Probes and Inhibitors for Bleomycin Hydrolase. Chemistry & biology van der Linden, W. A., Segal, E., Child, M. A., Byzia, A., Drag, M., Bogyo, M. 2015; 22 (8): 995-1001

  Abstract

  Bleomycin hydrolase (BLMH) is a neutral cysteine aminopeptidase that has been ascribed roles in many physiological and pathological processes, yet its primary biological function remains enigmatic. In this work, we describe the results of screening of a library of fluorogenic substrates to identify non-natural amino acids that are optimally recognized by BLMH. This screen identified several substrates with kcat/KM values that are substantially improved over the previously reported fluorogenic substrates for this enzyme. The substrate sequences were used to design activity-based probes that showed potent labeling of recombinant BLMH as well as endogenously expressed BLMH in cell extracts, and in intact cells. Importantly, we identify potent BLMH inhibitors that are able to fully inhibit endogenous BLMH activity in intact cells. These probes and inhibitors will be valuable new reagents to study BLMH function in cellular and animal models of human diseases where BLMH is likely to be involved.

  View details for DOI 10.1016/j.chembiol.2015.07.010

  View details for PubMedID 26256478

  View details for PubMedCentralID PMC4546515

• New chemical probe technologies for diagnostic and intra-operative imaging applications Ofori, L., Withana, N., Verdoes, M., Sorger, J., Bogyo, M. AMER CHEMICAL SOC. 2015

  View details for Web of Science ID 000432475501315

• Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination PLANT PHYSIOLOGY Lu, H., Chandrasekar, B., Oeljeklaus, J., Misas-Villamil, J. C., Wang, Z., Shindo, T., Bogyo, M., Kaiser, M., van der Hoorn, R. A. 2015; 168 (4): 1462-U1636

  Abstract

  Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.

  View details for DOI 10.1104/pp.114.254466

  View details for Web of Science ID 000359317400025

  View details for PubMedID 26048883

• Voices of chemical biology NATURE CHEMICAL BIOLOGY Bertozzi, C. R., Stockwell, B. R., Kubicek, S., Dickinson, B. C., Chang, C. J., Schultz, C., Silver, P. A., Gestwicki, J. E., Chiosis, G., Chattopadhyay, A., Butcher, R. A., Park, S., Shoichet, B. K., Flitsch, S. L., Zhang, J., Liu, D. R., Ohnishi, Y., Weerapana, E., Williams, A. H., He, C., Moroni, A., Thiel, G., Chang, Y., Waldmann, H., Bogyo, M., Oddershede, L. B., Christopoulos, A., Imperiali, B., Ehrlich, H., Chen, X., Prescher, J. A. 2015; 11 (6): 378-379

  View details for Web of Science ID 000354539400004

  View details for PubMedID 25978987

  View details for PubMedCentralID PMC5283386

• Inhibition of cathepsin proteases attenuates migration and sensitizes aggressive N-Myc amplified human neuroblastoma cells to doxorubicin ONCOTARGET Gangoda, L., Keerthikumar, S., Fonseka, P., Edgington, L. E., Ang, C., Ozcitti, C., Bogyo, M., Parker, B. S., Mathivanan, S. 2015; 6 (13): 11175-11190

  Abstract

  Neuroblastoma arises from the sympathetic nervous system and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is reported to occur in more than 20% of patients. While N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressive progression of the disease is poorly understood. N-Myc being a transcription factor can modulate the secretion of key proteins that may play a pivotal role in tumorigenesis. Characterising the soluble secreted proteins or secretome will aid in understanding their role in the tumour microenvironment, such as promoting cancer cell invasion and resistance to treatment. The aim of this study is to characterise the secretome of human malignant neuroblastoma SK-N-BE2 (N-Myc amplified, more aggressive) and SH-SY5Y (N-Myc non-amplified, less aggressive) cells. Conditioned media from SK-N-BE2 and SH-SY5Y cell lines were subjected to proteomics analysis. We report a catalogue of 894 proteins identified in the secretome isolated from the two neuroblastoma cell lines, SK-N-BE2 and SH-SY5Y. Functional enrichment analysis using FunRich software identified enhanced secretion of proteins implicated in cysteine peptidase activity in the aggressive N-Myc amplified SK-N-BE2 secretome compared to the less tumorigenic SH-SY5Y cells. Protein-protein interaction-based network analysis highlighted the enrichment of cathepsin and epithelial-to-mesenchymal transition sub-networks. For the first time, inhibition of cathepsins by inhibitors sensitized the resistant SK-N-BE2 cells to doxorubicin as well as decreased its migratory potential. The dataset of secretome proteins of N-Myc amplified (more aggressive) and non-amplified (less aggressive) neuroblastoma cells represent the first inventory of neuroblastoma secretome. The study also highlights the prominent role of cathepsins in the N-Myc amplified neuroblastoma pathogenesis. As N-Myc amplification correlates with aggressive neuroblastoma and chemotherapy-based treatment failure, co-treatment with cathepsin inhibitors might be a better avenue for disease management.

  View details for Web of Science ID 000359006400040

  View details for PubMedID 25883214

• Design of a Highly Selective Quenched Activity-Based Probe and Its Application in Dual Color Imaging Studies of Cathepsin S Activity Localization JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bender, K. O., Ofori, L., van der Linden, W. A., Mock, E. D., Datta, G. K., Chowdhury, S., Li, H., Segal, E., Lopez, M. S., Ellman, J. A., Figdor, C. G., Bogyo, M., Verdoes, M. 2015; 137 (14): 4771-4777

  Abstract

  The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity. This observation demonstrates the value of our complementary cathepsin probes and provides evidence for the existence of specific localization of cathepsin S activity in dendritic cells.

  View details for DOI 10.1021/jacs.5b00315

  View details for Web of Science ID 000353177100033

  View details for PubMedCentralID PMC4747655

• Design of a highly selective quenched activity-based probe and its application in dual color imaging studies of cathepsin S activity localization. Journal of the American Chemical Society Oresic Bender, K., Ofori, L., van der Linden, W. A., Mock, E. D., Datta, G. K., Chowdhury, S., Li, H., Segal, E., Sanchez Lopez, M., Ellman, J. A., Figdor, C. G., Bogyo, M., Verdoes, M. 2015; 137 (14): 4771-4777

  Abstract

  The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity. This observation demonstrates the value of our complementary cathepsin probes and provides evidence for the existence of specific localization of cathepsin S activity in dendritic cells.

  View details for DOI 10.1021/jacs.5b00315

  View details for PubMedID 25785540

• Probes to Monitor Activity of the Paracaspase MALT1. Chemistry & biology Hachmann, J., Edgington-Mitchell, L. E., Poreba, M., Sanman, L. E., Drag, M., Bogyo, M., Salvesen, G. S. 2015; 22 (1): 139-147

  Abstract

  The human paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) plays a central role in nuclear factor-κB (NF-κB) signaling as both a protease and scaffolding protein. Knocking out MALT1 leads to impaired NF-κB signaling and failure to mount an effective immune response. However, it is unclear to which degree it is the scaffolding function versus the proteolytic activity of MALT1 that is essential. Previous work involving a MALT1 inhibitor with low selectivity suggests that the enzymatic function plays an important role in different cell lines. To help elucidate this proteolytic role of MALT1, we have designed activity-based probes that inhibit its proteolytic activity. The probes selectively label active enzyme and can be used to inhibit MALT1 and trace its activity profile, helping to create a better picture of the significance of the proteolytic function of MALT1.

  View details for DOI 10.1016/j.chembiol.2014.11.011

  View details for PubMedID 25556944

  View details for PubMedCentralID PMC4304901

• Detection of intestinal cancer by local, topical application of a quenched fluorescence probe for cysteine cathepsins. Chemistry & biology Segal, E., Prestwood, T. R., van der Linden, W. A., Carmi, Y., Bhattacharya, N., Withana, N., Verdoes, M., Habtezion, A., Engleman, E. G., Bogyo, M. 2015; 22 (1): 148-158

  Abstract

  Early detection of colonic polyps can prevent up to 90% of colorectal cancer deaths. Conventional colonoscopy readily detects the majority of premalignant lesions, which exhibit raised morphology. However, lesions that are flat and depressed are often undetected using this method. Therefore, there is a need for molecular-based contrast agents to improve detection rates over conventional colonoscopy. We evaluated a quenched fluorescent activity-based probe (qABP; BMV109) that targets multiple cysteine cathepsins that are overexpressed in intestinal dysplasia in a genetic model of spontaneous intestinal polyp formation and in a chemically induced model of colorectal carcinoma. We found that the qABP selectively targets cysteine cathepsins, resulting in high sensitivity and specificity for intestinal tumors in mice and humans. Additionally, the qABP can be administered by either intravenous injection or by local delivery to the colon, making it a highly valuable tool for improved detection of colorectal lesions using fluorescence-guided colonoscopy.

  View details for DOI 10.1016/j.chembiol.2014.11.008

  View details for PubMedID 25579207

  View details for PubMedCentralID PMC4353655

• Multiple Cathepsins Promote Pro-IL-1β Synthesis and NLRP3-Mediated IL-1β Activation. Journal of immunology (Baltimore, Md. : 1950) Orlowski, G. M., Colbert, J. D., Sharma, S. n., Bogyo, M. n., Robertson, S. A., Rock, K. L. 2015; 195 (4): 1685–97

  Abstract

  Sterile particles induce robust inflammatory responses that underlie the pathogenesis of diseases like silicosis, gout, and atherosclerosis. A key cytokine mediating this response is IL-1β. The generation of bioactive IL-1β by sterile particles is mediated by the NOD-like receptor containing a pyrin domain 3 (NLRP3) inflammasome, although exactly how this occurs is incompletely resolved. Prior studies have found that the cathepsin B inhibitor, Ca074Me, suppresses this response, supporting a model whereby ingested particles disrupt lysosomes and release cathepsin B into the cytosol, somehow activating NLRP3. However, reports that cathepsin B-deficient macrophages have no defect in particle-induced IL-1β generation have questioned cathepsin B's involvement. In this study, we examine the hypothesis that multiple redundant cathepsins (not just cathepsin B) mediate this process by evaluating IL-1β generation in murine macrophages, singly or multiply deficient in cathepsins B, L, C, S and X. Using an activity-based probe, we measure specific cathepsin activity in living cells, documenting compensatory changes in cathepsin-deficient cells, and Ca074Me's dose-dependent cathepsin inhibition profile is analyzed in parallel with its suppression of particle-induced IL-1β secretion. Also, we evaluate endogenous cathepsin inhibitors cystatins C and B. Surprisingly, we find that multiple redundant cathepsins, inhibited by Ca074Me and cystatins, promote pro-IL-1β synthesis, and to our knowledge, we provide the first evidence that cathepsin X plays a nonredundant role in nonparticulate NLRP3 activation. Finally, we find cathepsin inhibitors selectively block particle-induced NLRP3 activation, independently of suppressing pro-IL-1β synthesis. Altogether, we demonstrate that both small molecule and endogenous cathepsin inhibitors suppress particle-induced IL-1β secretion, implicating roles for multiple cathepsins in both pro-IL-1β synthesis and NLRP3 activation.

  View details for PubMedID 26195813

  View details for PubMedCentralID PMC4530060

• Smart Imaging Probes for the Study of Protease Function OPTICAL PROBES IN BIOLOGY Segal, E., Bogyo, M., Zhang, J., Mehta, S., Schultz, C. 2015; 6: 377-409

  View details for Web of Science ID 000409474100017

• Loss of Prkar1a leads to Bcl-2 family protein induction and cachexia in mice CELL DEATH AND DIFFERENTIATION Gangoda, L., Doerflinger, M., Srivastava, R., Narayan, N., Edgington, L. E., Orian, J., Hawkins, C., O'Reilly, L. A., Gu, H., Bogyo, M., Ekert, P., Strasser, A., Puthalakath, H. 2014; 21 (11): 1815-1824

  Abstract

  Loss of function mutations in the Prkar1a gene are the cause of most cases of Carney complex disorder. Defects in Prkar1a are thought to cause hyper-activation of PKA signalling, which drives neoplastic transformation, and Prkar1a is therefore considered to be a tumour suppressor. Here we show that loss of Prkar1a in genetically modified mice caused transcriptional activation of several proapoptotic Bcl-2 family members and thereby caused cell death. Interestingly, combined loss of Bim and Prkar1a increased colony formation of fibroblasts in culture and promoted their growth as tumours in immune-deficient mice. Apart from inducing apoptosis, systemic deletion of Prkar1a caused cachexia with muscle loss, macrophage activation and increased lipolysis as well as serum triglyceride levels. Loss of single allele of Prkar1a did not enhance tumour development in a skin cancer model, but surprisingly, when combined with the loss of Bim, caused a significant delay in tumorigenesis and this was associated with upregulation of other BH3-only proteins, PUMA and NOXA. These results show that loss of Prkar1a can only promote tumorigenesis when Prkar1a-mediated apoptosis is somehow countered.

  View details for DOI 10.1038/cdd.2014.98

  View details for Web of Science ID 000343296800015

  View details for PubMedID 25012505

• Identification of Potent and Selective Non-covalent Inhibitors of the Plasmodium falciparum Proteasome JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Li, H., Tsu, C., Blackburn, C., Li, G., Hales, P., Dick, L., Bogyo, M. 2014; 136 (39): 13562-13565

  Abstract

  We have identified short N,C-capped peptides that selectively inhibit the proteasome of the malaria-causing pathogen Plasmodium falciparum. These compounds are highly potent in culture with no toxicity in host cells. One cyclic biphenyl ether compound inhibited intraerythrocytic growth of P. falciparum with an IC50 of 35 nM, and we show that even a pulse treatment with this cyclic peptide induced parasite death due to proteasome inhibition. These compounds represent promising new antimalarial agents that target the essential proteasomal machinery of the parasite without toxicity toward the host.

  View details for DOI 10.1021/ja507692y

  View details for Web of Science ID 000342608800024

  View details for PubMedCentralID PMC4183598

• Molecular imaging of tumor-associated cathepsins: implication for rapid detection of human nonmelanoma skin cancer Walker, E., Mann, M., Blum, G., Bogyo, M., Basilion, J. P. AMER ASSOC CANCER RESEARCH. 2014

  View details for DOI 10.1158/1538-7445.AM2014-4288

  View details for Web of Science ID 000349910202264

• Microscopic Detection of Quenched Activity-Based Optical Imaging Probes Using an Antibody Detection System: Localizing Protease Activity MOLECULAR IMAGING AND BIOLOGY Walker, E., Gopalakrishnan, R., Bogyo, M., Basilion, J. P. 2014; 16 (5): 608-618

  Abstract

  The family of cathepsin proteases plays an important physiological role in both normal physiology and in the physiology of many human diseases. This activity, which is upregulated in many cancers, can be exploited for tumor imaging both in vivo and ex vivo. To characterize the behavior of a topically applied quenched fluorescent activity-based probe, GB119, ex vivo, we developed a basic immunohistochemistry technique to identify unquenched GB119 within tissue.Immunoblot assays were used to validate the utility of an anit-Cy5 antibody for the detection of unquenched GB119 generated by cathepsin-L. Following validation of the anti-Cy5 antibody, an immunohistochemical procedure was developed to detect the presence of unquenched GB119 in frozen sections of brain tumors derived from an orthotopic mouse model.These studies demonstrate that the anti-Cy5 antibody preferentially recognizes unquenched GB119 and that this differential can be used to identify the regions within the brain and the tumor that contained unquenched GB119. Using H&E staining and antibodies against other biochemical markers, it was further determined that unquenched GB119 was localized to the peri-tumor space and co-localized with cathepsin-L expression.Our data indicate that this methodology allows high-resolution detection of unquenched GB119 that can be correlated with other immunohistological stains.

  View details for DOI 10.1007/s11307-014-0736-1

  View details for Web of Science ID 000342135800004

  View details for PubMedID 24705781

  View details for PubMedCentralID PMC4240007

• Identification of potent and selective non-covalent inhibitors of the Plasmodium falciparum proteasome. Journal of the American Chemical Society Li, H., Tsu, C., Blackburn, C., Li, G., Hales, P., Dick, L., Bogyo, M. 2014; 136 (39): 13562-13565

  Abstract

  We have identified short N,C-capped peptides that selectively inhibit the proteasome of the malaria-causing pathogen Plasmodium falciparum. These compounds are highly potent in culture with no toxicity in host cells. One cyclic biphenyl ether compound inhibited intraerythrocytic growth of P. falciparum with an IC50 of 35 nM, and we show that even a pulse treatment with this cyclic peptide induced parasite death due to proteasome inhibition. These compounds represent promising new antimalarial agents that target the essential proteasomal machinery of the parasite without toxicity toward the host.

  View details for DOI 10.1021/ja507692y

  View details for PubMedID 25226494

  View details for PubMedCentralID PMC4183598

• Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes. ACS chemical biology Li, H., van der Linden, W. A., Verdoes, M., Florea, B. I., McAllister, F. E., Govindaswamy, K., Elias, J. E., Bhanot, P., Overkleeft, H. S., Bogyo, M. 2014; 9 (8): 1869-1876

  Abstract

  The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

  View details for DOI 10.1021/cb5001263

  View details for PubMedID 24918547

  View details for PubMedCentralID PMC4136710

• Serine proteases and protease-activated receptor 2 mediate the proinflammatory and algesic actions of diverse stimulants BRITISH JOURNAL OF PHARMACOLOGY Cattaruzza, F., Amadesi, S., Carlsson, J. F., Murphy, J. E., Lyo, V., Kirkwood, K., Cottrell, G. S., Bogyo, M., Knecht, W., Bunnett, N. W. 2014; 171 (16): 3814-3826

  Abstract

  Although serine proteases and agonists of protease-activated receptor 2 (PAR2) cause inflammation and pain, the spectrum of proteases that are activated by proinflammatory and algesic stimuli and their contribution to inflammatory pain are uncertain.Enzymic assays and selective inhibitors were used to characterize protease activity in mice after intraplantar injections of formalin, bradykinin, PAR2 activating peptide (AP) or vehicle. The capacity of these proteases and of recombinant mouse trypsin 4 to cleave fragments of PAR2 and to activate PAR2 in cell lines was determined. Protease inhibitors and par2 (-/-) mice were used to assess the contributions of proteases and PAR2 to pain and inflammation.Intraplantar injection of formalin, bradykinin or PAR2-AP led to the activation of proteases that were susceptible to the serine protease inhibitor melagatran but resistant to soybean trypsin inhibitor (SBTI). Melagatran inhibited mouse trypsin 4, which degraded SBTI. Proteases generated in inflamed tissues cleaved PAR2-derived peptides. These proteases and trypsin 4 increased [Ca(2+) ]i in PAR2-transfected but not in untransfected cells, and melagatran suppressed this activity. Melagatran or PAR2 deletion suppressed oedema and mechanical hypersensitivity induced by intraplantar formalin, bradykinin and PAR2-AP, but had no effect on capsaicin-induced pain.Diverse proinflammatory and algesic agents activate melagatran-sensitive serine proteases that cause inflammation and pain by a PAR2-mediated mechanism. By inducing self-activating proteases, PAR2 amplifies and sustains inflammation and pain. Serine protease inhibitors can attenuate the inflammatory and algesic effects of diverse stimuli, representing a useful therapeutic strategy.

  View details for DOI 10.1111/bph.12738

  View details for Web of Science ID 000340268700004

  View details for PubMedID 24749982

  View details for PubMedCentralID PMC4128045

• Application of Small Molecule Probes to the Study of Protease Function Bogyo, M. WILEY-BLACKWELL. 2014: 69

  View details for Web of Science ID 000339545700036

• Activity-based profiling of proteases. Annual review of biochemistry Sanman, L. E., Bogyo, M. 2014; 83: 249-273

  Abstract

  Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.

  View details for DOI 10.1146/annurev-biochem-060713-035352

  View details for PubMedID 24905783

• Phosphoramidates as Novel Activity-Based Probes for Serine Proteases CHEMBIOCHEM Haedke, U. R., Frommel, S. C., Hansen, F., Hahne, H., Kuster, B., Bogyo, M., Verhelst, S. H. 2014; 15 (8): 1106-1110

  Abstract

  Activity-based probes (ABPs) are small molecules that exclusively form covalent bonds with catalytically active enzymes. In the last decade, they have especially been used in functional proteomics studies of proteases. Here, we present phosphoramidate peptides as a novel type of ABP for serine proteases. These molecules can be made in a straightforward manner by standard Fmoc-based solid-phase peptide synthesis, allowing rapid diversification. The resulting ABPs covalently bind different serine proteases, depending on the amino acid recognition element adjacent to the reactive group. A reporter tag enables downstream gel-based analysis or LC-MS/MS-mediated identification of the targeted proteases. Overall, we believe that these readily accessible probes will provide new avenues for the functional study of serine proteases in complex proteomes.

  View details for DOI 10.1002/cbic.201400013

  View details for Web of Science ID 000337582200008

  View details for PubMedID 24817682

• The Apoptosis Repressor with a CARD Domain (ARC) Gene Is a Direct Hypoxia-Inducible Factor 1 Target Gene and Promotes Survival and Proliferation of VHL-Deficient Renal Cancer Cells. Molecular and cellular biology Razorenova, O. V., Castellini, L., Colavitti, R., Edgington, L. E., Nicolau, M., Huang, X., Bedogni, B., Mills, E. M., Bogyo, M., Giaccia, A. J. 2014; 34 (4): 739-751

  Abstract

  The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp -190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.

  View details for DOI 10.1128/MCB.00644-12

  View details for PubMedID 24344197

  View details for PubMedCentralID PMC3911479

• A biocompatible "split luciferin" reaction and its application for non-invasive bioluminescent imaging of protease activity in living animals. Current protocols in chemical biology Godinat, A., Budin, G., Morales, A. R., Park, H. M., Sanman, L. E., Bogyo, M., Yu, A., Stahl, A., Dubikovskaya, E. A. 2014; 6 (3): 169-189

  Abstract

  The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real-time non-invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the "split luciferin" biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of the activity of multiple proteases, as well as identification of their new peptide-specific substrates. In this unit, we describe several applications of this "split luciferin" reaction for quantification of protease activities in test tube assays and living animals.

  View details for DOI 10.1002/9780470559277.ch140047

  View details for PubMedID 25205565

  View details for PubMedCentralID PMC4219325

• Toxoplasma gondii Chemical Biology TOXOPLASMA GONDII: THE MODEL APICOMPLEXAN - PERSPECTIVES AND METHODS, 2ND EDITION Bogyo, M., Ward, G., Weiss, L. M., Kim, K. 2014: 707-730

  View details for DOI 10.1016/B978-0-12-396481-6.00021-0

  View details for Web of Science ID 000327995800023

• Ferrous iron-dependent drug delivery enables controlled and selective release of therapeutic agents in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Deu, E., Chen, I. T., Lauterwasser, E. M., Valderramos, J., Li, H., Edgington, L. E., Renslo, A. R., Bogyo, M. 2013; 110 (45): 18244-18249

  Abstract

  The precise targeting of cytotoxic agents to specific cell types or cellular compartments is of significant interest in medicine, with particular relevance for infectious diseases and cancer. Here, we describe a method to exploit aberrant levels of mobile ferrous iron (Fe(II)) for selective drug delivery in vivo. This approach makes use of a 1,2,4-trioxolane moiety, which serves as an Fe(II)-sensitive "trigger," making drug release contingent on Fe(II)-promoted trioxolane fragmentation. We demonstrate in vivo validation of this approach with the Plasmodium berghei model of murine malaria. Malaria parasites produce high concentrations of mobile ferrous iron as a consequence of their catabolism of host hemoglobin in the infected erythrocyte. Using activity-based probes, we successfully demonstrate the Fe(II)-dependent and parasite-selective delivery of a potent dipeptidyl aminopeptidase inhibitor. We find that delivery of the compound in its Fe(II)-targeted form leads to more sustained target inhibition with greatly reduced off-target inhibition of mammalian cathepsins. This selective drug delivery translates into improved efficacy and tolerability. These findings demonstrate the utility of a purely chemical means to achieve selective drug targeting in vivo. This approach may find useful application in parasitic infections and more broadly in any disease state characterized by aberrant production of reactive ferrous iron.

  View details for DOI 10.1073/pnas.1312782110

  View details for Web of Science ID 000326550800056

  View details for PubMedID 24145449

  View details for PubMedCentralID PMC3831501

• Improved quenched fluorescent probe for imaging of cysteine cathepsin activity. Journal of the American Chemical Society Verdoes, M., Oresic Bender, K., Segal, E., van der Linden, W. A., Syed, S., Withana, N. P., Sanman, L. E., Bogyo, M. 2013; 135 (39): 14726-14730

  Abstract

  The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile. These reagents show enhanced in vivo properties and broad reactivity resulting in dramatically improved labeling and tumor imaging properties compared to those of previously reported ABPs.

  View details for DOI 10.1021/ja4056068

  View details for PubMedID 23971698

  View details for PubMedCentralID PMC3826460

• Improved Quenched Fluorescent Probe for Imaging of Cysteine Cathepsin Activity JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Verdoes, M., Bender, K. O., Segal, E., van der Linden, W. A., Syed, S., Withana, N. P., Sanman, L. E., Bogyo, M. 2013; 135 (39): 14726-14730

  Abstract

  The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile. These reagents show enhanced in vivo properties and broad reactivity resulting in dramatically improved labeling and tumor imaging properties compared to those of previously reported ABPs.

  View details for DOI 10.1021/ja4056068

  View details for Web of Science ID 000326300500042

  View details for PubMedCentralID PMC3826460

• Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion. Nature chemical biology Child, M. A., Hall, C. I., Beck, J. R., Ofori, L. O., Albrow, V. E., Garland, M., Bowyer, P. W., Bradley, P. J., Powers, J. C., Boothroyd, J. C., Weerapana, E., Bogyo, M. 2013; 9 (10): 651-656

  Abstract

  Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

  View details for DOI 10.1038/nchembio.1315

  View details for PubMedID 23934245

  View details for PubMedCentralID PMC3832678

• Acid-mediated tumor proteolysis: contribution of cysteine cathepsins. Neoplasia Rothberg, J. M., Bailey, K. M., Wojtkowiak, J. W., Ben-Nun, Y., Bogyo, M., Weber, E., Moin, K., Blum, G., Mattingly, R. R., Gillies, R. J., Sloane, B. F. 2013; 15 (10): 1125-1137

  Abstract

  One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8) affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D) cultures. For these studies, we used 1) 3D reconstituted basement membrane overlay cultures of human carcinomas, 2) live cell imaging assays to assess proteolysis, and 3) in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.

  View details for PubMedID 24204192

• Acid-Mediated Tumor Proteolysis: Contribution of Cysteine Cathepsins NEOPLASIA Rothberg, J. M., Bailey, K. M., Wojtkowiak, J. W., Ben-Nun, Y., Bogyo, M., Weber, E., Moin, K., Blum, G., Mattingly, R. R., Gillies, R. J., Sloane, B. F. 2013; 15 (10): 1111-1123

  Abstract

  One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8) affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D) cultures. For these studies, we used 1) 3D reconstituted basement membrane overlay cultures of human carcinomas, 2) live cell imaging assays to assess proteolysis, and 3) in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.

  View details for DOI 10.1593/neo.13946

  View details for Web of Science ID 000328136300001

  View details for PubMedCentralID PMC3819629

• Plasmodium Dipeptidyl Aminopeptidases as Malaria Transmission-Blocking Drug Targets ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Tanaka, T. Q., Deu, E., Molina-Cruz, A., Ashburne, M. J., Ali, O., Suri, A., Kortagere, S., Bogyo, M., Williamsona, K. C. 2013; 57 (10): 4645-4652

  Abstract

  The Plasmodium falciparum and P. berghei genomes each contain three dipeptidyl aminopeptidase (dpap) homologs. dpap1 and -3 are critical for asexual growth, but the role of dpap2, the gametocyte-specific homolog, has not been tested. If DPAPs are essential for transmission as well as asexual growth, then a DPAP inhibitor could be used for treatment and to block transmission. To directly analyze the role of DPAP2, a dpap2-minus P. berghei (Pbdpap2Δ) line was generated. The Pbdpap2Δ parasites grew normally, differentiated into gametocytes, and generated sporozoites that were infectious to mice when fed to a mosquito. However, Pbdpap1 transcription was >2-fold upregulated in the Pbdpap2Δ clonal lines, possibly compensating for the loss of Pbdpap2. The role of DPAP1 and -3 in the dpap2Δ parasites was then evaluated using a DPAP inhibitor, ML4118S. When ML4118S was added to the Pbdpap2Δ parasites just before a mosquito membrane feed, mosquito infectivity was not affected. To assess longer exposures to ML4118S and further evaluate the role of DPAPs during gametocyte development in a parasite that causes human malaria, the dpap2 deletion was repeated in P. falciparum. Viable P. falciparum dpap2 (Pfdpap2)-minus parasites were obtained that produced morphologically normal gametocytes. Both wild-type and Pfdpap2-negative parasites were sensitive to ML4118S, indicating that, unlike many antimalarials, ML4118S has activity against parasites at both the asexual and sexual stages and that DPAP1 and -3 may be targets for a dual-stage drug that can treat patients and block malaria transmission.

  View details for DOI 10.1128/AAC.02495-12

  View details for Web of Science ID 000324480300007

  View details for PubMedID 23836185

  View details for PubMedCentralID PMC3811399

• Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion. Nature chemical biology Child, M. A., Hall, C. I., Beck, J. R., Ofori, L. O., Albrow, V. E., Garland, M., Bowyer, P. W., Bradley, P. J., Powers, J. C., Boothroyd, J. C., Weerapana, E., Bogyo, M. 2013; 9 (10): 651-656

  Abstract

  Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

  View details for DOI 10.1038/nchembio.1315

  View details for PubMedID 23934245

  View details for PubMedCentralID PMC3832678

• Cathepsin C is a tissue-specific regulator of squamous carcinogenesis GENES & DEVELOPMENT Ruffell, B., Affara, N. I., Cottone, L., Junankar, S., Johansson, M., DeNardo, D. G., Korets, L., Reinheckel, T., Sloane, B. F., Bogyo, M., Coussens, L. M. 2013; 27 (19): 2086-2098

  Abstract

  Serine and cysteine cathepsin (Cts) proteases are an important class of intracellular and pericellular enzymes mediating multiple aspects of tumor development. Emblematic of these is CtsB, reported to play functionally significant roles during pancreatic islet and mammary carcinogenesis. CtsC, on the other hand, while up-regulated during pancreatic islet carcinogenesis, lacks functional significance in mediating neoplastic progression in that organ. Given that protein expression and enzymatic activity of both CtsB and CtsC are increased in numerous tumors, we sought to understand how tissue specificity might factor into their functional significance. Thus, whereas others have reported that CtsB regulates metastasis of mammary carcinomas, we found that development of squamous carcinomas occurs independently of CtsB. In contrast to these findings, our studies found no significant role for CtsC during mammary carcinogenesis but revealed squamous carcinogenesis to be functionally dependent on CtsC. In this context, dermal/stromal fibroblasts and bone marrow-derived cells expressed increased levels of enzymatically active CtsC that regulated the complexity of infiltrating immune cells in neoplastic skin, development of angiogenic vasculature, and overt squamous cell carcinoma growth. These studies highlight the important contribution of tissue/microenvironment context to solid tumor development and indicate that tissue specificity defines functional significance for these two members of the cysteine protease family.

  View details for DOI 10.1101/gad.224899.113

  View details for Web of Science ID 000325710800003

  View details for PubMedID 24065739

• Applications of Small Molecule Probes in Dissecting Mechanisms of Bacterial Virulence and Host Responses BIOCHEMISTRY Puri, A. W., Bogyo, M. 2013; 52 (35): 5985-5996

  Abstract

  Elucidating the molecular and biochemical details of bacterial infections can be challenging because of the many complex interactions that exist between a pathogen and its host. Consequently, many tools have been developed to aid the study of bacterial pathogenesis. Small molecules are a valuable complement to traditional genetic techniques because they can be used to rapidly perturb genetically intractable systems and to monitor post-translationally regulated processes. Activity-based probes are a subset of small molecules that covalently label an enzyme of interest based on its catalytic mechanism. These tools allow monitoring of enzyme activation within the context of a native biological system and can be used to dissect the biochemical details of enzyme function. This review describes the development and application of activity-based probes for examining aspects of bacterial infection on both sides of the host-pathogen interface.

  View details for DOI 10.1021/bi400854d

  View details for Web of Science ID 000330031000002

  View details for PubMedID 23937332

  View details for PubMedCentralID PMC3838904

• Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis MOLECULAR MICROBIOLOGY Gloeckl, S., Ong, V. A., Patel, P., Tyndall, J. D., Timms, P., Beagley, K. W., Allan, J. A., Armitage, C. W., Turnbull, L., Whitchurch, C. B., Merdanovic, M., Ehrmann, M., Powers, J. C., Oleksyszyn, J., Verdoes, M., Bogyo, M., Huston, W. M. 2013; 89 (4): 676-689

  Abstract

  The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.

  View details for DOI 10.1111/mmi.12306

  View details for Web of Science ID 000322948500007

  View details for PubMedID 23796320

• A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases. Journal of the American Chemical Society Xiao, J., Broz, P., Puri, A. W., Deu, E., Morell, M., Monack, D. M., Bogyo, M. 2013; 135 (24): 9130-9138

  Abstract

  Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization, and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active-site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8, and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild-type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation.

  View details for DOI 10.1021/ja403521u

  View details for PubMedID 23701470

  View details for PubMedCentralID PMC3722599

• Coupling protein engineering with probe design to inhibit and image matrix metalloproteinases with controlled specificity. Journal of the American Chemical Society Morell, M., Nguyen Duc, T., Willis, A. L., Syed, S., Lee, J., Deu, E., Deng, Y., Xiao, J., Turk, B. E., Jessen, J. R., Weiss, S. J., Bogyo, M. 2013; 135 (24): 9139-9148

  Abstract

  Matrix metalloproteinases (MMPs) are zinc endopeptidases that play roles in numerous pathophysiological processes and therefore are promising drug targets. However, the large size of this family and a lack of highly selective compounds that can be used for imaging or inhibition of specific MMPs members has limited efforts to better define their biological function. Here we describe a protein engineering strategy coupled with small-molecule probe design to selectively target individual members of the MMP family. Specifically, we introduce a cysteine residue near the active-site of a selected protease that does not alter its overall activity or function but allows direct covalent modification by a small-molecule probe containing a reactive electrophile. This specific engineered interaction between the probe and the target protease provides a means to both image and inhibit the modified protease with absolute specificity. Here we demonstrate the feasibility of the approach for two distinct MMP proteases, MMP-12 and MT1-MMP (or MMP-14).

  View details for DOI 10.1021/ja403523p

  View details for PubMedID 23701445

  View details for PubMedCentralID PMC3722588

• A Biocompatible in Vivo Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice ACS CHEMICAL BIOLOGY Godinat, A., Park, H. M., Miller, S. C., Cheng, K., Hanahan, D., Sanman, L. E., Bogyo, M., Yu, A., Nikitin, G. F., Stahl, A., Dubikovskaya, E. A. 2013; 8 (5): 987-999

  Abstract

  The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. The success of this "split luciferin" ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a "caged" luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH(2)-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting in vivo.

  View details for DOI 10.1021/cb3007314

  View details for Web of Science ID 000319720700018

  View details for PubMedID 23463944

• Real-time molecular imaging of cathepsins for rapid detection of cancer in human breast lumpectomy specimens. Vykhovanets, E., Gilmore, H., Blum, G., Bogyo, M., Basilion, J. AMER ASSOC CANCER RESEARCH. 2013

  View details for DOI 10.1158/1538-7445.AM2013-LB-85

  View details for Web of Science ID 000331220601332

• Applying small molecules to the study of parasite pathogenesis Bogyo, M., Child, M. A., Hall, C., Bowyer, P., Albrow, V., Weerapana, E. AMER CHEMICAL SOC. 2013

  View details for Web of Science ID 000323851300724

• In vivo imaging and biochemical characterization of protease function using fluorescent activity-based probes. Current protocols in chemical biology Edgington, L. E., Bogyo, M. 2013; 5 (1): 25-44

  Abstract

  Activity-based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this article, detailed protocols are provided for using fluorescent ABPs to biochemically characterize the activity of proteases in vitro. Furthermore, descriptions are provided of how these probes can be applied to image protease activity in live animals and tissues along with subsequent analysis by histology, flow cytometry, and SDS-PAGE. Curr. Protoc. Chem. Biol. 5:25-44 © 2013 by John Wiley & Sons, Inc.

  View details for DOI 10.1002/9780470559277.ch120235

  View details for PubMedID 23788323

  View details for PubMedCentralID PMC3691694

• A Substrate-Inspired Probe Monitors Translocation, Activation, and Subcellular Targeting of Bacterial Type III Effector Protease AvrPphB CHEMISTRY & BIOLOGY Lu, H., Wang, Z., Shabab, M., Oeljeklaus, J., Verhelst, S. H., Kaschani, F., Kaiser, M., Bogyo, M., van der Hoorn, R. A. 2013; 20 (2): 168-176

  Abstract

  The AvrPphB effector of Pseudomonas syringae is a papain-like protease that is injected into the host plant cell and cleaves specific kinases to disrupt immune signaling. Here, we used the unique substrate specificity of AvrPphB to generate a specific activity-based probe. This probe displays various AvrPphB isoforms in bacterial extracts, upon secretion and inside the host plant. We show that AvrPphB is secreted as a proprotease and that secretion requires the prodomain, but probably does not involve a pH-dependent unfolding mechanism. The prodomain removal is required for the ability of AvrPphB to trigger a hypersensitive cell death in resistant host plants, presumably since processing exposes a hidden acylation site required for subcellular targeting in the host cell. We detected two active isoforms of AvrPphB in planta, of which the major one localizes exclusively to membranes.

  View details for DOI 10.1016/j.chembiol.2012.11.007

  View details for Web of Science ID 000315978600009

  View details for PubMedID 23438746

• New technologies and their impact on 'omics' research CURRENT OPINION IN CHEMICAL BIOLOGY Bogyo, M., Rudd, P. M. 2013; 17 (1): 1-3

  View details for DOI 10.1016/j.cbpa.2013.01.005

  View details for Web of Science ID 000316503400001

  View details for PubMedID 23395431

• Target deconvolution techniques in modern phenotypic profiling CURRENT OPINION IN CHEMICAL BIOLOGY Lee, J., Bogyo, M. 2013; 17 (1): 118-126

  Abstract

  The past decade has seen rapid growth in the use of diverse compound libraries in classical phenotypic screens to identify modulators of a given process. The subsequent process of identifying the molecular targets of active hits, also called 'target deconvolution', is an essential step for understanding compound mechanism of action and for using the identified hits as tools for further dissection of a given biological process. Recent advances in 'omics' technologies, coupled with in silico approaches and the reduced cost of whole genome sequencing, have greatly improved the workflow of target deconvolution and have contributed to a renaissance of 'modern' phenotypic profiling. In this review, we will outline how both new and old techniques are being used in the difficult process of target identification and validation as well as discuss some of the ongoing challenges remaining for phenotypic screening.

  View details for DOI 10.1016/j.cbpa.2012.12.022

  View details for Web of Science ID 000316503400016

  View details for PubMedID 23337810

  View details for PubMedCentralID PMC3594516

• Activity profiling of vacuolar processing enzymes reveals a role for VPE during oomycete infection PLANT JOURNAL Misas-Villamil, J. C., Toenges, G., Kolodziejek, I., Sadaghiani, A. M., Kaschani, F., Colby, T., Bogyo, M., van der Hoorn, R. A. 2013; 73 (4): 689-700

  Abstract

  Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant-microbe interactions and development. Here, we introduce a specific, cell-permeable, activity-based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity-dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host-derived and EDS1-independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.

  View details for DOI 10.1111/tpj.12062

  View details for Web of Science ID 000315294300014

  View details for PubMedID 23134548

• Functional Imaging of Legumain in Cancer Using a New Quenched Activity-Based Probe JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Edgington, L. E., Verdoes, M., Ortega, A., Withana, N. P., Lee, J., Syed, S., Bachmann, M. H., Blum, G., Bogyo, M. 2013; 135 (1): 174-182

  Abstract

  Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.

  View details for DOI 10.1021/ja307083b

  View details for Web of Science ID 000313143000036

  View details for PubMedID 23215039

  View details for PubMedCentralID PMC4429797

• Self-Cleaving Bacterial Toxins HANDBOOK OF PROTEOLYTIC ENZYMES, VOLS 1 AND 2, 3RD EDITION Shen, A., Bogyo, M., Rawlings, N. D., Salvesen, G. S. 2013: 2350-2355

  View details for DOI 10.1016/B978-0-12-382219-2.00523-8

  View details for Web of Science ID 000328545103091

• Validation of the Proteasome as a Therapeutic Target in Plasmodium Using an Epoxyketone Inhibitor with Parasite-Specific Toxicity CHEMISTRY & BIOLOGY Li, H., Ponder, E. L., Verdoes, M., Asbjornsdottir, K. H., Deu, E., Edgington, L. E., Lee, J. T., Kirk, C. J., Demo, S. D., Williamson, K. C., Bogyo, M. 2012; 19 (12): 1535-1545

  Abstract

  The Plasmodium proteasome has been suggested to be a potential antimalarial drug target; however, toxicity of inhibitors has prevented validation of this enzyme in vivo. We report a screen of a library of 670 analogs of the recent US Food and Drug Administration-approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in Plasmodium berghei infected mice without host toxicity, thus validating the proteasome as a viable antimalarial drug target.

  View details for DOI 10.1016/j.chembiol.2012.09.019

  View details for PubMedID 23142757

• The Antimalarial Natural Product Symplostatin 4 Is a Nanomolar Inhibitor of the Food Vacuole Falcipains CHEMISTRY & BIOLOGY Stolze, S. C., Deu, E., Kaschani, F., Li, N., Florea, B. I., Richau, K. H., Colby, T., van der Hoom, R. A., Overkleeft, H. S., Bogyo, M., Kaiser, M. 2012; 19 (12): 1546-1555

  Abstract

  The marine natural product symplostatin 4 (Sym4) has been recognized as a potent antimalarial agent. However, its mode of action and, in particular, direct targets have to date remained elusive. We report a chemical synthesis of Sym4 and show that Sym4-treatment of P. falciparum-infected red blood cells (RBCs) results in the generation of a swollen food vacuole phenotype and a reduction of parasitemia at nanomolar concentrations. We furthermore demonstrate that Sym4 is a nanomolar inhibitor of the P. falciparum falcipains in infected RBCs, suggesting inhibition of the hemoglobin degradation pathway as Sym4's mode of action. Finally, we reveal a critical influence of the unusual methyl-methoxypyrrolinone (mmp) group of Sym4 for potent inhibition, indicating that Sym4 derivatives with such a mmp moiety might represent viable lead structures for the development of antimalarial falcipain inhibitors.

  View details for DOI 10.1016/j.chembiol.2012.09.020

  View details for Web of Science ID 000313087300008

  View details for PubMedID 23261598

  View details for PubMedCentralID PMC3601557

• Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins BIOLOGICAL CHEMISTRY Mullins, S. R., Sameni, M., Blum, G., Bogyo, M., Sloane, B. F., Moin, K. 2012; 393 (12): 1405-?

  Abstract

  The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

  View details for DOI 10.1515/hsz-2012-0252

  View details for Web of Science ID 000311051400004

  View details for PubMedID 23667900

  View details for PubMedCentralID PMC3789365

• Autophagic degradation of the BCR-ABL oncoprotein and generation of antileukemic responses by arsenic trioxide BLOOD Goussetis, D. J., Gounaris, E., Wu, E. J., Vakana, E., Sharma, B., Bogyo, M., Altman, J. K., Platanias, L. C. 2012; 120 (17): 3555-3562

  Abstract

  We provide evidence that arsenic trioxide (As(2)O(3)) targets the BCR-ABL oncoprotein via a novel mechanism involving p62/SQSTM1-mediated localization of the oncoprotein to the autolysosomes and subsequent degradation mediated by the protease cathepsin B. Our studies demonstrate that inhibitors of autophagy or cathepsin B activity and/or molecular targeting of p62/SQSTM1, Atg7, or cathepsin B result in partial reversal of the suppressive effects of AS(2)O(3) on BCR-ABL expressing leukemic progenitors, including primitive leukemic precursors from chronic myelogenous leukemia (CML) patients. Altogether, these findings indicate that autophagic degradation of BCR-ABL is critical for the induction of the antileukemic effects of As(2)O(3) and raise the potential for future therapeutic approaches to target BCR-ABL expressing cells by modulating elements of the autophagic machinery to promote BCR-ABL degradation.

  View details for DOI 10.1182/blood-2012-01-402578

  View details for Web of Science ID 000311623800022

  View details for PubMedID 22898604

  View details for PubMedCentralID PMC3482863

• Active cathepsins B, L, and S in murine and human pancreatitis AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Lyo, V., Cattaruzza, F., Kim, T. N., Walker, A. W., Paulick, M., Cox, D., Cloyd, J., Buxbaum, J., Ostroff, J., Bogyo, M., Grady, E. F., Bunnett, N. W., Kirkwood, K. S. 2012; 303 (8): G894-G903

  Abstract

  Cathepsins regulate premature trypsinogen activation within acinar cells, a key initial step in pancreatitis. The identity, origin, and causative roles of activated cathepsins in pancreatic inflammation and pain are not defined. By using a near infrared-labeled activity-based probe (GB123) that covalently modifies active cathepsins, we localized and identified activated cathepsins in mice with cerulein-induced pancreatitis and in pancreatic juice from patients with chronic pancreatitis. We used inhibitors of activated cathepsins to define their causative role in pancreatic inflammation and pain. After GB123 administration to mice with pancreatitis, reflectance and confocal imaging showed significant accumulation of the probe in inflamed pancreas compared with controls, particularly in acinar cells and macrophages, and in spinal cord microglia and neurons. Biochemical analysis of pancreatic extracts identified them as cathepsins B, L, and S (Cat-B, Cat-L, and Cat-S, respectively). These active cathepsins were also identified in pancreatic juice from patients with chronic pancreatitis undergoing an endoscopic procedure for the treatment of pain, indicating cathepsin secretion. The cathepsin inhibitor K11777 suppressed cerulein-induced activation of Cat-B, Cat-L, and Cat-S in the pancreas and ameliorated pancreatic inflammation, nocifensive behavior, and activation of spinal nociceptive neurons. Thus pancreatitis is associated with an increase in the active forms of the proteases Cat-B, Cat-L, and Cat-S in pancreatic acinar cells and macrophages, and in spinal neurons and microglial cells. Inhibition of cathepsin activation ameliorated pancreatic inflammation and pain. Activity-based probes permit identification of proteases that are predictive biomarkers of disease progression and response to therapy and may be useful noninvasive tools for the detection of pancreatic inflammation.

  View details for DOI 10.1152/ajpgi.00073.2012

  View details for Web of Science ID 000309980500002

  View details for PubMedID 22899821

  View details for PubMedCentralID PMC3469694

• Caspase-1 activity is required to bypass macrophage apoptosis upon Salmonella infection NATURE CHEMICAL BIOLOGY Puri, A. W., Broz, P., Shen, A., Monack, D. M., Bogyo, M. 2012; 8 (9): 745-747

  Abstract

  Here we report AWP28, an activity-based probe that can be used to biochemically monitor caspase-1 activation in response to proinflammatory stimuli. Using AWP28, we show that apoptosis is triggered upon Salmonella enterica var. Typhimurium infection in primary mouse bone marrow macrophages lacking caspase-1. Furthermore, we report that upon Salmonella infection, inflammasome-mediated caspase-1 activity is required to bypass apoptosis in favor of proinflammatory pyroptotic cell death.

  View details for DOI 10.1038/NCHEMBIO.1023

  View details for Web of Science ID 000308077600004

  View details for PubMedID 22797665

  View details for PubMedCentralID PMC3461347

• Highlight: The universe of proteolytic networks and mechanisms BIOLOGICAL CHEMISTRY Salvesen, G. S., Bogyo, M. 2012; 393 (9): 841

  View details for DOI 10.1515/hsz-2012-0262

  View details for Web of Science ID 000307509500001

  View details for PubMedID 22944685

• Disruption of gingipain oligomerization into non-covalent cell-surface attached complexes BIOLOGICAL CHEMISTRY Sztukowska, M., Veillard, F., Potempa, B., Bogyo, M., Enghild, J. J., Thogersen, I. B., Ky-Anh Nguyen, K. A., Potempa, J. 2012; 393 (9): 971-977

  Abstract

  RgpA and Kgp gingipains are non-covalent complexes of endoprotease catalytic and hemagglutinin-adhesin domains on the surface of Porphyromonas gingivalis. A motif conserved in each domain has been suggested to function as an oligomerization motif. We tested this hypothesis by mutating motif residues to hexahistidine or insertion of hexahistidine tag to disrupt the motif within the Kgp catalytic domain. All modifications led to the secretion of entire Kgp activity into the growth media, predominantly in a form without functional His-tag. This confirmed the role of the conserved motif in correct posttranslational proteolytic processing and assembly of the multidomain complexes.

  View details for DOI 10.1515/hsz-2012-0175

  View details for Web of Science ID 000307509500012

  View details for PubMedID 22944696

  View details for PubMedCentralID PMC3488288

• The Apoptosis Repressor With a CARD Domain (ARC) is Essential for the Survival of VHL Deficient Renal Cancer Cells 22nd Biennial Congress of the European-Association-for-Cancer-Research Razorenova, O. V., Colavitti, R., Castellini, L., Edgington, L. E., Huang, X., Nicolau, M., Bedogni, B., Mills, E. M., Bogyo, M., Giaccia, A. J. ELSEVIER SCI LTD. 2012: S120–S120

  View details for Web of Science ID 000313036501011

• A Nonpeptidic Cathepsin S Activity-Based Probe for Noninvasive Optical Imaging of Tumor-Associated Macrophages CHEMISTRY & BIOLOGY Verdoes, M., Edgington, L. E., Scheeren, F. A., Leyva, M., Blum, G., Weiskopf, K., Bachmann, M. H., Ellman, J. A., Bogyo, M. 2012; 19 (5): 619-628

  Abstract

  Macrophage infiltration into tumors has been correlated with poor clinical outcome in multiple cancer types. Therefore, tools to image tumor-associated macrophages could be valuable for diagnosis and prognosis of cancer. Herein, we describe the synthesis and characterization of a cathepsin S-directed, quenched activity-based probe (qABP), BMV083. This probe makes use of an optimized nonpeptidic scaffold leading to enhanced in vivo properties relative to previously reported peptide-based probes. In a syngeneic breast cancer model, BMV083 provides high tumor-specific fluorescence that can be visualized using noninvasive optical imaging methods. Furthermore, analysis of probe-labeled cells demonstrates that the probe primarily targets macrophages with an M2 phenotype. Thus, BMV083 is a potential valuable in vivo reporter for tumor-associated macrophages that could greatly facilitate the future studies of macrophage function in the process of tumorigenesis.

  View details for DOI 10.1016/j.chembiol.2012.03.012

  View details for Web of Science ID 000304794600013

  View details for PubMedID 22633413

  View details for PubMedCentralID PMC3361968

• Caspase-3 feeds back on caspase-8, Bid and XIAP in type I Fas signaling in primary mouse hepatocytes APOPTOSIS Ferreira, K., Kreutz, C., MacNelly, S., Neubert, K., Haber, A., Bogyo, M., Timmer, J., Borner, C. 2012; 17 (5): 503-515

  Abstract

  The TNF-R1 like receptor Fas is highly expressed on the plasma membrane of hepatocytes and plays an essential role in liver homeostasis. We recently showed that in collagen-cultured primary mouse hepatocytes, Fas stimulation triggers apoptosis via the so-called type I extrinsic signaling pathway. Central to this pathway is the direct caspase-8-mediated cleavage and activation of caspase-3 as compared to the type II pathway which first requires caspase-8-mediated Bid cleavage to trigger mitochondrial cytochrome c release for caspase-3 activation. Mathematical modeling can be used to understand complex signaling systems such as crosstalks and feedback or feedforward loops. A previously published model predicted a positive feedback loop between active caspases-3 and -8 in both type I and type II FasL signaling in lymphocytes and Hela cells, respectively. Here we experimentally tested this hypothesis in our hepatocytic type I Fas signaling pathway by using wild-type and XIAP-deficient primary hepatocytes and two recently characterized, selective caspase-3/-7 inhibitors (AB06 and AB13). Caspase-3/-7 activity assays and quantitative western blotting confirmed that fully processed, active p17 caspase-3 feeds back on caspase-8 by cleaving its partially processed p43 form into the fully processed p18 species. Our data do not discriminate if p18 positively or negatively influences FasL-induced apoptosis or is responsible for non-apoptotic aspects of FasL signaling. However, we found that caspase-3 also feeds back on Bid and degrades its own inhibitor XIAP, both events that may enhance caspase-3 activity and apoptosis. Thus, potent, selective caspase-3 inhibitors are useful tools to understand complex signaling circuitries in apoptosis.

  View details for DOI 10.1007/s10495-011-0691-0

  View details for Web of Science ID 000302569700007

  View details for PubMedID 22246639

• Autophagic degradation of BCR/ABL by arsenic trioxide and the role of cysteine cathepsins Goussetis, D., Gounaris, E., Wu, E. J., Vakana, E., Sharma, B., Altman, J. K., Platanais, L. C., Bogyo, M. AMER ASSOC CANCER RESEARCH. 2012

  View details for DOI 10.1158/1538-7445.AM2012-LB-81

  View details for Web of Science ID 000209701501191

• Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics PLANT PHYSIOLOGY Richau, K. H., Kaschani, F., Verdoes, M., Pansuriya, T. C., Niessen, S., Stueber, K., Colby, T., Overkleeft, H. S., Bogyo, M., van der Hoorn, R. A. 2012; 158 (4): 1583-1599

  Abstract

  Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.

  View details for DOI 10.1104/pp.112.194001

  View details for Web of Science ID 000303001400010

  View details for PubMedID 22371507

  View details for PubMedCentralID PMC3320171

• Treatment of Arthritis by Macrophage Depletion and Immunomodulation Testing an Apoptosis-Mediated Therapy in a Humanized Death Receptor Mouse Model ARTHRITIS AND RHEUMATISM Li, J., Hsu, H., Yang, P., Wu, Q., Li, H., Edgington, L. E., Bogyo, M., Kimberly, R. P., Mountz, J. D. 2012; 64 (4): 1098-1109

  Abstract

  To determine the therapeutic efficacy and immunomodulatory effect of an anti-human death receptor 5 (DR5) antibody, TRA-8, in eliminating macrophage subsets in a mouse model of type II collagen-induced arthritis (CIA).A human/mouse-chimeric DR5-transgenic mouse, under the regulation of a mouse 3-kb promoter and a loxP-flanked STOP cassette, was generated and crossed with an ubiquitous Cre (Ubc.Cre) mouse and a lysozyme M-Cre (LysM.Cre)-transgenic mouse to achieve inducible or macrophage-specific expression. Chicken type II collagen was used to induce CIA in mice, which were then treated with an anti-human DR5 antibody, TRA-8. Clinical scores, histopathologic severity, macrophage apoptosis and depletion, and T cell subset development were evaluated.In human/mouse DR5-transgenic Ubc.Cre mice with CIA, transgenic DR5 was most highly expressed on CD11b+ macrophages, with lower expression on CD4+ T cells. In human/mouse DR5-transgenic LysM.Cre mice, transgenic DR5 was restrictively expressed on macrophages. Both in vivo near-infrared imaging of caspase activity and TUNEL staining demonstrated that TRA-8 rapidly induced apoptosis of macrophages in inflamed synovium. Depletion of pathogenic macrophages by TRA-8 led to significantly reduced clinical scores for arthritis; decreased macrophage infiltration, synovial hyperplasia, osteoclast formation, joint destruction, cathepsin activity, and inflammatory cytokine expression in joints; reduced numbers of Th17 cells; and an increased number of Treg cells in draining lymph nodes.The anti-human DR5 antibody TRA-8 was efficacious in reducing the severity of arthritis via targeted depletion of macrophages and immunomodulation. Our data provide preclinical evidence that TRA-8 is a potential novel biologic agent for rheumatoid arthritis therapy.

  View details for DOI 10.1002/art.33423

  View details for Web of Science ID 000302475500019

  View details for PubMedID 22006294

  View details for PubMedCentralID PMC3596268

• Small molecule probes of protease function: Applications to molecular imaging and drug discovery Bogyo, M. AMER CHEMICAL SOC. 2012

  View details for Web of Science ID 000324475100532

• An Optimized Activity-Based Probe for the Study of Caspase-6 Activation CHEMISTRY & BIOLOGY Edgington, L. E., van Raam, B. J., Verdoes, M., Wierschem, C., Salvesen, G. S., Bogyo, M. 2012; 19 (3): 340-352

  Abstract

  Although significant efforts have been made to understand the mechanisms of caspase activation during apoptosis, many questions remain regarding how and when executioner caspases get activated. We describe the design and synthesis of an activity-based probe that labels caspase-3/-6/-7, allowing direct monitoring of all executioner caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe, AB50. Using this probe, we find that caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the proenzyme. We also demonstrate that caspase-6 activation does not require active caspase-3/-7, suggesting that it may autoactivate or be cleaved by other proteases. Together, our results suggest that caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions.

  View details for DOI 10.1016/j.chembiol.2011.12.021

  View details for Web of Science ID 000302588900007

  View details for PubMedID 22444589

  View details for PubMedCentralID PMC3314226

• Topical Application of Activity-based Probes for Visualization of Brain Tumor Tissue PLOS ONE Cutter, J. L., Cohen, N. T., Wang, J., Sloan, A. E., Cohen, A. R., Panneerselvam, A., Schluchter, M., Blum, G., Bogyo, M., Basilion, J. P. 2012; 7 (3)

  Abstract

  Several investigators have shown the utility of systemically delivered optical imaging probes to image tumors in small animal models of cancer. Here we demonstrate an innovative method for imaging tumors and tumor margins during surgery. Specifically, we show that optical imaging probes topically applied to tumors and surrounding normal tissue rapidly differentiate between tissues. In contrast to systemic delivery of optical imaging probes which label tumors uniformly over time, topical probe application results in rapid and robust probe activation that is detectable as early as 5 minutes following application. Importantly, labeling is primarily associated with peri-tumor spaces. This methodology provides a means for rapid visualization of tumor and potentially infiltrating tumor cells and has potential applications for directed surgical excision of tumor tissues. Furthermore, this technology could find use in surgical resections for any tumors having differential regulation of cysteine cathepsin activity.

  View details for DOI 10.1371/journal.pone.0033060

  View details for Web of Science ID 000303129700030

  View details for PubMedID 22427947

  View details for PubMedCentralID PMC3302795

• Cathepsin B Inhibition Limits Bone Metastasis in Breast Cancer CANCER RESEARCH Withana, N. P., Blum, G., Sameni, M., Slaney, C., Anbalagan, A., Olive, M. B., Bidwell, B. N., Edgington, L., Wang, L., Moin, K., Sloane, B. F., Anderson, R. L., Bogyo, M. S., Parker, B. S. 2012; 72 (5): 1199-1209

  Abstract

  Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.

  View details for DOI 10.1158/0008-5472.CAN-11-2759

  View details for Web of Science ID 000300989100019

  View details for PubMedID 22266111

  View details for PubMedCentralID PMC3538126

• Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Lee, J., Bogyo, M. 2012; 22 (3): 1340-1343

  Abstract

  Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10(4)M(-1)s(-1) and IC(50) values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.

  View details for DOI 10.1016/j.bmcl.2011.12.079

  View details for PubMedID 22243962

• Substrate specificity of Staphylococcus aureus cysteine proteases - Staphopains A, B and C BIOCHIMIE Kalinska, M., Kantyka, T., Greenbaum, D. C., Larsen, K. S., Wladyka, B., Jabaiah, A., Bogyo, M., Daugherty, P. S., Wysocka, M., Jaros, M., Lesner, A., Rolka, K., Schaschke, N., Stennicke, H., Dubin, A., Potempa, J., Dubin, G. 2012; 94 (2): 318-327

  Abstract

  Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2-Gly↓Ala(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.

  View details for DOI 10.1016/j.biochi.2011.07.020

  View details for Web of Science ID 000300270900006

  View details for PubMedID 21802486

• Proteomic Analysis of Fractionated Toxoplasma Oocysts Reveals Clues to Their Environmental Resistance PLOS ONE Fritz, H. M., Bowyer, P. W., Bogyo, M., Conrad, P. A., Boothroyd, J. C. 2012; 7 (1)

  Abstract

  Toxoplasma gondii is an obligate intracellular parasite that is unique in its ability to infect a broad range of birds and mammals, including humans, leading to an extremely high worldwide prevalence and distribution. This work focuses on the environmentally resistant oocyst, which is the product of sexual replication in felids and an important source of human infection. Due to the difficulty in producing and working with oocysts, relatively little is known about how this stage is able to resist extreme environmental stresses and how they initiate a new infection, once ingested. To fill this gap, the proteome of the wall and sporocyst/sporozoite fractions of mature, sporulated oocysts were characterized using one-dimensional gel electrophoresis followed by LC-MS/MS on trypsin-digested peptides. A combined total of 1021 non-redundant T. gondii proteins were identified in the sporocyst/sporozoite fraction and 226 were identified in the oocyst wall fraction. Significantly, 172 of the identified proteins have not previously been identified in Toxoplasma proteomic studies. Among these are several of interest for their likely role in conferring environmental resistance including a family of small, tyrosine-rich proteins present in the oocyst wall fractions and late embryogenesis abundant domain-containing (LEA) proteins in the cytosolic fractions. The latter are known from other systems to be key to enabling survival against desiccation.

  View details for DOI 10.1371/journal.pone.0029955

  View details for Web of Science ID 000299771900033

  View details for PubMedID 22279555

  View details for PubMedCentralID PMC3261165

• New approaches for dissecting protease functions to improve probe development and drug discovery NATURE STRUCTURAL & MOLECULAR BIOLOGY Deu, E., Verdoes, M., Bogyo, M. 2012; 19 (1): 9-16

  Abstract

  Proteases are well-established targets for pharmaceutical development because of their known enzymatic mechanism and their regulatory roles in many pathologies. However, many potent clinical lead compounds have been unsuccessful either because of a lack of specificity or because of our limited understanding of the biological roles of the targeted protease. In order to successfully develop protease inhibitors as drugs, it is necessary to understand protease functions and to expand the platform of inhibitor development beyond active site-directed design and in vitro optimization. Several newly developed technologies will enhance assessment of drug selectivity in living cells and animal models, allowing researchers to focus on compounds with high specificity and minimal side effects in vivo. In this review, we highlight advances in the development of chemical probes, proteomic methods and screening tools that we feel will help facilitate this paradigm shift in drug discovery.

  View details for DOI 10.1038/nsmb.2203

  View details for Web of Science ID 000299046000004

  View details for PubMedID 22218294

  View details for PubMedCentralID PMC3513415

• Proteases as regulators of pathogenesis: Examples from the Apicomplexa BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS Li, H., Child, M. A., Bogyo, M. 2012; 1824 (1): 177-185

  Abstract

  The diverse functional roles that proteases play in basic biological processes make them essential for virtually all organisms. Not surprisingly, proteolysis is also a critical process required for many aspects of pathogenesis. In particular, obligate intracellular parasites must precisely coordinate proteolytic events during their highly regulated life cycle inside multiple host cell environments. Advances in chemical, proteomic and genetic tools that can be applied to parasite biology have led to an increased understanding of the complex events centrally regulated by proteases. In this review, we outline recent advances in our knowledge of specific proteolytic enzymes in two medically relevant apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii. Efforts over the last decade have begun to provide a map of key proteotolyic events that are essential for both parasite survival and propagation inside host cells. These advances in our molecular understanding of proteolytic events involved in parasite pathogenesis provide a foundation for the validation of new networks and enzyme targets that could be exploited for therapeutic purposes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

  View details for DOI 10.1016/j.bbapap.2011.06.002

  View details for Web of Science ID 000298715900018

  View details for PubMedID 21683169

  View details for PubMedCentralID PMC3232290

• Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes CURRENT OPINION IN CHEMICAL BIOLOGY Edgington, L. E., Verdoes, M., Bogyo, M. 2011; 15 (6): 798-805

  Abstract

  Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few 'druggable' classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.

  View details for DOI 10.1016/j.cbpa.2011.10.012

  View details for Web of Science ID 000300034200008

  View details for PubMedID 22098719

  View details for PubMedCentralID PMC3237724

• Non-Invasive Imaging of Cysteine Cathepsin Activity in Solid Tumors Using a Cu-64-Labeled Activity-Based Probe PLOS ONE Ren, G., Blum, G., Verdoes, M., Liu, H., Syed, S., Edgington, L. E., Gheysens, O., Miao, Z., Jiang, H., Gambhir, S. S., Bogyo, M., Cheng, Z. 2011; 6 (11)

  Abstract

  The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.

  View details for DOI 10.1371/journal.pone.0028029

  View details for Web of Science ID 000297789900039

  View details for PubMedID 22132198

  View details for PubMedCentralID PMC3221694

• Cathepsin S Is Activated During Colitis and Causes Visceral Hyperalgesia by a PAR(2)-Dependent Mechanism in Mice GASTROENTEROLOGY Cattaruzza, F., Lyo, V., Jones, E., Pham, D., Hawkins, J., Kirkwood, K., Valdez-Morales, E., Ibeakanma, C., Vanner, S. J., Bogyo, M., Bunnett, N. W. 2011; 141 (5): 1864-U440

  Abstract

  Although proteases control inflammation and pain, the identity, cellular origin, mechanism of action, and causative role of proteases that are activated during disease are not defined. We investigated the activation and function of cysteine cathepsins (Cat) in colitis.Because protease activity, rather than expression, is regulated, we treated mice with fluorescent activity-based probes that covalently modify activated cathepsins. Activated proteases were localized by tomographic imaging of intact mice and confocal imaging of tissues, and were identified by electrophoresis and immunoprecipitation. We examined the effects of activated cathepsins on excitability of colonic nociceptors and on colonic pain, and determined their role in colonic inflammatory pain by gene deletion.Tomography and magnetic resonance imaging localized activated cathepsins to the inflamed colon of piroxicam-treated il10(-/-) mice. Confocal imaging detected activated cathepsins in colonic macrophages and spinal neurons and microglial cells of mice with colitis. Gel electrophoresis and immunoprecipitation identified activated Cat-B, Cat-L, and Cat-S in colon and spinal cord, and Cat-S was preferentially secreted into the colonic lumen. Intraluminal Cat-S amplified visceromotor responses to colorectal distension and induced hyperexcitability of colonic nociceptors, which required expression of protease-activated receptor-2. Cat-S deletion attenuated colonic inflammatory pain induced with trinitrobenzene sulfonic acid.Activity-based probes enable noninvasive detection, cellular localization, and proteomic identification of proteases activated during colitis and are potential diagnostic tools for detection of predictive disease biomarkers. Macrophage cathepsins are activated during colitis, and Cat-S activates nociceptors to induce visceral pain via protease-activated receptor-2. Cat-S mediates colitis pain and is a potential therapeutic target.

  View details for DOI 10.1053/j.gastro.2011.07.035

  View details for Web of Science ID 000296512200051

  View details for PubMedID 21802389

  View details for PubMedCentralID PMC4041343

• Ferri-liposomes as an MRI-visible drug-delivery system for targeting tumours and their microenvironment NATURE NANOTECHNOLOGY Mikhaylov, G., Mikac, U., Magaeva, A. A., Itin, V. I., Naiden, E. P., Psakhye, I., Babes, L., Reinheckel, T., Peters, C., Zeiser, R., Bogyo, M., Turk, V., Psakhye, S. G., Turk, B., Vasiljeva, O. 2011; 6 (9): 594-602

  Abstract

  The tumour microenvironment regulates tumour progression and the spread of cancer in the body. Targeting the stromal cells that surround cancer cells could, therefore, improve the effectiveness of existing cancer treatments. Here, we show that magnetic nanoparticle clusters encapsulated inside a liposome can, under the influence of an external magnet, target both the tumour and its microenvironment. We use the outstanding T2 contrast properties (r2=573-1,286 s(-1) mM(-1)) of these ferri-liposomes, which are ∼95 nm in diameter, to non-invasively monitor drug delivery in vivo. We also visualize the targeting of the tumour microenvironment by the drug-loaded ferri-liposomes and the uptake of a model probe by cells. Furthermore, we used the ferri-liposomes to deliver a cathepsin protease inhibitor to a mammary tumour and its microenvironment in a mouse, which substantially reduced the size of the tumour compared with systemic delivery of the same drug.

  View details for DOI 10.1038/NNANO.2011.112

  View details for Web of Science ID 000294550000017

  View details for PubMedID 21822252

• Identification of a myeloid-derived suppressor cell cystatin-like protein that inhibits metastasis FASEB JOURNAL Boutte, A. M., Friedman, D. B., Bogyo, M., Min, Y., Yang, L., Lin, P. C. 2011; 25 (8): 2626-2637

  Abstract

  Myeloid-derived suppressor cells (MDSCs) are significantly increased in cancer patients and tumor bearing-animals. MDSCs infiltrate into tumors and promote tumor invasion and metastasis. To identify the mediator responsible for the prometastatic property of MDSCs, we used proteomics. We found neutrophilic granule protein (NGP) was decreased >2-fold in MDSCs from metastatic 4T1 tumor-bearing mice compared to nonmetastatic 67NR controls. NGP mRNA levels were decreased in bone marrow and in tumor-infiltrating MDSCs by 45 and 66%, respectively, in 4T1 tumor-bearing mice compared to 67NR controls. Interestingly, 4T1-conditioned medium reduced myeloid cell NGP expression by ∼ 40%, suggesting that a secreted factor mediates gene reduction. Sequence analysis shows a putative cystatin domain in NGP, and biochemical analysis confirms NGP a novel cathepsin inhibitor. It inhibited cathepsin B activity by nearly 40% in vitro. NGP expression in 4T1 tumor cells suppressed cell invasion, delayed primary tumor growth, and greatly reduced lung metastasis in vivo. A 2.8-fold reduction of cathepsin activity was found in tumors expressing NGP compared to controls. NGP significantly reduced tumor angiogenesis to 12.6 from 19.6 and lymphangiogenesis to 4.6 from 9.1 vessels/field. Necrosis was detectable only in NGP-expressing tumors, and the number of apoptotic cells increased to 22.4 from 8.3 in controls. Taken together, this study identifies a negative regulator of tumor metastasis in MDSCs, NGP, which is down-regulated in metastatic conditions. The finding suggests that malignant tumors promote invasion/metastasis not only through up-regulation of proteases but also down-regulation of protease inhibitors.

  View details for DOI 10.1096/fj.10-180604

  View details for Web of Science ID 000293337800013

  View details for PubMedID 21518852

  View details for PubMedCentralID PMC3136340

• Application of fluorogenic substrate libraries containing natural and unnatural amino acids for profiling of proteolytic enzymes Drag, M., Gajda, A., Kasperkiewicz, P., Poreba, M., Latajka, R., Pawelczak, M., Marschner, A., Klein, C., Bogyo, M., Ellman, J. A., Salvesen, G. S. SPRINGER WIEN. 2011: S17

  View details for Web of Science ID 000306273000053

• Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hall, C. I., Reese, M. L., Weerapana, E., Child, M. A., Bowyer, P. W., Albrow, V. E., Haraldsen, J. D., Phillips, M. R., Sandoval, E. D., Ward, G. E., Cravatt, B. F., Boothroyd, J. C., Bogyo, M. 2011; 108 (26): 10568-10573

  Abstract

  Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.

  View details for DOI 10.1073/pnas.1105622108

  View details for Web of Science ID 000292251000042

  View details for PubMedID 21670272

  View details for PubMedCentralID PMC3127939

• Functional Characterization of a SUMO Deconjugating Protease of Plasmodium falciparum Using Newly Identified Small Molecule Inhibitors CHEMISTRY & BIOLOGY Ponder, E. L., Albrow, V. E., Leader, B. A., Bekes, M., Mikolajczyk, J., Fonovic, U. P., Shen, A., Drag, M., Xiao, J., Deu, E., Campbell, A. J., Powers, J. C., Salvesen, G. S., Bogyo, M. 2011; 18 (6): 711-721

  Abstract

  Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum.

  View details for DOI 10.1016/j.chembiol.2011.04.010

  View details for Web of Science ID 000292583800006

  View details for PubMedID 21700207

  View details for PubMedCentralID PMC3131532

• Development of Small Molecule Inhibitors and Probes of Human SUMO Deconjugating Proteases CHEMISTRY & BIOLOGY Albrow, V. E., Ponder, E. L., Fasci, D., Bekes, M., Deu, E., Salvesen, G. S., Bogyo, M. 2011; 18 (6): 722-732

  Abstract

  Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation.

  View details for DOI 10.1016/j.chembiol.2011.05.008

  View details for Web of Science ID 000292583800007

  View details for PubMedID 21700208

  View details for PubMedCentralID PMC3131534

• Development of Activity-Based Probes for Cathepsin X ACS CHEMICAL BIOLOGY Paulick, M. G., Bogyo, M. 2011; 6 (6): 563-572

  Abstract

  Cathepsin X is a lysosomal cysteine protease that functions as a carboxypeptidase with broad substrate specificity. Cathepsin X was discovered only recently, and its physiological roles are still not well understood. A number of studies suggest that cathepsin X may be involved in a variety of biological processes, including cancer, aging and degenerative conditions of the brain, inflammation, and cellular communication. Here we present the synthesis and characterization of several activity-based probes (ABPs) that target active cathepsin X. These ABPs were used to label cathepsin X in complex lysates, whole cells, and in vivo. Furthermore, we have developed a method for selectively labeling and visualizing active cathepsin X in vitro and in vivo. Overall, the probes developed in this study are valuable tools for the study of cathepsin X function.

  View details for DOI 10.1021/cb100392r

  View details for Web of Science ID 000291896400006

  View details for PubMedID 21322635

  View details for PubMedCentralID PMC3117957

• Global Profiling of Proteolysis during Rupture of Plasmodium falciparum from the Host Erythrocyte MOLECULAR & CELLULAR PROTEOMICS Bowyer, P. W., Simon, G. M., Cravatt, B. F., Bogyo, M. 2011; 10 (5)

  Abstract

  The obligate intracellular parasite pathogen Plasmodium falciparum is the causative agent of malaria, a disease that results in nearly one million deaths per year. A key step in disease pathology in the human host is the parasite-mediated rupture of red blood cells, a process that requires extensive proteolysis of a number of host and parasite proteins. However, only a relatively small number of specific proteolytic processing events have been characterized. Here we describe the application of the Protein Topography and Migration Analysis Platform (PROTOMAP) (Dix, M. M., Simon, G. M., and Cravatt, B. F. (2008) Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell 134, 679-691; Simon, G. M., Dix, M. M., and Cravatt, B. F. (2009) Comparative assessment of large-scale proteomic studies of apoptotic proteolysis. ACS Chem. Biol. 4, 401-408) technology to globally profile proteolytic events occurring over the last 6-8 h of the intraerythrocytic cycle of P. falciparum. Using this method, we were able to generate peptographs for a large number of proteins at 6 h prior to rupture as well as at the point of rupture and in purified merozoites after exit from the host cell. These peptographs allowed assessment of proteolytic processing as well as changes in both protein localization and overall stage-specific expression of a large number of parasite proteins. Furthermore, by using a highly selective inhibitor of the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) that has been shown to be a key regulator of host cell rupture, we were able to identify specific substrates whose processing may be of particular importance to the process of host cell rupture. These results provide the first global map of the proteolytic processing events that take place as the human malarial parasite extracts itself from the host red blood cell. These data also provide insight into the biochemical events that take place during host cell rupture and are likely to be valuable for the study of proteases that could potentially be targeted for therapeutic gain.

  View details for DOI 10.1074/mcp.M110.001636

  View details for Web of Science ID 000290216700002

  View details for PubMedID 20943600

  View details for PubMedCentralID PMC3098579

• Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase JOURNAL OF EXPERIMENTAL MEDICINE Ewald, S. E., Engel, A., Lee, J., Wang, M., Bogyo, M., Barton, G. M. 2011; 208 (4): 643-651

  Abstract

  Toll-like receptor (TLR) 9 requires proteolytic processing in the endolysosome to initiate signaling in response to DNA. However, recent studies conflict as to which proteases are required for receptor cleavage. We show that TLR9 proteolysis is a multistep process. The first step removes the majority of the ectodomain and can be performed by asparagine endopeptidase (AEP) or cathepsin family members. This initial cleavage event is followed by a trimming event that is solely cathepsin mediated and required for optimal receptor signaling. This dual requirement for AEP and cathepsins is observed in all cell types that we have analyzed, including mouse macrophages and dendritic cells. In addition, we show that TLR7 and TLR3 are processed in an analogous manner. These results define the core proteolytic steps required for TLR9 function and suggest that receptor proteolysis may represent a general regulatory strategy for all TLRs involved in nucleic acid recognition.

  View details for DOI 10.1084/jem.20100682

  View details for Web of Science ID 000289404800002

  View details for PubMedID 21402738

  View details for PubMedCentralID PMC3135342

• Increased activities of cysteine cathepsins in mouse models of asthma Yin, F., Yu, M., Liu, C., Paulick, M., Verdoes, M., Stein, P., Galli, S., D'Andrea, A., Bogyo, M. AMER ASSOC IMMUNOLOGISTS. 2011

  View details for Web of Science ID 000209751706050

• Development for Taspase1 inhibitors 241st National Meeting and Exposition of the American-Chemical-Society (ACS) Lee, J. T., Chen, D. Y., Yang, Z., Ramos, A. D., Hsieh, J. J., Bogyo, M. AMER CHEMICAL SOC. 2011

  View details for Web of Science ID 000291982806099

• A Fragmenting Hybrid Approach for Targeted Delivery of Multiple Therapeutic Agents to the Malaria Parasite CHEMMEDCHEM Mahajan, S. S., Deu, E., Lauterwasser, E. M., Leyva, M. J., Ellman, J. A., Bogyo, M., Renslo, A. R. 2011; 6 (3): 415-419

  View details for DOI 10.1002/cmdc.201100002

  View details for Web of Science ID 000288599600003

  View details for PubMedID 21360816

  View details for PubMedCentralID PMC3265971

• Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins NATURE STRUCTURAL & MOLECULAR BIOLOGY Shen, A., Lupardus, P. J., Gersch, M. M., Puri, A. W., Albrow, V. E., Garcia, K. C., Bogyo, M. 2011; 18 (3): 364-U158

  Abstract

  An internal cysteine protease domain (CPD) autoproteolytically regulates Clostridium difficile glucosylating toxins by releasing a cytotoxic effector domain into target cells. CPD activity is itself allosterically regulated by the eukaryote-specific molecule inositol hexakisphosphate (InsP(6)). Although allostery controls the function of most proteins, the molecular details underlying this regulatory mechanism are often difficult to characterize. Here we use chemical probes to show that apo-CPD is in dynamic equilibrium between active and inactive states. InsP(6) markedly shifts this equilibrium toward an active conformer that is further restrained upon binding a suicide substrate. Structural analyses combined with systematic mutational and disulfide bond engineering studies show that residues within a β-hairpin region functionally couple the InsP(6)-binding site to the active site. Collectively, our results identify an allosteric circuit that allows bacterial virulence factors to sense and respond to the eukaryotic environment.

  View details for DOI 10.1038/nsmb.1990

  View details for Web of Science ID 000288072200019

  View details for PubMedID 21317893

  View details for PubMedCentralID PMC3076311

• Contribution of tumor cysteine cathepsin B to breast cancer metastasis to lung and bone Withana, N. P., Blum, G., Bidwell, B. N., Anderson, R. L., Bogyo, M. S., Parker, B. S. SPRINGER. 2011: 240–40

  View details for Web of Science ID 000289796200255

• Cathepsin X is activated by cathepsin L, in-activates the chemokine SDF-1 and reduces adhesion of hematopoietic stem and progenitor cells to osteoblasts Staudt, N., Aicher, W. K., Kalbacher, H., Stevanovic, S., Carmona, A., Bogyo, M., Klein, G. MARY ANN LIEBERT INC. 2011: 575-576

  View details for Web of Science ID 000286661600136

• Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1 MOLECULAR AND BIOCHEMICAL PARASITOLOGY Wang, F., Krai, P., Deu, E., Bibb, B., Lauritzen, C., Pedersen, J., Bogyo, M., Klemba, M. 2011; 175 (1): 10-20

  Abstract

  Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.

  View details for DOI 10.1016/j.molbiopara.2010.08.004

  View details for Web of Science ID 000284789500002

  View details for PubMedID 20833209

  View details for PubMedCentralID PMC3514014

• Cathepsin B trafficking in thyroid carcinoma cells. Thyroid research Tedelind, S., Jordans, S., Resemann, H., Blum, G., Bogyo, M., Führer, D., Brix, K. 2011; 4: S2-?

  Abstract

  The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in prohormone processing initiated in the follicle lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space in thyroid cancer tissue, and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through e.g. extracellular matrix degradation.Transport of cathepsin B in normal thyroid epithelial and carcinoma cells was investigated through immunolocalization of endogenous cathepsin B in combination with probing protease activity. Transport analyses of cathepsin B-eGFP and its active-site mutant counterpart cathepsin B-C29A-eGFP were used to test whether intrinsic sequences of a protease influence its trafficking.Our approach employing activity based probes, which distinguish between active and inactive cysteine proteases, demonstrated that both eGFP-tagged normal and active-site mutated cathepsin B chimeras reached the endo-lysosomal compartments of thyroid epithelial cells, thereby ruling out alterations of sorting signals by mutagenesis of the active-site cysteine. Analysis of chimeric protein trafficking further showed that GFP-tagged cathepsin B was transported to the expected compartments, i.e. endoplasmic reticulum, Golgi apparatus and endo-lysosomes of normal and thyroid carcinoma cell lines. However, the active-site mutated cathepsin B chimera was mostly retained in the endoplasmic reticulum and Golgi of KTC-1 and HTh7 cells. Hence the latter, as the least polarized of the three carcinoma cell lines analyzed, exhibited severe transport defects in that it retained chimeras in pre-endolysosomal compartments. Furthermore, secretion of endogenous cathepsin B and of other cysteine peptidases, which occurs at the apical pole of normal thyroid epithelial cells, was most prominent and occurred in a non-directed fashion in thyroid carcinoma cells.Transport of endogenous and eGFP-tagged active and inactive cathepsin B in the cultured thyroid carcinoma cells reflected the distribution patterns of this protease in thyroid carcinoma tissue. Hence, our studies showed that sub-cellular localization of proteolysis is a crucial step in regulation of tissue homeostasis. We conclude that any interference with protease trafficking resulting in altered regulation of proteolytic events leads to, or is a consequence of the onset and progression of thyroid cancer.

  View details for DOI 10.1186/1756-6614-4-S1-S2

  View details for PubMedID 21835049

  View details for PubMedCentralID PMC3155108

• Rational Design of Inhibitors and Activity-Based Probes Targeting Clostridium difficile Virulence Factor TcdB CHEMISTRY & BIOLOGY Puri, A. W., Lupardus, P. J., Deu, E., Albrow, V. E., Garcia, K. C., Bogyo, M., Shen, A. 2010; 17 (11): 1201-1211

  Abstract

  Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0 Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.

  View details for DOI 10.1016/j.chembiol.2010.09.011

  View details for Web of Science ID 000285405000009

  View details for PubMedID 21095570

  View details for PubMedCentralID PMC3005307

• Carbon Nanotubes Enable Noninvasive Optical Imaging of Macrophages in Mouse Atherosclerosis and Have Intrinsic Fluorescence for Near Infrared Imaging Scientific Sessions on Arteriosclerosis, Thrombosis and Vascular Biology Kitagawa, T., Kosuge, H., Sherlock, S., Bogyo, M., Dai, H., McConnell, M. LIPPINCOTT WILLIAMS & WILKINS. 2010: E298–E298

  View details for Web of Science ID 000283234800519

• Cathepsin X is secreted by human osteoblasts, digests CXCL-12 and impairs adhesion of hematopoietic stem and progenitor cells to osteoblasts HAEMATOLOGICA-THE HEMATOLOGY JOURNAL Staudt, N. D., Aicher, W. K., Kalbacher, H., Stevanovic, S., Carmona, A. K., Bogyo, M., Klein, G. 2010; 95 (9): 1452-1460

  Abstract

  Hematopoietic stem cells are retained within discrete bone marrow niches through the effects of cell adhesion molecules and chemokine gradients. However, a small proportion of hematopoietic stem cells can also be found trafficking in the peripheral blood. During induced stem cell mobilization a proteolytic microenvironment is generated, but whether proteases are also involved in physiological trafficking of hematopoietic stem cells is not known. In the present study we examined the expression, secretion and function of the cysteine protease cathepsin X by cells of the human bone marrow.Human osteoblasts, bone marrow stromal cells and hematopoietic stem and progenitor cells were analyzed for the secretion of cathepsin X by western blotting, active site labeling, immunofluorescence staining and activity assays. A possible involvement of cathepsin X in cell adhesion and CXCL-12-mediated cell migration was studied in functional assays. Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) analysis revealed the digestion mechanism of CXCL-12 by cathepsin X.Osteoblasts and stromal cells secrete cathepsin X, whereas hematopoietic stem and progenitor cells do not. Using a cathepsin X-selective substrate, we detected the catalytic activity of cathepsin X in cell culture supernatants of osteoblasts. Activated cathepsin X is able to reduce cellular adhesive interactions between CD34(+) hematopoietic stem and progenitor cells and adherent osteoblasts. The chemokine CXCL-12, a highly potent chemoattractant for hematopoietic stem cells secreted by osteoblasts, is readily digested by cathepsin X.The exo-peptidase cathepsin X has been identified as a new member of the group of CXCL-12-degrading enzymes secreted by non-hematopoietic bone marrow cells. Functional data indicate that cathepsin X can influence hematopoietic stem and progenitor cell trafficking in the bone marrow.

  View details for DOI 10.3324/haematol.2009.018671

  View details for Web of Science ID 000281933200005

  View details for PubMedID 20494937

  View details for PubMedCentralID PMC2930944

• Functional Studies of Plasmodium falciparum Dipeptidyl Aminopeptidase I Using Small Molecule Inhibitors and Active Site Probes CHEMISTRY & BIOLOGY Deu, E., Leyva, M. J., Albrow, V. E., Rice, M. J., Ellman, J. A., Bogyo, M. 2010; 17 (8): 808-819

  Abstract

  The widespread resistance of malaria parasites to all affordable drugs has made the identification of new targets urgent. Dipeptidyl aminopeptidases (DPAPs) represent potentially valuable new targets that are involved in hemoglobin degradation (DPAP1) and parasite egress (DPAP3). Here we use activity-based probes to demonstrate that specific inhibition of DPAP1 by a small molecule results in the formation of an immature trophozoite that leads to parasite death. Using computational methods, we designed stable, nonpeptidic covalent inhibitors that kill Plasmodium falciparum at low nanomolar concentrations. These compounds show signs of slowing parasite growth in a murine model of malaria, which suggests that DPAP1 might be a viable antimalarial target. Interestingly, we found that resynthesis and activation of DPAP1 after inhibition is rapid, suggesting that effective drugs would need to sustain DPAP1 inhibition for a period of 2-3 hr.

  View details for DOI 10.1016/j.chembiol.2010.06.007

  View details for Web of Science ID 000281721800007

  View details for PubMedID 20797610

  View details for PubMedCentralID PMC2929396

• Small molecule probes of protease function: Applications to molecular imaging Bogyo, M., Blum, G., Edgington, L., Yin, F., Berger, A., Lee, J., Terashima, M., Kosuge, H., McConnell, M. AMER CHEMICAL SOC. 2010

  View details for Web of Science ID 000208164700716

• Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts PLOS ONE Deu, E., Yang, Z., Wang, F., Klemba, M., Bogyo, M. 2010; 5 (8)

  Abstract

  High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

  View details for DOI 10.1371/journal.pone.0011985

  View details for Web of Science ID 000280605400018

  View details for PubMedID 20700487

  View details for PubMedCentralID PMC2916841

• Nuclear cysteine cathepsin variants in thyroid carcinoma cells 6th General Meeting of the International-Proteolysis-Society Tedelind, S., Poliakova, K., Valeta, A., Hunegnaw, R., Yemanaberhan, E. L., Heldin, N., Kurebayashi, J., Weber, E., Kopitar-Jerala, N., Turk, B., Bogyo, M., Brix, K. WALTER DE GRUYTER & CO. 2010: 923–35

  Abstract

  The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.

  View details for DOI 10.1515/BC.2010.109

  View details for Web of Science ID 000281198700010

  View details for PubMedID 20536394

  View details for PubMedCentralID PMC3518386

• Increased nucleolar localization of SpiA3G in classically but not alternatively activated macrophages FEBS LETTERS Konjar, S., Yin, F., Bogyo, M., Turk, B., Kopitar-Jerala, N. 2010; 584 (11): 2201-2206

  Abstract

  Macrophages play a key role in innate immune response to pathogens and in tissue homeostasis, inflammation and repair. A serpin A3G (SpiA3G) is highly induced in classically activated macrophages. We show increased localization of SpiA3G in the nucleolus and co-localization with cathepsin L, upon classical, but not alternative activation of macrophages. Despite the increased expression of cathepsin L in the nuclei of classically activated macrophages, no cathepsin activity was detected. Since only pro-inflammatory, but not anti-inflammatory stimuli induce increased nucleolar localization of SpiA3G, we propose that SpiA3g translocation into the nucleolus is important in host defense against pathogens.

  View details for DOI 10.1016/j.febslet.2010.03.031

  View details for Web of Science ID 000277793200005

  View details for PubMedID 20338168

• Finding enzymes that are actively involved in cancer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bogyo, M. 2010; 107 (6): 2379-2380

  View details for DOI 10.1073/pnas.0914955107

  View details for Web of Science ID 000274408100006

  View details for PubMedID 20133631

  View details for PubMedCentralID PMC2823869

• Development of Near-Infrared Fluorophore (NIRF)-Labeled Activity-Based Probes for in Vivo Imaging of Legumain ACS CHEMICAL BIOLOGY Lee, J., Bogyo, M. 2010; 5 (2): 233-243

  Abstract

  Asparaginyl endopeptidase, or legumain, is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers, and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for legumain. We demonstrate that optimized aza-peptidyl Asn epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label legumain in various normal tissues as well as in solid tumors when applied in vivo. Interestingly, addition of cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of legumain function in more complex models of human disease.

  View details for DOI 10.1021/cb900232a

  View details for Web of Science ID 000274747400010

  View details for PubMedID 20017516

  View details for PubMedCentralID PMC3222276

• Aminopeptidase Fingerprints, an Integrated Approach for Identification of Good Substrates and Optimal Inhibitors JOURNAL OF BIOLOGICAL CHEMISTRY Drag, M., Bogyo, M., Ellman, J. A., Salvesen, G. S. 2010; 285 (5): 3310-3318

  Abstract

  Aminopeptidases process the N-terminal amino acids of target substrates by sequential cleavage of one residue at a time. They are found in all cell compartments of prokaryotes and eukaryotes, being implicated in the major proteolytic events of cell survival, defense, growth, and development. We present a new approach for the fast and reliable evaluation of the substrate specificity of individual aminopeptidases. Using solid phase chemistry with the 7-amino-4-carbamoylmethylcoumarin fluorophore, we have synthesized a library of 61 individual natural and unnatural amino acids substrates, chosen to cover a broad spectrum of the possible interactions in the S1 pocket of this type of protease. As proof of concept, we determined the substrate specificity of human, pig, and rat orthologs of aminopeptidase N (CD13), a highly conserved cell surface protease that inactivates enkephalins and other bioactive peptides. Our data reveal a large and hydrophobic character for the S1 pocket of aminopeptidase N that is conserved with aminopeptidase Ns. Our approach, which can be applied in principle to all aminopeptidases, yields useful information for the design of specific inhibitors, and more importantly, reveals a relationship between the kinetics of substrate hydrolysis and the kinetics of enzyme inhibition.

  View details for DOI 10.1074/jbc.M109.060418

  View details for Web of Science ID 000273829000046

  View details for PubMedID 19948737

  View details for PubMedCentralID PMC2823418

• Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag PLOS ONE Shen, A., Lupardus, P. J., Morell, M., Ponder, E. L., Sadaghiani, A. M., Garcia, K. C., Bogyo, M. 2009; 4 (12)

  Abstract

  We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP(6)), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His(6)-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP(6) to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.

  View details for DOI 10.1371/journal.pone.0008119

  View details for Web of Science ID 000272828800015

  View details for PubMedID 19956581

  View details for PubMedCentralID PMC2780291

• 4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion PLOS ONE Ravindran, S., Lodoen, M. B., Verhelst, S. H., Bogyo, M., Boothroyd, J. C. 2009; 4 (12)

  Abstract

  Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa that is able to infect a wide variety of host cells. During its active invasion process it secretes proteins from discrete secretory organelles: the micronemes, rhoptries and dense granules. Although a number of rhoptry proteins have been shown to be involved in important interactions with the host cell, very little is known about the mechanism of secretion of any Toxoplasma protein into the host cell. We used a chemical inhibitor of phospholipase A2s, 4-bromophenacyl bromide (4-BPB), to look at the role of such lipases in the secretion of Toxoplasma proteins. We found that 4-BPB was a potent inhibitor of rhoptry secretion in Toxoplasma invasion. This drug specifically blocked rhoptry secretion but not microneme secretion, thus effectively showing that the two processes can be de-coupled. It affected parasite motility and invasion, but not attachment or egress. Using propargyl- or azido-derivatives of the drug (so-called click chemistry derivatives) and a series of 4-BPB-resistant mutants, we found that the drug has a very large number of target proteins in the parasite that are involved in at least two key steps: invasion and intracellular growth. This potent compound, the modified "click-chemistry" forms of it, and the resistant mutants should serve as useful tools to further study the processes of Toxoplasma early invasion, in general, and rhoptry secretion, in particular.

  View details for DOI 10.1371/journal.pone.0008143

  View details for Web of Science ID 000272828800031

  View details for PubMedID 19956582

  View details for PubMedCentralID PMC2780294

• Cathepsin X proteolytically cleaves CXCL-12 and alters adhesive interactions in the hematopoietic stem cell niche Staudt, N., Aicher, W., Kalbacher, H., Carmora, A., Bogyo, M., Klein, G. MARY ANN LIEBERT INC. 2009: 1468

  View details for Web of Science ID 000271441000341

• Hemoglobin Digestion in Blood-Feeding Ticks: Mapping a Multipeptidase Pathway by Functional Proteomics CHEMISTRY & BIOLOGY Horn, M., Nussbaumerova, M., Sanda, M., Kovarova, Z., Srba, J., Franta, Z., Sojka, D., Bogyo, M., Caffrey, C. R., Kopacek, P., Mares, M. 2009; 16 (10): 1053-1063

  Abstract

  Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.

  View details for DOI 10.1016/j.chembiol.2009.09.009

  View details for Web of Science ID 000271894000008

  View details for PubMedID 19875079

  View details for PubMedCentralID PMC2801564

• Intracellular inhibitors of cysteine cathepsins in activated macrophages Tri-Society Annual Conference of the International-Cytokine-Society/International-Society-of-Interferon-and-Cytokine-Research/Society-of-Leukocyte-Biology Maher, K., Konjar, S., Ceru, S., Bogyo, M., Turk, B., Kopitar-Jerala, N. ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. 2009: 24–24

  View details for DOI 10.1016/j.cyto.2009.07.095

  View details for Web of Science ID 000270855100074

• Toxoplasma gondii Cathepsin L Is the Primary Target of the Invasion-inhibitory Compound Morpholinurea-leucyl-homophenyl-vinyl Sulfone Phenyl JOURNAL OF BIOLOGICAL CHEMISTRY Larson, E. T., Parussini, F., Huynh, M., Giebel, J. D., Kelley, A. M., Zhang, L., Bogyo, M., Merritt, E. A., Carruthers, V. B. 2009; 284 (39): 26839–50

  Abstract

  The protozoan parasite Toxoplasma gondii relies on post-translational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS), which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 A resolution crystal structure of TgCPL in complex with its propeptide. We discuss possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion.

  View details for DOI 10.1074/jbc.M109.003780

  View details for Web of Science ID 000269969600065

  View details for PubMedID 19596863

  View details for PubMedCentralID PMC2785372

• Rab35 Controls Actin Bundling by Recruiting Fascin as an Effector Protein SCIENCE Zhang, J., Fonovic, M., Suyama, K., Bogyo, M., Scott, M. P. 2009; 325 (5945): 1250-1254

  Abstract

  Actin filaments are key components of the eukaryotic cytoskeleton that provide mechanical structure and generate forces during cell shape changes, growth, and migration. Actin filaments are dynamically assembled into higher-order structures at specified locations to regulate diverse functions. The Rab family of small guanosine triphosphatases is evolutionarily conserved and mediates intracellular vesicle trafficking. We found that Rab35 regulates the assembly of actin filaments during bristle development in Drosophila and filopodia formation in cultured cells. These effects were mediated by the actin-bundling protein fascin, which directly associated with active Rab35. Targeting Rab35 to the outer mitochondrial membrane triggered actin recruitment, demonstrating a role for an intracellular trafficking protein in localized actin assembly.

  View details for DOI 10.1126/science.1174921

  View details for Web of Science ID 000269523200039

  View details for PubMedID 19729655

• Design, syntheses, and evaluation of Taspase1 inhibitors BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Lee, J. T., Chen, D. Y., Yang, Z., Ramos, A. D., Hsieh, J. J., Bogyo, M. 2009; 19 (17): 5086-5090

  Abstract

  Taspase1 is a threonine protease responsible for cleaving MLL (Mixed-Lineage Leukemia) to achieve proper HOX gene expression. Subsequent studies identified additional Taspase1 substrates including Transcription Factor IIA (TFIIA) and Drosophila HCF. Taspase1 is essential for cell proliferation and is overexpressed in many cancer cell lines. Currently no small molecule inhibitors of this enzyme have been described. Here, we report the synthesis and evaluation of vinyl sulfone, vinyl ketone, epoxy ketone, and boronic acid inhibitors designed based on the preferred Taspase1 cleavage site (Ac-Ile-Ser-Gln-Leu-Asp). Specifically, we evaluated compounds in which the reactive warhead is positioned in place of the P1 aspartic acid side chain as well as at the C-terminus of the peptide. Interestingly, both classes of inhibitors were effective and vinyl ketones and vinyl sulfones showed the greatest potency for the target protease. These results suggest that Taspase1 has unique substrate recognition properties that could potentially be exploited in the design of potent and selective inhibitors of this enzyme.

  View details for DOI 10.1016/j.bmcl.2009.07.045

  View details for Web of Science ID 000268863800044

  View details for PubMedID 19631530

  View details for PubMedCentralID PMC3513416

• Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes NATURE MEDICINE Edgington, L. E., Berger, A. B., Blum, G., Albrow, V. E., Paulick, M. G., Lineberry, N., Bogyo, M. 2009; 15 (8): 967-U177

  Abstract

  Imaging agents that enable direct visualization and quantification of apoptosis in vivo have great potential value for monitoring chemotherapeutic response as well as for early diagnosis and disease monitoring. We describe here the development of fluorescently labeled activity-based probes (ABPs) that covalently label active caspases in vivo. We used these probes to monitor apoptosis in the thymi of mice treated with dexamethasone as well as in tumor-bearing mice treated with the apoptosis-inducing monoclonal antibody Apomab (Genentech). Caspase ABPs provided direct readouts of the kinetics of apoptosis in live mice, whole organs and tissue extracts. The probes produced a maximum fluorescent signal that could be monitored noninvasively and that coincided with the peak in caspase activity, as measured by gel analysis. Overall, these studies demonstrate that caspase-specific ABPs have the potential to be used for noninvasive imaging of apoptosis in both preclinical and clinical settings.

  View details for DOI 10.1038/nm.1938

  View details for Web of Science ID 000268770400044

  View details for PubMedID 19597506

  View details for PubMedCentralID PMC3196344

• Using Small Molecules To Dissect Mechanisms of Microbial Pathogenesis ACS CHEMICAL BIOLOGY Puri, A. W., Bogyo, M. 2009; 4 (8): 603-616

  Abstract

  Understanding the ways in which pathogens invade and neutralize their hosts is of great interest from both an academic and a clinical perspective. However, in many cases genetic tools are unavailable or insufficient to fully characterize the detailed mechanisms of pathogenesis. Small molecule approaches are particularly powerful due to their ability to modulate specific biological functions in a highly controlled manner and their potential to broadly target conserved processes across species. Recently, two approaches that make use of small molecules, activity-based protein profiling and high-throughput phenotypic screening, have begun to find applications in the study of pathways involved in pathogenesis. In this Review we highlight ways in which these techniques have been applied to examine bacterial and parasitic pathogenesis and discuss possible ways in which these efforts can be expanded in the near future.

  View details for DOI 10.1021/cb9001409

  View details for Web of Science ID 000269087500003

  View details for PubMedID 19606820

  View details for PubMedCentralID PMC2766775

• Comparative Assessment of Substrates and Activity Based Probes as Tools for Non-Invasive Optical Imaging of Cysteine Protease Activity PLOS ONE Blum, G., Weimer, R. M., Edgington, L. E., Adams, W., Bogyo, M. 2009; 4 (7)

  Abstract

  Recent advances in the field of non-invasive optical imaging have included the development of contrast agents that report on the activity of enzymatic targets associated with disease pathology. In particular, proteases have proven to be ideal targets for development of optical sensors for cancer. Recently developed contrast agents for protease activity include both small peptides and large polymer-based quenched fluorescent substrates as well as fluorescently labeled activity based probes (ABPs). While substrates produce a fluorescent signal as a result of processing by a protease, ABPs are retained at the site of proteolysis due to formation of a permanent covalent bond with the active site catalytic residue. Both methods have potential advantages and disadvantages yet a careful comparison of substrates and ABPs has not been performed. Here we present the results of a direct comparison of commercially available protease substrates with several recently described fluorescent ABPs in a mouse model of cancer. The results demonstrate that fluorescent ABPs show more rapid and selective uptake into tumors as well as overall brighter signals compared to substrate probes. These data suggest that the lack of signal amplification for an ABP is offset by the increased kinetics of tissue uptake and prolonged retention of the probes once bound to a protease target. Furthermore, fluorescent ABPs can be used as imaging reagents with similar or better results as the commercially available protease substrates.

  View details for DOI 10.1371/journal.pone.0006374

  View details for Web of Science ID 000268404900009

  View details for PubMedID 19636372

  View details for PubMedCentralID PMC2712068

• Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin NATURE CHEMICAL BIOLOGY Shen, A., Lupardus, P. J., Albrow, V. E., Guzzetta, A., Powers, J. C., Garcia, K. C., Bogyo, M. 2009; 5 (7): 469-478

  Abstract

  MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a new allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small-molecule inhibitors of this protease domain and present the 2.35-A structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising new antitoxin strategies for a number of bacterial pathogens.

  View details for DOI 10.1038/nchembio.178

  View details for Web of Science ID 000267266100012

  View details for PubMedID 19465933

  View details for PubMedCentralID PMC2783333

• A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY Beckham, S. A., Piedrafita, D., Phillips, C. I., Samarawickrema, N., Law, R. H., Smooker, P. M., Quinsey, N. S., Irving, J. A., Greenwood, D., Verhelst, S. H., Bogyo, M., Turk, B., Coetzer, T. H., Wijeyewickrema, L. C., Spithill, T. W., Pike, R. N. 2009; 41 (7): 1601-1612

  Abstract

  The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.

  View details for DOI 10.1016/j.biocel.2009.02.003

  View details for Web of Science ID 000265768000019

  View details for PubMedID 19401154

  View details for PubMedCentralID PMC3514016

• VEGF-A Induces Angiogenesis by Perturbing the Cathepsin-Cysteine Protease Inhibitor Balance in Venules, Causing Basement Membrane Degradation and Mother Vessel Formation CANCER RESEARCH Chang, S., Kanasaki, K., Gocheva, V., Blum, G., Harper, J., Moses, M. A., Shih, S., Nagy, J. A., Joyce, J., Bogyo, M., Kalluri, R., Dvorak, H. F. 2009; 69 (10): 4537-4544

  Abstract

  Tumors initiate angiogenesis primarily by secreting vascular endothelial growth factor (VEGF-A(164)). The first new vessels to form are greatly enlarged, pericyte-poor sinusoids, called mother vessels (MV), that originate from preexisting venules. We postulated that the venular enlargement necessary to form MV would require a selective degradation of their basement membranes, rigid structures that resist vascular expansion. To identify the specific proteases responsible for MV formation, we induced angiogenesis in mouse tissues with an adenoviral vector expressing VEGF-A(164) (Ad-VEGF-A(164)) or with VEGF-A-secreting TA3/St mammary tumors. We found that MV formation resulted from greatly increased activity of cathepsins (B>S>L) in venules transitioning into MV, as well as from a reciprocal decrease in the expression of several cysteine protease inhibitors (CPI), stefin A and cystatins B and C, by these same venules. Using a fluorescence probe that selectively binds cellular sites of cathepsin protease activity in vivo, we showed that increased cathepsin activity was localized exclusively to perivenular cells, not to venule endothelial cells. CPI strikingly inhibited angiogenesis in the Matrigel assay, and Ad-VEGF-A(164)-induced angiogenesis was reduced by approximately 50% in cathepsin B-null mice. Thus, VEGF-A, whether expressed by interstitial cells infected with an adenoviral vector or by tumor cells, upsets the normal cathepsin-CPI balance in nearby venules, leading to degradation of their basement membranes, an important first step in angiogenesis.

  View details for DOI 10.1158/0008-5472.CAN-08-4539

  View details for Web of Science ID 000266214400055

  View details for PubMedID 19435903

  View details for PubMedCentralID PMC2683911

• Caspase-8 Association with the Focal Adhesion Complex Promotes Tumor Cell Migration and Metastasis CANCER RESEARCH Barbero, S., Mielgo, A., Torres, V., Teitz, T., Shields, D. J., Mikolon, D., Bogyo, M., Barila, D., Lahti, J. M., Schlaepfer, D., Stupack, D. G. 2009; 69 (9): 3755-3763

  Abstract

  Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

  View details for DOI 10.1158/0008-5472.CAN-08-3937

  View details for Web of Science ID 000265761900006

  View details for PubMedID 19383910

  View details for PubMedCentralID PMC2684981

• Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation EXPERIMENTAL CELL RESEARCH Cavallo-Medved, D., Rudy, D., Blum, G., Bogyo, M., Caglic, D., Sloane, B. F. 2009; 315 (7): 1234-1246

  Abstract

  Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

  View details for DOI 10.1016/j.yexcr.2009.01.021

  View details for Web of Science ID 000265126900014

  View details for PubMedID 19331819

  View details for PubMedCentralID PMC2677760

• Substrate specificity of transthyretin: identification of natural substrates in the nervous system BIOCHEMICAL JOURNAL Liz, M. A., Fleming, C. E., Nunes, A. F., Almeida, M. R., Mar, F. M., Choe, Y., Craik, C. S., Powers, J. C., Bogyo, M., Sousa, M. M. 2009; 419: 467-474

  Abstract

  Besides functioning as the plasma transporter of retinol and thyroxine, TTR (transthyretin) is a protease, cleaving apoA-I (apolipoprotein A-I) after a phenylalanine residue. In the present study, we further investigated TTR substrate specificity. By using both P-diverse libraries and a library of phosphonate inhibitors, a TTR preference for a lysine residue in P1 was determined, suggesting that TTR might have a dual specificity and that, in addition to apoA-I, other TTR substrates might exist. Previous studies revealed that TTR is involved in the homoeostasis of the nervous system, as it participates in neuropeptide maturation and enhances nerve regeneration. We investigated whether TTR proteolytic activity is involved in these functions. Both wild-type TTR and TTR(prot-) (proteolytically inactive TTR) had a similar effect in the expression of peptidylglycine alpha-amidating mono-oxygenase, the rate-limiting enzyme in neuropeptide amidation, excluding the involvement of TTR proteolytic activity in neuropeptide maturation. However, TTR was able to cleave amidated NPY (neuropeptide Y), probably contributing to the increased NPY levels reported in TTR-knockout mice. To assess the involvement of TTR proteolytic activity in axonal regeneration, neurite outgrowth of cells cultivated with wild-type TTR or TTR(prot-), was measured. Cells grown with TTR(prot-) displayed decreased neurite length, thereby suggesting that TTR proteolytic activity is important for its function as a regeneration enhancer. By showing that TTR is able to cleave NPY and that its proteolytic activity affects axonal growth, the present study shows that TTR has natural substrates in the nervous system, establishing further its relevance in neurobiology.

  View details for DOI 10.1042/BJ20082090

  View details for Web of Science ID 000265196900023

  View details for PubMedID 19138167

• Cathepsin X-mediated beta(2) integrin activation results in nanotube outgrowth CELLULAR AND MOLECULAR LIFE SCIENCES Obermajer, N., Jevnikar, Z., Doljak, B., Sadaghiani, A. M., Bogyo, M., Kos, J. 2009; 66 (6): 1126-1134

  Abstract

  Membrane nanotubes were recently described as a new principle of cell-cell communication enabling complex and specific messaging to distant cells. Calcium fluxes, vesicles, and cell-surface components can all traffic between cells connected by nanotubes. Here we report for the first time the mechanism of membrane nanotube formation in T cells through LFA-1 (CD11a/CD18; alpha(L)beta(2)) integrin activation by the cysteine protease cathepsin X. Cathepsin X is shown to induce persistent LFA-1 activation. Cathepsin X-upregulated T cells exhibit increased homotypic aggregation and polarized, migration-associated morphology in 2D and 3D models, respectively. In these cells, extended uropods are frequently formed, which subsequently elongate to nanotubes connecting T lymphocytes. Our results demonstrate that LFA-1 activation with subsequent cytoskeletal reorganization induces signal transmission through a physically connected network of T lymphocytes for better coordination of their action at various stages of the immune response.

  View details for DOI 10.1007/s00018-009-8829-8

  View details for Web of Science ID 000264174800014

  View details for PubMedID 19194656

• Evaluation of alpha,beta-unsaturated ketone-based probes for papain-family cysteine proteases BIOORGANIC & MEDICINAL CHEMISTRY Yang, Z., Fonovic, M., Verhelst, S. H., Blum, G., Bogyo, M. 2009; 17 (3): 1071-1078

  Abstract

  The field of activity-based proteomics makes use of small molecule active site probes to monitor distinct subsets of enzymatic proteins. While a number of reactive functional groups have been applied to activity-based probes (ABPs) that target diverse families of proteases, there remains a continual need for further evaluation of new probe scaffolds and reactive functional groups for use in ABPs. In this study we evaluate the utility of the, alpha,beta-unsaturated ketone reactive group for use in ABPs targeting the papain-family of cysteine proteases. We find that this reactive group shows highly selective labeling of cysteine cathepsins in both intact cells and total cell extracts. We observed a variable degree of background labeling that depended on the type of tag and linker used in the probe synthesis. The relative ease of synthesis of this class of compounds provides the potential for further derivatization to generate new families of cysteine protease ABPs with unique specificity and labeling properties.

  View details for DOI 10.1016/j.bmc.2008.02.089

  View details for Web of Science ID 000262980500016

  View details for PubMedID 18343672

  View details for PubMedCentralID PMC2659416

• Autocatalytic processing of procathepsin B is triggered by proenzyme activity FEBS JOURNAL Pungercar, J. R., Caglic, D., Sajid, M., Dolinar, M., Vasiljeva, O., Pozgan, U., Turk, D., Bogyo, M., Turk, V., Turk, B. 2009; 276 (3): 660-668

  Abstract

  Cathepsin B (EC 3.4.22.1) and other cysteine proteases are synthesized as zymogens, which are processed to their mature forms autocatalytically or by other proteases. Autocatalytic processing was suggested to be a bimolecular process, whereas initiation of the processing has not yet been clarified. Procathepsin B was shown by zymography to hydrolyze the synthetic substrate 7-N-benzyloxycarbonyl-L-arginyl-L-arginylamide-4-methylcoumarin (Z-Arg-Arg-NH-MEC), suggesting that procathepsin B is catalytically active. The activity-based probe DCG-04, which is an E-64-type inhibitor, was found to label both mature cathepsin B and its zymogen, confirming the zymography data. Mutation analyses in the linker region between the propeptide and the mature part revealed that autocatalytic processing of procathepsin B is largely unaffected by mutations in this region, including mutations to prolines. On the basis of these results, a model for autocatalytic activation of cysteine cathepsins is proposed, involving propeptide dissociation from the active-site cleft as the first step during zymogen activation. This unimolecular conformational change is followed by a bimolecular proteolytic removal of the propeptide, which can be accomplished in one or more steps. Such activation, which can be also facilitated by glycosaminoglycans or by binding to negatively charged surfaces, may have important physiological consequences because cathepsin zymogens were often found secreted in various pathological states.

  View details for DOI 10.1111/j.1742-4658.2008.06815.x

  View details for Web of Science ID 000262468200007

  View details for PubMedID 19143833

• Metabolomics cuts to the chase to chase the cuts NATURE CHEMICAL BIOLOGY Bogyo, M. 2009; 5 (1): 5-6

  View details for DOI 10.1038/nchembio0109-5

  View details for Web of Science ID 000261935500004

  View details for PubMedID 19088710

• Minitags for small molecules: detecting targets of reactive small molecules in living plant tissues using 'click chemistry' PLANT JOURNAL Kaschani, F., Verhelst, S. H., van Swieten, P. F., Verdoes, M., Wong, C., Wang, Z., Kaiser, M., Overkleeft, H. S., Bogyo, M., Van Der Hoorn, R. A. 2009; 57 (2): 373-385

  Abstract

  Small molecules offer unprecedented opportunities for plant research since plants respond to, metabolize, and react with a diverse range of endogenous and exogenous small molecules. Many of these small molecules become covalently attached to proteins. To display these small molecule targets in plants, we introduce a two-step labelling method for minitagged small molecules. Minitags are small chemical moieties (azide or alkyne) that are inert under biological conditions and have little influence on the membrane permeability and specificity of the small molecule. After labelling, proteomes are extracted under denaturing conditions and minitagged proteins are coupled to reporter tags through a 'click chemistry' reaction. We introduce this two-step labelling procedure in plants by studying the well-characterized targets of E-64, a small molecule cysteine protease inhibitor. In contrast to biotinylated E-64, minitagged E-64 efficiently labels vacuolar proteases in vivo. We displayed, purified and identified targets of a minitagged inhibitor that targets the proteasome and cysteine proteases in living plant cells. Chemical interference assays with inhibitors showed that MG132, a frequently used proteasome inhibitor, preferentially inhibits cysteine proteases in vivo. The two-step labelling procedure can be applied on detached leaves, cell cultures, seedlings and other living plant tissues and, when combined with photoreactive groups, can be used to identify targets of herbicides, phytohormones and reactive small molecules selected from chemical genetic screens.

  View details for DOI 10.1111/j.1365-313X.2008.03683.x

  View details for Web of Science ID 000262488300015

  View details for PubMedID 18786180

• Maturation of dendritic cells depends on proteolytic cleavage by cathepsin X JOURNAL OF LEUKOCYTE BIOLOGY Obermajer, N., Svajger, U., Bogyo, M., Jeras, M., Kos, J. 2008; 84 (5): 1306-1315

  Abstract

  The maturation status of dendritic cells (DCs) is crucial for effective antigen presentation and initiation of the primary immune response. Maturation stimuli cause the adhesion of immature DCs to the extracellular matrix, which is accompanied by recruitment of the CD11b/CD18 [macrophage antigen-1 (Mac-1)] integrin receptor, cytoskeleton reorganization, and podosome formation. Cathepsin X, a cysteine protease expressed in DCs and other APCs, is involved in Mac-1 activation. We have shown that during maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac-1 activation and consequently, cell adhesion. In mature DCs, cathepsin X redistributes from the membrane to the perinuclear region, which coincides with the de-adhesion of DCs, formation of cell clusters, and acquisition of the mature phenotype. Inhibition of cathepsin X activity during DC differentiation and maturation resulted in an altered phenotype and function of mature DCs. It reduced surface expression of costimulatory molecules, increased expression of inhibitory Ig-like transcripts 3 and 4 (ILT3 and ILT4), almost completely abolished cytokine production, diminished migration, and reduced the capacity of DCs to stimulate T lymphocytes. These results stress the importance of cathepsin X in regulating DC adhesion, a crucial event for their maturation and T cell activation.

  View details for DOI 10.1189/jlb.0508285

  View details for Web of Science ID 000260016300011

  View details for PubMedID 18701767

• Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain SCIENCE Lupardus, P. J., Shen, A., Bogyo, M., Garcia, K. C. 2008; 322 (5899): 265-268

  Abstract

  Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP6), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP6. InsP6 binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP6 binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  View details for DOI 10.1126/science.1162403

  View details for Web of Science ID 000259902300050

  View details for PubMedID 18845756

  View details for PubMedCentralID PMC3272704

• Activity-based probes as a tool for functional proteomic analysis of proteases EXPERT REVIEW OF PROTEOMICS Fonovic, M., Bogyo, M. 2008; 5 (5): 721-730

  Abstract

  Traditional proteomics methodology allows global analysis of protein abundance but does not provide information on the regulation of protein activity. Proteases, in particular, are known for their multilayered post-translational activity regulation that can lead to a significant difference between protease abundance levels and their enzyme activity. To address these issues, the field of activity-based proteomics has been established in order to characterize protein activity and monitor the functional regulation of enzymes in complex proteomes. In this review, we present structural features of activity-based probes for proteases and discuss their applications in proteomic profiling of various catalytic classes of proteases.

  View details for DOI 10.1586/14789450.5.5.721

  View details for Web of Science ID 000260701000013

  View details for PubMedID 18937562

  View details for PubMedCentralID PMC2997944

• Friend or Foe? Turning a Host Defense Protein Into a Pathogen's Accomplice CHEMISTRY & BIOLOGY Shen, A., Bogyo, M. 2008; 15 (9): 879-880

  Abstract

  Cystatins are cysteine protease inhibitors that are at the front-line of defense against pathogens that secrete proteases as virulence factors. In this issue, Vincents et al. (2008) reveal how the bacterial protease IdeS from Streptococcus pyogenes hijacks normal cystatin C function to convert it into a cofactor that enhances proteolytic destruction of host-defense antibodies.

  View details for DOI 10.1016/j.chembiol.2008.09.001

  View details for Web of Science ID 000259918200002

  View details for PubMedID 18804024

• ORGN 4-Small molecule probes of protease function: Applications to in vivo molecular imaging Bogyo, M., Blum, G., Berger, A., Phillips, C., Ponder, E., Arastu-Kapur, S., Fonovic, M., Fonovic, U. AMER CHEMICAL SOC. 2008

  View details for Web of Science ID 000270256309673

• The role of cathepsin X in the migration and invasiveness of T lymphocytes JOURNAL OF CELL SCIENCE Jevnikar, Z., Obermajer, N., Bogyo, M., Kos, J. 2008; 121 (16): 2652-2661

  Abstract

  Cathepsin X is a lysosomal cysteine protease exhibiting carboxypeptidase activity. Its expression is high in the cells of immune system and its function has been related to the processes of inflammatory and immune responses. It regulates processes such as adhesion, T lymphocyte activation and phagocytosis through its interaction with beta2 integrins. To investigate the role of cathepsin X in the migration of T lymphocytes, Jurkat T lymphocytes were stably transfected with a pcDNA3 expression vector containing cathepsin X cDNA. The cathepsin-X-overexpressing T lymphocytes exhibited polarised migration-associated morphology, enhanced migration on 2D and 3D models using intercellular adhesion molecule 1 (ICAM1)- and Matrigel-coated surfaces, and increased homotypic aggregation. The increased invasiveness of cathepsin-X-overexpressing cells does not involve proteolytic degradation of extracellular matrix. Confocal microscopy showed that the active mature form of cathepsin X was colocalised in migrating cells together with lymphocyte-function-associated antigen 1 (LFA-1). The colocalisation was particularly evident at the trailing edge protrusion, the uropod, that has an important role in T lymphocyte migration and cell-cell interactions. We propose that cathepsin X causes cytoskeletal rearrangements and stimulates migration of T lymphocytes by modulating the activity of the beta2 integrin receptor LFA-1.

  View details for DOI 10.1242/jcs.023721

  View details for Web of Science ID 000258243900006

  View details for PubMedID 18664495

• Trial of the cysteine cathepsin inhibitor JPM-OEt on early and advanced mammary cancer stages in the MMTV-PyMT-transgenic mouse model 5th General Meeting of the International-Proteolysis-Society Schurigt, U., Sevenich, L., Vannier, C., Gajda, M., Schwinde, A., Werner, F., Stahl, A., von Elverfeldt, D., Becker, A., Bogyo, M., Peters, C., Reinheckel, T. WALTER DE GRUYTER & CO. 2008: 1067–74

  Abstract

  Recent data suggest proteases of the papain-like cysteine cathepsin family as molecular targets for cancer therapy. Here, we report the treatment of polyoma middle T oncogene-induced breast cancers in mice with the cell-permeable broad-spectrum cysteine cathepsin inhibitor JPM-OEt. Up to 100 mg/kg inhibitor was intraperitoneally injected once per day in two trials on early and advanced cancers. In both trials, transient delays in tumour growth were observed. However, at the endpoint of both experiments no significant differences in tumour weights, histopathology and lung metastasis were found between the inhibitor and the control group. The invasive strand formation of collagen I-embedded tumour cell spheroids generated from primary tumours of inhibitor-treated mice in the early cancer trial could be inhibited in vitro by JPM-OEt; a result arguing against induction of resistance to the inhibitor. Measurement of cysteine cathepsin activities in tissue extracts after intraperitoneal injection of JPM-OEt revealed effective inhibition of cysteine cathepsins in pancreas, kidneys and liver, while activities in mammary cancers and in lungs were not significantly affected. We conclude that the pharmacokinetic properties of JPM-OEt, which result in poor bioavailability, may prohibit its use for stand-alone treatment of solid mammary cancers and their lung metastases.

  View details for DOI 10.1515/BC.2008.115

  View details for Web of Science ID 000258262300011

  View details for PubMedID 18710344

• Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum NATURE CHEMICAL BIOLOGY Arastu-Kapur, S., Ponder, E. L., Fonovic, U. P., Yeoh, S., Yuan, F., Fonovic, M., Grainger, M., Phillips, C. I., Powers, J. C., Bogyo, M. 2008; 4 (3): 203-213

  Abstract

  Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.

  View details for Web of Science ID 000253417400017

  View details for PubMedID 18246061

• Application of activity-based probes to the study of enzymes involved in cancer progression CURRENT OPINION IN GENETICS & DEVELOPMENT Paulick, M. G., Bogyo, M. 2008; 18 (1): 97-106

  Abstract

  Many tumor cells have elevated levels of hydrolytic and proteolytic enzymes, presumably to aid in key processes such as angiogenesis, cancer cell invasion, and metastasis. Functional roles of enzymes in cancer progression are difficult to study using traditional genomic and proteomic methods because the activities of these enzymes are often regulated by post-translational mechanisms. Thus, methods that allow for the direct monitoring of enzyme activity in a physiologically relevant environment are required to better understand the roles of specific players in the complex process of tumorigenesis. This review highlights advances in the field of activity-based proteomics, which uses small molecules known as activity-based probes (ABPs) that covalently bind to the catalytic site of target enzymes. We discuss the application of ABPs to cancer biology, especially to the discovery of tumor biomarkers, the screening of enzyme inhibitors, and the imaging of enzymes implicated in cancer.

  View details for DOI 10.1016/j.gde.2007.12.001

  View details for Web of Science ID 000256954100015

  View details for PubMedID 18294838

  View details for PubMedCentralID PMC2474457

• A chemical genetic screen in Plasmodium falciparum identifies proteases that regulate erythrocyte rupture. Arastu-Kapur, S., Ponder, E. L., Fonovic, U., Yeoh, S., Yuan, F., Fonovi, M., Grainger, M., Phillips, C. I., Powers, J. C., Bogyo, M. PERGAMON-ELSEVIER SCIENCE LTD. 2008: S86

  View details for Web of Science ID 000253127600245

• Ubiquitin-like modifiers and their deconjugating enzymes in medically important parasitic protozoa EUKARYOTIC CELL Ponder, E. L., Bogyo, M. 2007; 6 (11): 1943-1952

  View details for DOI 10.1128/EC.00282-07

  View details for Web of Science ID 000251410200002

  View details for PubMedID 17905920

  View details for PubMedCentralID PMC2168404

• Proteomics evaluation of chemically cleavable activity-based probes MOLECULAR & CELLULAR PROTEOMICS Fonovic, M., Verhelst, S. H., Sorum, M. T., Bogyo, M. 2007; 6 (10): 1761-1770

  Abstract

  Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.

  View details for DOI 10.1074/mcp.M700124-MCP200

  View details for Web of Science ID 000250092600009

  View details for PubMedID 17615255

• Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes NATURE CHEMICAL BIOLOGY Blum, G., von Degenfeld, G., Merchant, M. J., Blau, H. M., Bogyo, M. 2007; 3 (10): 668-677

  Abstract

  We have generated a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit a fluorescent signal only after covalently modifying a specific protease target. After intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive, whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to identify specific proteases and to correlate their activity with whole-body images. Finally, we demonstrate that these probes can be used to monitor small-molecule inhibition of protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo.

  View details for Web of Science ID 000249642700017

  View details for PubMedID 17828252

• Finding the needles in the haystack: mapping constitutive proteolytic events in vivo. Biochemical journal Bogyo, M. 2007; 407 (1): e1-2

  Abstract

  Our quest to understand the complex inner workings of the cell depends on the development of new technologies that allow the study of global regulatory events as they happen within their native cellular environment. Post-translational processing of proteins by proteases is one such regulatory process that can control many aspects of basic cell biology. In this issue of the Biochemical Journal, Timmer et al. describe a new proteomic approach that can be used to globally monitor constitutive proteolytic events in vivo. Using bacterial, human, yeast and mouse cells, the authors show that this methodology provides a comprehensive map of constitutive trimming events mediated by regulatory proteases such as methionine aminopeptidase. This study also identifies previously uncharacterized processing events that highlight potential novel regulatory mechanisms mediated by proteolysis.

  View details for PubMedID 17822382

• Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation MOLECULAR CANCER RESEARCH Goulet, B., Sansregret, L., Leduy, L., Bogyo, M., Weber, E., Chauhan, S. S., Nepveu, A. 2007; 5 (9): 899-907

  Abstract

  It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non-cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus.

  View details for DOI 10.1158/1541-7786.MCR-07-0160

  View details for Web of Science ID 000249512000004

  View details for PubMedID 17855659

• Insulin-like growth factor II receptor-mediated intracellular retention of cathepsin B is essential for transformation of endothelial cells by Kaposi's sarcoma-associated herpesvirus JOURNAL OF VIROLOGY Rose, P. P., Bogyo, M., Moses, A. V., Fruh, K. 2007; 81 (15): 8050-8062

  Abstract

  Kaposi's sarcoma-associated herpesvirus (KSHV) is the pathological agent of Kaposi's sarcoma (KS), a tumor characterized by aberrant proliferation of endothelial-cell-derived spindle cells. Since in many cancers tumorigenesis is associated with an increase in the activity of the cathepsin family, we studied the role of cathepsins in KS using an in vitro model of KSHV-mediated endothelial cell transformation. Small-molecule inhibitors and small interfering RNA (siRNA) targeting CTSB, but not other cathepsins, inhibited KSHV-induced postconfluent proliferation and the formation of spindle cells and foci of dermal microvascular endothelial cells. Interestingly, neither CTSB mRNA nor CTSB protein levels were induced in endothelial cells latently infected with KSHV. Secretion of CTSB was strongly diminished upon KSHV infection. Increased targeting of CTSB to endosomes was caused by the induction by KSHV of the expression of insulin-like growth factor-II receptor (IGF-IIR), a mannose-6-phosphate receptor (M6PR) that binds to cathepsins. Inhibition of IGF-IIR/M6PR expression by siRNA released CTSB for secretion. In contrast to the increased cathepsin secretion observed in most other tumors, viral inhibition of CTSB secretion via induction of an M6PR is crucial for the transformation of endothelial cells.

  View details for DOI 10.1128/JVI.00249-07

  View details for PubMedID 17507477

• Inhibition of cysteine cathepsin protease activity enhances chemotherapy regimens by decreasing tumor growth and invasiveness in a mouse model of multistage cancer CANCER RESEARCH Bell-McGuinn, K. M., Garfall, A. L., Bogyo, M., Hanahan, D., Joyce, J. A. 2007; 67 (15): 7378-7385

  Abstract

  Increases in protease expression and activity are associated with malignant progression and poor patient prognosis in a number of human cancers. Members of the papain family of cysteine cathepsins are among the protease classes that have been functionally implicated in cancer. Inhibition of the cysteine cathepsin family using a pan-cathepsin inhibitor, JPM-OEt, led to tumor regression in the RIP1-Tag2 (RT2) mouse model of pancreatic islet cell tumorigenesis. The present study was designed to determine whether this cathepsin inhibitor, when used in combination with chemotherapy, would increase antitumor efficacy. RT2 mice were treated in a late-stage regression trial with three different chemotherapy regimens, alone or in combination with the cathepsin inhibitor, JPM-OEt. Cyclophosphamide was administered in either a maximum tolerated dose (MTD) regimen, a "metronomic" continuous low-dose regimen, or a "chemo-switch" regimen consisting of MTD followed by metronomic dosing. Mice were sacrificed at a defined end point and tumor burden was assessed followed by a detailed analysis of cell proliferation, apoptosis, vascularization, and invasiveness in the treated and control lesions. An additional cohort of mice was followed for survival analysis. The cathepsin inhibitor plus the chemo-switch regimen of cyclophosphamide led to the most pronounced reduction in tumor burden and greatest increase in overall survival. Cysteine cathepsin inhibition resulted in a significant decrease in tumor invasiveness, which was further augmented in combination with each of the chemotherapy dosing regimens. These results encourage the development and continuing evaluation of cysteine cathepsin inhibitors as cancer therapeutics.

  View details for DOI 10.1158/0008-5472.CAN-07-0602

  View details for Web of Science ID 000248529300041

  View details for PubMedID 17671208

• IrAE - An asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus INTERNATIONAL JOURNAL FOR PARASITOLOGY Sojka, D., Hajdusek, O., Dvorak, J., Sajid, M., Franta, Z., Schneider, E. L., Craik, C. S., Vancova, M., Buresova, V., Bogyo, M., Sexton, K. B., McKerrow, J. H., Caffrey, C. R., Kopacek, P. 2007; 37 (7): 713-724

  Abstract

  Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite's ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterised a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which we believe is the first such characterisation of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and EM localised IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH > or = 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 -- an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.

  View details for DOI 10.1016/j.ijpara.2006.12.020

  View details for Web of Science ID 000246870100002

  View details for PubMedID 17336985

  View details for PubMedCentralID PMC2587490

• Design, synthesis, and evaluation of in vivo potency and selectivity of epoxysuccinyl-based inhibitors of papain-family cysteine proteases CHEMISTRY & BIOLOGY Sadaghiani, A. M., Verhelst, S. H., Gocheva, V., Hill, K., Majerova, E., Stinson, S., Joyce, J. A., Bogyo, M. 2007; 14 (5): 499-511

  Abstract

  The papain-family cathepsins are cysteine proteases that are emerging as promising therapeutic targets for a number of human disease conditions ranging from osteoporosis to cancer. Relatively few selective inhibitors for this family exist, and the in vivo selectivity of most existing compounds is unclear. We present here the synthesis of focused libraries of epoxysuccinyl-based inhibitors and their screening in crude tissue extracts. We identified a number of potent inhibitors that display selectivity for endogenous cathepsin targets both in vitro and in vivo. Importantly, the selectivity patterns observed in crude extracts were generally retained in vivo, as assessed by active-site labeling of tissues from treated animals. Overall, this study identifies several important compound classes and highlights the use of activity-based probes to assess pharmacodynamic properties of small-molecule inhibitors in vivo.

  View details for DOI 10.1016/j.chembiol.2007.03.010

  View details for Web of Science ID 000246946000005

  View details for PubMedID 17524981

• Influenza A virus elevates active cathepsin B in primary murine DC INTERNATIONAL IMMUNOLOGY Burster, T., Giffon, T., Dahl, M. E., Bjorck, P., Bogy, M., Weber, E., Mahmood, K., Lewis, D. B., Mellins, E. D. 2007; 19 (5): 645-655

  Abstract

  Dendritic cells (DCs) act as a first-line recognition system for invading pathogens, such as influenza A. The interaction of DC with influenza A virus results in DC activation via endosomal Toll-like receptors and also leads to presentation of viral peptides on MHC class II molecules. Prior work demonstrated that influenza A virus (A/HKx31; H3N2) infection of BALB/c mice activates lung DCs for antigen presentation, and that the enhanced function of these cells persists long after viral clearance and resolution of the virus-induced inflammatory response. Whether influenza A virus has acute or longer-lasting effects on the endo/lysosomal antigen-processing machinery of DCs has not been studied. Here, we show that antigen presentation from intact protein antigen, but not peptide presentation, results in increased T cell stimulation by influenza-exposed lung DCs, suggesting increased antigen processing/loading in these DCs. We find that cathepsin (Cat) B levels and activity are substantially up-regulated in murine lung DCs, harvested 30 days after A/HKx31 infection. CatB levels and activity are also increased in murine splenic and bone marrow-derived DCs, following short-term in vitro exposure to UV-inactivated influenza A virus. Modest effects on CatX are also seen during in vivo and in vitro exposure to influenza A virus. Using a cell permeable Cat inhibitor, we show Cats in influenza-exposed DCs to be functional and required for generation of a T cell epitope from intact ovalbumin. Our findings indicate that influenza A virus affects the MHC class II antigen-processing pathway, an essential pathway for CD4(+) T cell activation.

  View details for DOI 10.1093/intimm/dxm030

  View details for Web of Science ID 000246964500007

  View details for PubMedID 17446210

• Specificity of aza-peptide electrophile activity-based probes of caspases CELL DEATH AND DIFFERENTIATION Sexton, K. B., Kato, D., Berger, A. B., Fonovic, M., Verhelst, S. H., Bogyo, M. 2007; 14 (4): 727-732

  Abstract

  Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here.

  View details for DOI 10.1038/sj.cdd.4402074

  View details for Web of Science ID 000245102900009

  View details for PubMedID 17170749

• Development of calpain-specific inactivators by screening of positional scanning epoxide libraries JOURNAL OF BIOLOGICAL CHEMISTRY Cuerrier, D., Moldoveanu, T., Campbell, R. L., Kelly, J., Yoruk, B., Verhelst, S. H., Greenbaum, D., Bogyo, M., Davies, P. L. 2007; 282 (13): 9600-9611

  Abstract

  Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

  View details for DOI 10.1074/jbc.M610372200

  View details for Web of Science ID 000245421700033

  View details for PubMedID 17218315

• Tagging and detection strategies for activity-based proteomics CURRENT OPINION IN CHEMICAL BIOLOGY Sadaghiani, A. M., Verhelst, S. H., Bogyo, M. 2007; 11 (1): 20-28

  Abstract

  The field of activity-based proteomics is a relatively new discipline that makes use of small molecules, termed activity-based probes (ABPs), to tag and monitor distinct sets of proteins within a complex proteome. These activity-dependant labels facilitate analysis of systems-wide changes at the level of enzyme activity rather than simple protein abundance. While the use of small molecule inhibitors to label enzyme targets is not a new concept, the past ten years have seen a rapid expansion in the diversity of probe families that have been developed. In addition to increasing the number and types of enzymes that can be targeted by this method, there has also been an increase in the number of methods used to visualize probes once they are bound to target enzymes. In particular, the use of small organic fluorophores has created a wealth of applications for ABPs that range from biochemical profiling of diverse proteomes to direct imaging of active enzymes in live cells and even whole animals. In addition, the advent of new bioorthogonal coupling chemistries now enables a diverse array of tags to be added after targets are labeled with an ABP. This strategy has opened the door to new in vivo applications for activity-based proteomic methods.

  View details for DOI 10.1016/j.cbpa.2006.11.030

  View details for Web of Science ID 000244807600004

  View details for PubMedID 17174138

• From genes to function: advances in applications of chemical and systems biology CURRENT OPINION IN CHEMICAL BIOLOGY Bogyo, M., Cravatt, B. F. 2007; 11 (1): 1-3

  View details for DOI 10.1016/j.cbpa.2006.12.029

  View details for Web of Science ID 000244807600001

• Cysteine protease inhibitors block Toxoplasma gondii microneme secretion and cell invasion ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Teo, C. F., Zhou, X. W., Bogyo, M., Carruthers, V. B. 2007; 51 (2): 679-688

  Abstract

  Toxoplasma gondii enters host cells via an active, self-driven process to fulfill its need for intracellular replication and survival. Successful host cell invasion is governed by sequential release of secretory proteins from three specialized organelles, including the micronemes, which contribute adhesive proteins necessary for parasite attachment and penetration. Cumulative evidence from studies of Trypanosoma species and malaria parasites has shown that cysteine protease inhibitors represent potent anti-parasitic agents capable of curing infections in vivo. In this study, we screened a series of selective cysteine protease inhibitors for their effects on T. gondii cell invasion. Two of these compounds, morpholinourea-leucyl-homophenolalaninyl-phenyl-vinyl-sulfone and N-benzoxycarbonyl-(leucyl)3-phenyl-vinyl-sulfone, impaired T. gondii invasion and gliding motility at low-micromolar concentrations. Unexpectedly, these inhibitors did not affect surface proteolysis of microneme products but instead impaired an earlier step by precluding the secretion of microneme-derived adhesins to the parasite surface. Our findings suggest that cysteine protease activity is required for microneme secretion and cell invasion by T. gondii.

  View details for DOI 10.1128/AAC.01059-06

  View details for Web of Science ID 000243900600039

  View details for PubMedID 17145790

  View details for PubMedCentralID PMC1797762

• Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl endopeptidase (AEP)/legumain BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Sexton, K. B., Witte, M. D., Blum, G., Bogyo, M. 2007; 17 (3): 649-653

  Abstract

  Asparaginyl endopeptidase (AEP), also known as legumain, is a cysteine protease that has been ascribed roles in antigen presentation yet its exact role in human biology remains poorly understood. We report here, the use of a positional scanning combinatorial library of peptide AOMKs containing a P1 aspartic acid to probe the P2, P3, and P4 subsite specificity of endogenous legumain. Using inhibitor specificity profiles of cathepsin B and legumain, we designed fluorescent ABPs that are highly selective, cell-permeable reagents for monitoring legumain activity in complex proteomes.

  View details for DOI 10.1016/j.bmcl.2006.10.100

  View details for Web of Science ID 000244170700015

  View details for PubMedID 17189693

  View details for PubMedCentralID PMC1832115

• Activity based probes for proteases: Applications to biomarker discovery, molecular imaging and drug screening CURRENT PHARMACEUTICAL DESIGN Fonovic, M., Bogyo, M. 2007; 13 (3): 253-261

  Abstract

  Recent advances in global genomic and proteomic methods have lead to a greater understanding of how genes and proteins function in complex networks within a cell. One of the major limitations in these methodologies is their inability to provide information on the dynamic, post-translational regulation of enzymatic proteins. In particular proteases are often synthesized as inactive zymogens that need to be activated in order to carry out specific biological processes. Thus, methods that allow direct monitoring of protease activity in the context of a living cell or whole animal will be required to begin to understand the systems-wide functional roles of proteases. In this review, we discuss the development and applications of activity based probes (ABPs) to study proteases and their role in pathological processes. Specifically we focus on application of this technique for biomarker discovery, in vivo imaging and drug screening.

  View details for Web of Science ID 000244880000002

  View details for PubMedID 17313359

• A mild chemically cleavable linker system for functional proteomic applications ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Verhelst, S. H., Fonovic, M., Bogyo, M. 2007; 46 (8): 1284-1286

  View details for DOI 10.1002/anie.200603811

  View details for Web of Science ID 000244382200017

  View details for PubMedID 17205587

• Commonly used caspase inhibitors designed based on substrate specificity profiles lack selectivity CELL RESEARCH Berger, A. B., Sexton, K. B., Bogyo, M. 2006; 16 (12): 961-963

  View details for DOI 10.1038/sj.cr.7310112

  View details for Web of Science ID 000243777300006

  View details for PubMedID 17117159

• Solid-phase methods for the preparation of epoxysuccinate-based inhibitors of cysteine proteases JOURNAL OF COMBINATORIAL CHEMISTRY Sadaghiani, A. M., Verhelst, S. H., Bogyo, M. 2006; 8 (6): 802-804

  View details for DOI 10.1021/cc0601027

  View details for Web of Science ID 000241942100002

  View details for PubMedID 17096566

• Falstatin, a cysteine protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion PLOS PATHOGENS Pandey, K. C., Singh, N., Arastu-Kapur, S., Bogyo, M., Rosenthal, P. J. 2006; 2 (11): 1031-1041

  Abstract

  Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.

  View details for DOI 10.1371/journal.ppat.0020117

  View details for Web of Science ID 000242787100003

  View details for PubMedID 17083274

  View details for PubMedCentralID PMC1630708

• Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes MOLECULAR CELL Berger, A. B., Witte, M. D., Denault, J., Sadaghiani, A. M., Sexton, K. M., Salvesen, G. S., Bogyo, M. 2006; 23 (4): 509-521

  Abstract

  Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.

  View details for DOI 10.1016/j.molcel.2006.06.021

  View details for Web of Science ID 000240155000007

  View details for PubMedID 16916639

• Engineered hybrid dimers: Tracking the activation pathway of caspase-7 MOLECULAR CELL Denault, J., Bekes, M., Scott, F. L., Sexton, K. M., Bogyo, M., Salvesen, G. S. 2006; 23 (4): 523-533

  Abstract

  Caspase-7 is an obligate dimer of catalytic domains, with generation of activity requiring limited proteolysis within a region that separates the large and small chains of each domain. Using hybrid dimers we distinguish the relative contribution of each domain to catalysis by the whole molecule. We demonstrate that the zymogen arises from direct dimerization and not domain swapping. In contrast to previous conclusions, we show that only one of the catalytic domains must be proteolyzed to enable activation. The processed domain of this singly cleaved zymogen has the same catalytic activity as a domain of fully active caspase-7. A transient intermediate of singly cleaved dimeric caspase-7 can be found in a cell-free model of apoptosis induction. However, we see no evidence for an analogous intermediate of the related executioner caspase-3. Our study demonstrates the efficiency by which the executioner caspases are activated in vivo.

  View details for DOI 10.1016/j.molcel.2006.06.020

  View details for Web of Science ID 000240155000008

  View details for PubMedID 16916640

• Novel aza peptide inhibitors and active-site probes of papain-family cysteine proteases CHEMBIOCHEM Verhelst, S. H., Witte, M. D., Arastu-Kapur, S., Fonovic, M., Bogyo, M. 2006; 7 (6): 943-950

  Abstract

  Recent characterization of multiple classes of functionalized azapeptides as effective covalent inhibitors of cysteine proteases prompted us to investigate O-acyl hydroxamates and their azapeptide analogues for use as activity-based probes (ABPs). We report here a new class of azaglycine-containing O-acylhydroxamates that form stable covalent adducts with target proteases. This allows them to be used as ABPs for papain family cysteine proteases. A second class of related analogues containing a novel O-acyl hydroxyurea warhead was found to function as covalent inhibitors of papain-like proteases. These inhibitors can be easily synthesized on solid support, which allows rapid optimization of compounds with improved selectivity and potency for a given target enzyme. We present here one such optimized inhibitor that showed selective inhibition of falcipain 1, a protease of the malaria-causing parasite, Plasmodium falciparum.

  View details for DOI 10.1002/cbic.200600001

  View details for Web of Science ID 000238171400014

  View details for PubMedID 16607671

• Development of activity-based probes for trypsin-family serine proteases BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Pan, Z. Y., Jeffery, D. A., Chehade, K., Beltman, J., Clark, J. M., Grothaus, P., Bogyo, M., Baruch, A. 2006; 16 (11): 2882-2885

  Abstract

  A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.

  View details for DOI 10.1016/j.bmcl.2006.03.012

  View details for Web of Science ID 000237407800011

  View details for PubMedID 16554154

• Tumor cell-derived and macrophage-derived cathepsin B promotes progression and lung metastasis of mammary cancer CANCER RESEARCH Vasiljeva, O., Papazoglou, A., Krueger, A., Brodoefel, H., Korovin, M., Deussing, J., Augustin, N., Nielsen, B. S., Almholt, K., Bogyo, M., Peters, C., Reinheckel, T. 2006; 66 (10): 5242-5250

  Abstract

  Proteolysis in close vicinity of tumor cells is a hallmark of cancer invasion and metastasis. We show here that mouse mammary tumor virus-polyoma middle T antigen (PyMT) transgenic mice deficient for the cysteine protease cathepsin B (CTSB) exhibited a significantly delayed onset and reduced growth rate of mammary cancers compared with wild-type PyMT mice. Lung metastasis volumes were significantly reduced in PyMT;ctsb(+/-), an effect that was not further enhanced in PyMT;ctsb(-/-) mice. Furthermore, lung colonization studies of PyMT cells with different CTSB genotypes injected into congenic wild-type mice and in vitro Matrigel invasion assays confirmed a specific role for tumor-derived CTSB in invasion and metastasis. Interestingly, cell surface labeling of cysteine cathepsins by the active site probe DCG-04 detected up-regulation of cathepsin X on PyMT;ctsb(-/-) cells. Treatment of cells with a neutralizing anti-cathepsin X antibody significantly reduced Matrigel invasion of PyMT;ctsb(-/-) cells but did not affect invasion of PyMT;ctsb(+/+) or PyMT;ctsb(+/-) cells, indicating a compensatory function of cathepsin X in CTSB-deficient tumor cells. Finally, an adoptive transfer model, in which ctsb(+/+), ctsb(+/-), and ctsb(-/-) recipient mice were challenged with PyMT;ctsb(+/+) cells, was used to address the role of stroma-derived CTSB in lung metastasis formation. Notably, ctsb(-/-) mice showed reduced number and volume of lung colonies, and infiltrating macrophages showed a strongly up-regulated expression of CTSB within metastatic cell populations. These results indicate that both cancer cell-derived and stroma cell-derived (i.e., macrophages) CTSB plays an important role in tumor progression and metastasis.

  View details for DOI 10.1158/0008-5472.CAN-05-4463

  View details for Web of Science ID 000237679900034

  View details for PubMedID 16707449

• Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities JOURNAL OF BIOLOGICAL CHEMISTRY Choe, Y., Leonetti, F., Greenbaum, D. C., Lecaille, F., Bogyo, M., Bromme, D., Ellman, J. A., Craik, C. S. 2006; 281 (18): 12824-12832

  Abstract

  The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.

  View details for DOI 10.1074/jbc.M513331200

  View details for Web of Science ID 000237134700077

  View details for PubMedID 16520377

• A selective activity-based probe for the papain family cysteine protease dipeptidyl peptidase I cathepsin C JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yuan, F., Verhelst, S. H., Blum, G., Coussens, L. M., Bogyo, M. 2006; 128 (17): 5616-5617

  Abstract

  Dipeptidyl peptidase I is involved in the activation of a number of disease-related proteases by removal of N-terminal prodipeptides. We here report a selective activity-based probe for monitoring dipeptidyl peptidase I activity in whole proteomes as well as in intact cells, without labeling of closely related enzyme family members.

  View details for DOI 10.1021/ja060835v

  View details for Web of Science ID 000237389900014

  View details for PubMedID 16637611

• Metalloproteases see the light NATURE CHEMICAL BIOLOGY Bogyo, M. 2006; 2 (5): 229-230

  View details for Web of Science ID 000236957900004

  View details for PubMedID 16619018

• Cysteine cathepsin inhibition in combination with a maximum-tolerated dose and metronomic dose "chemo-switch" regimen leads to tumor regression and an overall survival benefit in a murine model of cancer Bell-Mcguinn, K. M., Bogyo, M., Hanahan, D., Joyce, J. AMER ASSOC CANCER RESEARCH. 2006

  View details for Web of Science ID 000454606204276

• Inhibitors of cathepsin B reduce production of beta-amyloid in regulated secretory vesicles: A novel cysteine protease pathway as beta-secretase for generating beta-amyloid of Alzheimer's disease Hook, Toneff, T., Bogyo, M., Greenbaum, D., Lane, W., Hook, G., Reisine, T. FEDERATION AMER SOC EXP BIOL. 2006: A1135

  View details for Web of Science ID 000236326202434

• A general solid phase method for the preparation of diverse azapeptide probes directed against cysteine proteases ORGANIC LETTERS Kato, D., Verhelst, S. H., Sexton, K. B., Bogyo, M. 2005; 7 (25): 5649-5652

  Abstract

  [chemical reaction: see text]. A solid phase approach is presented for the synthesis of azapeptide inhibitors and activity based probes (ABPs) for cysteine proteases. This synthetic method allows the incorporation of diverse reactive warheads linked to different peptide recognition elements. Application of this method to the synthesis of a series of caspase probes is described.

  View details for DOI 10.1021/ol052275v

  View details for Web of Science ID 000233778300027

  View details for PubMedID 16321013

• Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease (vol 386, pg 931, 2005) BIOLOGICAL CHEMISTRY Hook, Toneff, T., Bogyo, M., Greenbaum, D., Medzihradszky, K. F., Neveu, J., Lane, W., Hook, G., Reisine, T. 2005; 386 (12): 1325

  View details for DOI 10.1515/BC.2005.151

  View details for Web of Science ID 000233965400014

• Dynamic imaging of protease activity with fluorescently quenched activity-based probes NATURE CHEMICAL BIOLOGY Blum, G., Mullins, S. R., Keren, K., Fonovic, M., Jedeszko, C., RICE, M. J., Sloane, B. F., Bogyo, M. 2005; 1 (4): 203-209

  Abstract

  Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.

  View details for DOI 10.1038/nchembo728

  View details for Web of Science ID 000232649000011

  View details for PubMedID 16408036

• Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease BIOLOGICAL CHEMISTRY Hook, V., Toneff, T., Bogyo, M., Greenbaum, D., Medzihradszky, K. R., Neveu, J., Lane, W., Hook, G., Reisine, T. 2005; 386 (9): 931-940

  Abstract

  The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.

  View details for DOI 10.1515/BC.2005.108

  View details for Web of Science ID 000232274100012

  View details for PubMedID 16164418

• Proteomics meets microbiology: technical advances in the global mapping of protein expression and function CELLULAR MICROBIOLOGY Phillips, C. I., Bogyo, M. 2005; 7 (8): 1061-1076

  Abstract

  The availability of complete genome sequences for a large number of pathogenic organisms has opened the door for large-scale proteomic studies to dissect both protein expression/regulation and function. This review highlights key proteomic methods including two-dimensional gel electrophoresis, reference mapping, protein expression profiling and recent advances in gel-free separation techniques that have made a significant impact on the resolution of complex proteomes. In addition, we highlight recent developments in the field of chemical proteomics, a branch of proteomics aimed at functionally profiling a proteome. These techniques include the development of activity-based probes and activity-based protein profiling methods as well as the use of synthetic small molecule libraries to screen for pharmacological tools to perturb basic biological processes. This review will focus on the applications of these technologies to the field of microbiology.

  View details for DOI 10.1111/j.1462-5822.2005.00554.x

  View details for Web of Science ID 000230343300002

  View details for PubMedID 16008574

• Activity-based probes that target diverse cysteine protease families NATURE CHEMICAL BIOLOGY Kato, D., Boatright, K. M., Berger, A. B., Nazif, T., Blum, G., Ryan, C., Chehade, K. A., Salvesen, G. S., Bogyo, M. 2005; 1 (1): 33-38

  Abstract

  Proteases are one of the largest and best-characterized families of enzymes in the human proteome. Unfortunately, the understanding of protease function in the context of complex proteolytic cascades remains in its infancy. One major reason for this gap in understanding is the lack of technologies that allow direct assessment of protease activity. We report here an optimized solid-phase synthesis protocol that allows rapid generation of activity-based probes (ABPs) targeting a range of cysteine protease families. These reagents selectively form covalent bonds with the active-site thiol of a cysteine protease, allowing direct biochemical profiling of protease activities in complex proteomes. We present a number of probes containing either a single amino acid or an extended peptide sequence that target caspases, legumains, gingipains and cathepsins. Biochemical studies using these reagents highlight their overall utility and provide insight into the biochemical functions of members of these protease families.

  View details for DOI 10.1038/nchembio707

  View details for Web of Science ID 000232621100010

  View details for PubMedID 16407991

• Solid-phase synthesis of double-headed epoxysuccinyl activity-based probes for selective targeting of papain family cysteine proteases CHEMBIOCHEM Verhelst, S. H., Bogyo, M. 2005; 6 (5): 824-?

  View details for DOI 10.1002/cbic.200400377

  View details for Web of Science ID 000229171800009

  View details for PubMedID 15776409

• Dissecting protein function using chemical proteomic methods QSAR & COMBINATORIAL SCIENCE Verhelst, S. H., Bogyo, M. 2005; 24 (2): 261-269

  View details for DOI 10.1002/qsar.200420070

  View details for Web of Science ID 000228251600008

• Chemical proteomics applied to target identification and drug discovery BIOTECHNIQUES Verhelst, S. H., Bogyo, M. 2005; 38 (2): 175-177

  View details for Web of Science ID 000226996200002

  View details for PubMedID 15727120

• Small-molecule inhibitors and probes for ubiquitin- and ubiquitin-like-specific proteases CHEMBIOCHEM Borodovsky, A., Ovaa, H., Meester, W. J., Venanzi, E. S., Bogyo, M. S., Hekking, B. G., Ploegh, H. L., Kessler, B. M., Overkleeft, H. S. 2005; 6 (2): 287-291

  View details for DOI 10.1002/cbic.200400236

  View details for Web of Science ID 000226957100008

  View details for PubMedID 15651044

• An improved preparation of the activity-based probe JPM-OEt and in situ applications SYNTHESIS-STUTTGART Chehade, K. A., Baruch, A., Verhelst, S. H., Bogyo, M. 2005: 240-244

  View details for DOI 10.1055/s-2004-837295

  View details for Web of Science ID 000226842300010

• Screening for selective small molecule inhibitors of the proteasome using activity-based probes UBIQUITIN AND PROTEIN DEGRADATION, PT B Bogyo, M. 2005; 399: 609-?

  Abstract

  The proteasome's role in fundamental biological processes ranging from control of the cell cycle to production of peptides for display to immune cells has been uncovered with the help of small molecule inhibitors. Most of the commonly used inhibitors have been designed and synthesized by organic chemists or by Nature. To continue to develop new inhibitors and reagents for the proteasome, a rapid screening method is required that allows not only assessment of potency but also selectivity of inhibitors for each of the primary catalytic sites in the complex. This chapter outlines methods for the solid-phase synthesis of diverse peptide vinyl sulfone libraries and a rapid screen for potent and selective inhibitors that makes use of an active site label (Nazif and Bogyo, 2001). This assay can be performed with small quantities of total cellular extracts as a source of enzyme and can be used to rapidly screen virtually any potential inhibitor.

  View details for DOI 10.1016/S0076-6879(05)99040-X

  View details for Web of Science ID 000233597300040

  View details for PubMedID 16338384

• Imaging protease activity using novel fluorescently quenched probes Blum, G., von Degenfeld, G., Keren, K., Blau, H. M., Bogyo, M. JOHN WILEY & SONS INC. 2005: 501

  View details for Web of Science ID 000229901200082

• Selective targeting of papain-like proteases by double-headed epoxysuccinyl probes 19th American Peptide Symposium Verhelst, S. H., Sadaghiani, A. M., Arastu-Kapur, S., Bogyo, M. JOHN WILEY & SONS INC. 2005: 603–

  View details for Web of Science ID 000229901200555

• Characteristics of the caspase-like catalytic domain of human paracaspase BIOLOGICAL CHEMISTRY Snipas, S. J., Wildfang, E., Nazif, T., Christensen, L., Boatright, K. M., Bogyo, M., Stennicke, H. R., Salvesen, G. S. 2004; 385 (11): 1093-1098

  Abstract

  Human paracaspase has been predicted to be a member of the protein structural fold that encompasses protease clan CD. To determine whether paracaspase has catalytic activity we have expressed the region corresponding to the catalytic domain and used protease activity-based chemical probes to profile the putative active site. A leucine-based acyloxymethyl ketone probe that covalently labels cysteine proteases discloses a hydrophobic P 1 preference in the putative active site. The probe covalently labels Cys539, which is not the predicted catalytic site based on structural and sequence comparisons with other clan CD proteases. Using a combinatorial peptide substrate library approach we have been unable to detect amidolytic activity of paracaspase, implying that if it is a protease it must be very specific. We suggest a switch in the use of catalytic residues to generate an enzyme overlapping the canonical clan CD protease active site.

  View details for DOI 10.1515/BC.2004.142

  View details for Web of Science ID 000225438200015

  View details for PubMedID 15576331

• Applications for chemical probes of proteolytic activity. Current protocols in protein science / editorial board, John E. Coligan ... [et al.] Bogyo, M., Baruch, A., Jeffery, D. A., Greenbaum, D., Borodovsky, A., Ovaa, H., Kessler, B. 2004; Chapter 21: Unit 21 17-?

  Abstract

  Recent genome sequencing projects have identified new peptidases in multiple organisms, many with unknown functions, suggesting the need for new tools to study these enzymes. This unit outlines selection and use of small-molecule and protein-based probes to covalently modify peptidases in complex cellular environments. These activity-based probes (ABPs) have been designed based on well characterized peptidase inhibitor scaffolds, but make use of new techniques to greatly enhance their utility for studying families of related peptidases. In particular, ABPs can be used to track activity of peptidases in crude cell extracts, intact cells, and in vivo, allowing rapid purification and identification of labeled targets. They can be used with libraries of small molecules to rapidly assess potency and selectivity of compounds in complex, physiologically relevant samples. Probe selection, probe tagging using reporters, labeling of recombinant targets, crude protein extracts, and peptidase targets in cell culture systems, affinity purification of targets, and inhibitor screening using affinity probes are outlined.

  View details for DOI 10.1002/0471140864.ps2117s36

  View details for PubMedID 18429259

• Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages JOURNAL OF BIOLOGICAL CHEMISTRY Yasuda, Y., Li, Z. Q., Greenbaum, D., Bogyo, M., Weber, E., Bromme, D. 2004; 279 (35): 36761-36770

  Abstract

  Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.

  View details for DOI 10.1074/jbc.M403986200

  View details for Web of Science ID 000223453600077

  View details for PubMedID 15192101

• Chemical probes for proteomic analysis of cysteine protease function. Bogyo, M., Greenbaum, D., Hanahan, D., Joyce, J., Baruch, A., Hunter, C., Vitorino, P. AMER CHEMICAL SOC. 2004: U181

  View details for Web of Science ID 000223712800759

• Targeted disruption of Plasmodium falciparum cysteine protease, falcipain 1, reduces oocyst production, not erythrocytic stage growth MOLECULAR MICROBIOLOGY Eksi, S., Czesny, B., Greenbaum, D. C., Bogyo, M., Williamson, K. C. 2004; 53 (1): 243-250

  Abstract

  Cysteine proteases are currently targets for drug development in a number of parasitic diseases, including malaria. In Plasmodium falciparum, the parasite responsible for the most virulent form of human malaria, there are four members of the cathepsin L-like family of cysteine proteases. Three of these (falcipains 2A, 2B and 3) are thought to be primarily involved in haemoglobin digestion, whereas falcipain 1 has recently been linked to erythrocyte invasion. Neither their expression nor their role in P. falciparum gametocytogenesis, which is required for malaria transmission, has been evaluated. In this study, RNA transcripts for the falcipain family members were identified as the parasite developed through all five stages of gametocytogenesis. Falcipain 1 transcript was upregulated in gametocytes, while levels of falcipain 2A/2B decreased in late-stage gametocytes and gametes. To evaluate the function of falcipain 1, the gene was disrupted, and clones from independent transformations were isolated. The asexual growth of the falcipain 1 minus clones was not overtly affected, and they produced morphologically normal gametocytes and gametes. However, when falcipain 1 minus parasites were fed to a mosquito, oocyst production was reduced by 70-90%, suggesting an important role for falcipain 1 during parasite development in the mosquito midgut.

  View details for DOI 10.1111/j.1365-2958.2004.04108.x

  View details for Web of Science ID 000222208100021

  View details for PubMedID 15225318

• Activity profiling of papain-like cysteine proteases in plants PLANT PHYSIOLOGY van der Hoorn, R. A., Leeuwenburgh, M. A., Bogyo, M., Joosten, M. H., Peck, S. C. 2004; 135 (3): 1170-1178

  Abstract

  Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.

  View details for Web of Science ID 000222692700004

  View details for PubMedID 15266051

  View details for PubMedCentralID PMC519038

• Growth phase-dependent production of a cell wall-associated elastinolytic cysteine proteinase by Staphylococcus epidermidis 3rd General Meeting of the International-Proteolysis-Society/International Conference on Protease Inhibitors Oleksy, A., Golonka, E., Banbula, A., Szmyd, G., Moon, J., Kubica, M., Greenbaum, D., Bogyo, M., Foster, T. J., Travis, J., Potempa, J. WALTER DE GRUYTER & CO. 2004: 525–35

  Abstract

  Staphylococcus epidermidis, a Gram-positive, coagulase-negative bacterium is a predominant inhabitant of human skin and mucous membranes. Recently, however, it has become one of the most important agents of hospital-acquired bacteriemia, as it has been found to be responsible for surgical wound infections developed in individuals with indwelling catheters or prosthetic devices, as well as in immunosupressed or neutropenic patients. Despite their medical significance, little is known about proteolytic enzymes of S. epidermidis and their possible contribution to the bacterium's pathogenicity; however, it is likely that they function as virulence factors in a manner similar to that proposed for the proteases of Staphylococcus aureus. Here we describe the purification of a cell wall-associated cysteine protease from S. epidermidis, its biochemical properties and specificity. A homology search using N-terminal sequence data revealed similarity to staphopain A (ScpA) and staphopain B (SspB), cysteine proteases from S. aureus. Moreover, the gene encoding S. epidermidis cysteine protease (Ecp) and a downstream gene coding for a putative inhibitor of the protease form an operon structure which resembles that of staphopain A in S. aureus. The active cysteine protease was detected on the bacterial cell surface as well as in the culture media and is apparently produced in a growth phase-dependent manner, with initial expression occurring in the mid-logarithmic phase. This enzyme, with elastinolytic properties, as well as the ability to cleave alpha1PI, fibrinogen and fibronectin, may possibly contribute to the invasiveness and pathogenic potential of S. epidermidis.

  View details for Web of Science ID 000222500200011

  View details for PubMedID 15255185

• Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones 3rd General Meeting of the International-Proteolysis-Society/International Conference on Protease Inhibitors Hook, V., Yasothornsrikul, S., Greenbaum, D., Medzihradszky, K. F., Troutner, K., Toneff, T., Bundey, R., Logrinova, A., Reinheckel, T., Peters, C., Bogyo, M. WALTER DE GRUYTER & CO. 2004: 473–80

  Abstract

  Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.

  View details for Web of Science ID 000222500200004

  View details for PubMedID 15255178

• O-sulfonation of serine and threonine - Mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes MOLECULAR & CELLULAR PROTEOMICS Medzihradszky, K. F., Darula, Z., Perlson, E., Fainzilber, M., Chalkley, R. J., Ball, H., Greenbaum, D., Bogyo, M., Tyson, D. R., BRADSHAW, R. A., Burlingame, A. L. 2004; 3 (5): 429-440

  Abstract

  Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.

  View details for DOI 10.1074/mcp.M300140-MCP200

  View details for Web of Science ID 000221242300001

  View details for PubMedID 14752058

• Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis CANCER CELL Joyce, J. A., Baruch, A., Chehade, K., Meyer-Morse, N., Giraudo, E., Tsai, F. Y., Greenbaum, D. C., Hager, J. H., Bogyo, M., Hanahan, D. 2004; 5 (5): 443-453

  Abstract

  Tumors develop through successive stages characterized by changes in gene expression and protein function. Gene expression profiling of pancreatic islet tumors in a mouse model of cancer revealed upregulation of cathepsin cysteine proteases. Cathepsin activity was assessed using chemical probes allowing biochemical and in vivo imaging, revealing increased activity associated with the angiogenic vasculature and invasive fronts of carcinomas, and differential expression in immune, endothelial, and cancer cells. A broad-spectrum cysteine cathepsin inhibitor was used to pharmacologically knock out cathepsin function at different stages of tumorigenesis, impairing angiogenic switching in progenitor lesions, as well as tumor growth, vascularity, and invasiveness. Cysteine cathepsins are also upregulated during HPV16-induced cervical carcinogenesis, further encouraging consideration of this protease family as a therapeutic target in human cancers.

  View details for Web of Science ID 000222016300008

  View details for PubMedID 15144952

• A cathepsin L isoform that is devoid of a signal peptide localizes to the nucleus in S phase and processes the CDP/Cux transcription factor MOLECULAR CELL Goulet, B., Baruch, A., Moon, N. S., Poirier, M., Sansregret, L. L., Erickson, A., Bogyo, M., Nepveu, A. 2004; 14 (2): 207-219

  Abstract

  The subclass of cysteine proteases termed lysosomal cathepsins has long been thought to be primarily involved in end-stage protein breakdown within lysosomal compartments. Furthermore, few specific protein substrates for these proteases have been identified. We show here that cathepsin L functions in the regulation of cell cycle progression through proteolytic processing of the CDP/Cux transcription factor. CDP/Cux processing in situ was increased following ectopic expression of cathepsin L but was reduced in Cat L(-/-) cells. Furthermore, catalytically active cathepsin L was localized to the nucleus during the G1-S transition as detected by immunofluorescence imaging and labeling using activity-based probes. Trafficking of cathepsin L to the nucleus is accomplished through a mechanism involving translation initiation at downstream AUG sites and the synthesis of proteases that are devoid of a signal peptide. Overall, these results uncover an as yet unsuspected role for cysteine proteases in the control of cell cycle progression.

  View details for Web of Science ID 000221051400009

  View details for PubMedID 15099520

• Regulation of collagenase activities of human cathepsins by glycosaminoglycans JOURNAL OF BIOLOGICAL CHEMISTRY Li, Z. Q., Yasuda, Y., Li, W. J., Bogyo, M., Katz, N., Gordon, R. E., Fields, G. B., Bromme, D. 2004; 279 (7): 5470-5479

  Abstract

  Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.

  View details for DOI 10.1074/jbc.M310349200

  View details for Web of Science ID 000188776500054

  View details for PubMedID 14645229

• Chemical proteomics and its application to drug discovery DRUG DISCOVERY TODAY Jeffery, D. A., Bogyo, M. 2004; 9 (2): S19-S26

  Abstract

  The completion of the human genome sequencing project has provided a flood of new information that is likely to change the way scientists approach the study of complex biological systems. A major challenge lies in translating this information into new and better ways to treat human disease. The multidisciplinary science of chemical proteomics can be used to distill this flood of new information. This approach makes use of synthetic small molecules that can be used to covalently modify a set of related enzymes and subsequently allow their purification and/or identification as valid drug targets. Furthermore, such methods enable rapid biochemical analysis and small-molecule screening of targets thereby accelerating the often difficult process of target validation and drug discovery.

  View details for Web of Science ID 000188081100003

  View details for PubMedID 23573640

• Enzyme activity - it's all about image TRENDS IN CELL BIOLOGY Baruch, A., Jeffery, D. A., Bogyo, M. 2004; 14 (1): 29-35

  Abstract

  Unraveling the functional roles of proteins is a major challenge facing the postgenome researcher. Advances towards this goal have been made through the development of both chemical and biochemical tools for monitoring protein activity. Recently, a myriad of fluorescence-based imaging tools have emerged for in vitro, in vivo and whole animal applications. These tools have provided methods to monitor the spatial and temporal distribution of proteins and bioorganic molecules dynamically. Here, recent advances in chemical and biochemical techniques that allow the detection of enzymatic activity within intact cells and in vivo are reviewed. Such technologies have the potential to be integrated into drug-development programs to facilitate both the functional validation of pharmaceutical targets and the treatment of human disease.

  View details for DOI 10.1016/j.tcb.2003.11.002

  View details for Web of Science ID 000188599000006

  View details for PubMedID 14729178

• Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. American journal of pharmacogenomics Berger, A. B., Vitorino, P. M., Bogyo, M. 2004; 4 (6): 371-381

  Abstract

  The genomic revolution has created a wealth of information regarding the fundamental genetic code that defines the inner workings of a cell. However, it has become clear that analyzing genome sequences alone will not lead to new therapies to fight human disease. Rather, an understanding of protein function within the context of complex cellular networks will be required to facilitate the discovery of novel drug targets and, subsequently, new therapies directed against them. The past ten years has seen a dramatic increase in technologies that allow large-scale, systems-based methods for analysis of global biological processes and disease states. In the field of proteomics, several well-established methods persist as a means to resolve and analyze complex mixtures of proteins derived from cells and tissues. However, the resolving power of these methods is often challenged by the diverse and dynamic nature of the proteome. The field of activity-based proteomics, or chemical proteomics, has been established in an attempt to focus proteomic efforts on subsets of physiologically important protein targets. This new approach to proteomics is centered around the use of small molecules termed activity-based probes (ABPs) as a means to tag, enrich, and isolate, distinct sets of proteins based on their enzymatic activity. Chemical probes can be 'tuned' to react with defined enzymatic targets through the use of chemically reactive warhead groups, fused to selective binding elements that control their overall specificity. As a result, ABPs function as highly specific, mechanism-based reagents that provide a direct readout of enzymatic activity within complex proteomes. Modification of protein targets by an ABP facilitates their purification and isolation, thereby eliminating many of the confounding issues of dynamic range in protein abundance. In this review, we outline recent advances in the field of chemical proteomics. Specifically, we highlight how this technology can be applied to advance the fields of biomarker discovery, in vivo imaging, and small molecule screening and drug target discovery.

  View details for PubMedID 15651898

• Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase MOLECULAR AND BIOCHEMICAL PARASITOLOGY Sajid, M., McKerrow, J. H., Hansell, E., Mathieu, M. A., Lucas, K. D., Hsieh, I., Greenbaum, D., Bogyo, M., Salter, J. P., Lim, K. C., Franklin, C., Kim, J. H., Caffrey, C. R. 2003; 131 (1): 65-75

  Abstract

  Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence. The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo. The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles. Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded. Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu). This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo.

  View details for DOI 10.1016/S0166-6851(03)00194-4

  View details for Web of Science ID 000185726000007

  View details for PubMedID 12967713

• Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yasothornsrikul, S., Greenbaum, D., Medzihradszky, K. F., Toneff, T., Bundey, R., Miller, R., Schilling, B., Petermann, I., Dehnert, J., Logvinova, A., Goldsmith, P., Neveu, J. M., Lane, W. S., Gibson, B., Reinheckel, T., Peters, C., Bogyo, M., Hook, V. 2003; 100 (16): 9590-9595

  Abstract

  Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of [Met]enkephalin by cathepsin L occurred by proteolytic processing at dibasic and monobasic prohormone-processing sites. Cellular studies showed the colocalization of cathepsin L with [Met]enkephalin in secretory vesicles of neuroendocrine chromaffin cells by immunofluorescent confocal and immunoelectron microscopy. Functional localization of cathepsin L to the regulated secretory pathway was demonstrated by its cosecretion with [Met]enkephalin. Finally, in cathepsin L gene knockout mice, [Met]enkephalin levels in brain were reduced significantly; this occurred with an increase in the relative amounts of enkephalin precursor. These findings indicate a previously uncharacterized biological role for secretory vesicle cathepsin L in the production of [Met]enkephalin, an endogenous peptide neurotransmitter.

  View details for DOI 10.1073/pnas.1531542100

  View details for Web of Science ID 000184620000087

  View details for PubMedID 12869695

• Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity CELL DEATH AND DIFFERENTIATION Rozman-Pungercar, J., Kopitar-Jerala, N., Bogyo, M., Turk, D., Vasiljeva, O., Stefe, I., Vandenabeele, P., Bromme, D., Puizdar, V., Fonovic, M., Trstenjak-Prebanda, M., Dolenc, I., Turk, V., Turk, B. 2003; 10 (8): 881-888

  Abstract

  We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

  View details for DOI 10.1038/sj.cdd.4401247

  View details for Web of Science ID 000184224100004

  View details for PubMedID 12867995

• Biochemical analysis of the 20 S proteasome of Trypanosoma brucei JOURNAL OF BIOLOGICAL CHEMISTRY Wang, C. C., Bozdech, Z., Liu, C. I., Shipway, A., Backes, B. J., Harris, J. L., Bogyo, M. 2003; 278 (18): 15800-15808

  Abstract

  We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.

  View details for DOI 10.1074/jbc.M300195200

  View details for Web of Science ID 000182680000045

  View details for PubMedID 12600991

• Sequential autolytic processing activates the zymogen of Arg-gingipain JOURNAL OF BIOLOGICAL CHEMISTRY Mikolajczyk, J., Boatright, K. M., Stennicke, H. R., Nazif, T., Potempa, J., Bogyo, M., Salvesen, G. S. 2003; 278 (12): 10458-10464

  Abstract

  Most proteases are synthesized as inactive precursors to protect the synthetic machinery of the cell and allow timing of activation. The mechanisms used to render latency are varied but tend to be conserved within protease families. Proteases belonging to the caspase family have a unique mechanism mediated by transitions of two surface loops, and on the basis of conservation of mechanism one would expect this to be preserved by caspase relatives. We have been able to express the full-length precursor of the Arg-specific caspase relative from the bacterium Porphyromonas gingivalis, Arg-gingipain-B, and we show that it contains N- and C-terminal extensions that render a low amount of latency, meaning that the zymogen is substantially active. Three sequential autolytic processing steps at the N and C terminus are required for full activity, and the N-propeptide may serve as an intramolecular chaperone rather than an inhibitory peptide. Each step in activation requires the previous step, and an affinity probe reveals that incremental activity enhancements are achieved in a stepwise manner.

  View details for DOI 10.1074/jbc.M210564200

  View details for Web of Science ID 000181777500063

  View details for PubMedID 12533545

• Pathways accessory to proteasomal proteolysis are less efficient in major histocompatibility complex class I antigen production JOURNAL OF BIOLOGICAL CHEMISTRY Kessler, B., Hong, X., Petrovic, J., Borodovsky, A., Dantuma, N. P., Bogyo, M., Overkleeft, H. S., Ploegh, H., Glas, R. 2003; 278 (12): 10013-10021

  Abstract

  Degradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal beta-subunits (beta(1), beta(2), and beta(5)) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2D(b) molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways.

  View details for DOI 10.1074/jbc.M211221200

  View details for Web of Science ID 000181777500004

  View details for PubMedID 12488316

• Chemical proteomics and its application to drug discovery CURRENT OPINION IN BIOTECHNOLOGY Jeffery, D. A., Bogyo, M. 2003; 14 (1): 87-95

  Abstract

  The completion of the human genome sequencing project has provided a flood of new information that is likely to change the way scientists approach the study of complex biological systems. A major challenge lies in translating this information into new and better ways to treat human disease. The multidisciplinary science of chemical proteomics can be used to distill this flood of new information. This approach makes use of synthetic small molecules that can be used to covalently modify a set of related enzymes and subsequently allow their purification and/or identification as valid drug targets. Furthermore, such methods enable rapid biochemical analysis and small-molecule screening of targets thereby accelerating the often difficult process of target validation and drug discovery.

  View details for DOI 10.1016/S0958-1669(02)00010-1

  View details for Web of Science ID 000181006300013

  View details for PubMedID 12566007

• A role for the protease falcipain 1 in host cell invasion by the human malaria parasite SCIENCE Greenbaum, D. C., Baruch, A., Grainger, M., Bozdech, Z., Medzihradszky, K. F., Engel, J., DeRisi, J., Holder, A. A., Bogyo, M. 2002; 298 (5600): 2002-2006

  Abstract

  Cysteine proteases of Plasmodium falciparum are required for survival of the malaria parasite, yet their specific cellular functions remain unclear. We used a chemical proteomic screen with a small-molecule probe to characterize the predominant cysteine proteases throughout the parasite life cycle. Only one protease, falcipain 1, was active during the invasive merozoite stage. Falcipain 1-specific inhibitors, identified by screening of chemical libraries, blocked parasite invasion of host erythrocytes, yet had no effect on normal parasite processes such as hemoglobin degradation. These results demonstrate a specific role for falcipain 1 in host cell invasion and establish a potential new target for antimalarial therapeutics.

  View details for Web of Science ID 000179629200049

  View details for PubMedID 12471262

• Small molecule affinity fingerprinting: a tool for enzyme family subclassification, target identification, and inhibitor design CHEMISTRY & BIOLOGY Greenbaum, D. C., Arnold, W. D., Lu, F., Hayrapetian, L., Baruch, A., Krumrine, J., Toba, S., Chehade, K., Bromme, D., Kuntz, I. D., Bogyo, M. 2002; 9 (10): 1085-1094

  Abstract

  Classifying proteins into functionally distinct families based only on primary sequence information remains a difficult task. We describe here a method to generate a large data set of small molecule affinity fingerprints for a group of closely related enzymes, the papain family of cysteine proteases. Binding data was generated for a library of inhibitors based on the ability of each compound to block active-site labeling of the target proteases by a covalent activity based probe (ABP). Clustering algorithms were used to automatically classify a reference group of proteases into subfamilies based on their small molecule affinity fingerprints. This approach was also used to identify cysteine protease targets modified by the ABP in complex proteomes by direct comparison of target affinity fingerprints with those of the reference library of proteases. Finally, experimental data were used to guide the development of a computational method that predicts small molecule inhibitors based on reported crystal structures. This method could ultimately be used with large enzyme families to aid in the design of selective inhibitors of targets based on limited structural/function information.

  View details for Web of Science ID 000178895200005

  View details for PubMedID 12401493

• Probing structural determinants distal to the site of hydrolysis that control substrate specificity of the 20S proteasome CHEMISTRY & BIOLOGY Groll, M., Nazif, T., Huber, R., Bogyo, M. 2002; 9 (5): 655-662

  Abstract

  The 20S proteasome is a large multicomponent protease complex. Relatively little is known about the mechanisms that control substrate specificity of its multiple active sites. We present here the crystal structure at 2.95 A resolution of a beta2-selective inhibitor (MB1) bound to the yeast 20S proteasome core particle (CP). This structure is compared to the structure of the CP bound to a general inhibitor (MB2) that covalently modified all three (beta1, beta2, beta5) catalytic subunits. These two inhibitors differ only in their P3 and P4 residues, thereby highlighting binding interactions distal to the active site threonine that control absolute substrate specificity of the complex. Comparisons of the CP-bound structures of MB1, MB2, and the natural products epoxomycin and TMC-95A also provide information regarding general binding modes for several classes of proteasome inhibitors.

  View details for Web of Science ID 000175777800015

  View details for PubMedID 12031672

• Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries MOLECULAR AND BIOCHEMICAL PARASITOLOGY Mathieu, M. A., Bogyo, M., Caffrey, C. R., Choe, Y., Lee, J., Chapman, H., Sajid, M., CRAIK, C. S., McKerrow, J. H. 2002; 121 (1): 99-105

  Abstract

  Asparaginyl endopeptidases, or 'legumains' have been identified and characterized in plants, the blood fluke parasite Schistosoma, and mammals. The legumains are a novel family of cysteine proteases and display restricted specificity for peptide hydrolysis on the carboxyl side of asparagine residues. Two forms of recombinant asparaginyl endopeptidase from Schistosoma mansoni (C197 Sm32 and N197C Sm32), expressed in Pichia pastoris, have been analyzed for substrate specificity using a positional-scanning synthetic combinatorial library (PS-SCL). We first screened Sm32 using a P1-diverse library. This library demonstrated the absolute specificity of Sm32 for asparagine at P1. To determine the P2-P3 preferences of Sm32, we constructed a library with asparagine fixed at P1, and the P2-P3 positions randomized. The library was screened using the two forms of Sm32, human asparaginyl endopeptidase, and to confirm its diversity, cruzain from Trypanosoma cruzi. The schistosome legumain showed a preference for P3: Thr>Ala>Val>Ile, and P2: Ala>Thr>Val>Asn, with an overall broader specificity at P3 than at P2. Both human and schistosome legumain can accommodate Thr and Ala at P2 and P3. However, optimal substrate sequences differ, with Sm32 preferring Thr-Ala-Asn, and human legumain preferring Pro-Thr-Asn. Predictions of substrate specificity from the library screen were confirmed using single peptide substrates for kinetic assays.

  View details for Web of Science ID 000175841200009

  View details for PubMedID 11985866

• Proteasome inhibitors: Complex tools for a complex enzyme PROTEASOME-UBIQUITIN PROTEIN DEGRADATION PATHWAY Bogyo, M., Wang, E. W. 2002; 268: 185-208

  Abstract

  As the dominant protease dedicated to protein turnover, the proteasome shapes the cellular protein repertoire. Our knowledge of proteasome regulation and activity has improved considerably over the past decade. Novel inhibitors, in particular, have helped to advance our understanding of proteasome biology. They range from small peptide-based structures that can be modified to vary target specificity, to large macromolecular inhibitors that include proteins. While these reagents have played an important role in establishing our current knowledge of the proteasome's catalytic mechanism, many questions remain. Rapid advances in the synthesis and identification of new classes of proteasome inhibitors over the last 10 years serve as a positive indicator that many of these questions will soon be resolved. The future lies in designing compounds that can function as drugs to target processes involved in disease progression. It may only be a short while before the products of such research have safe application in a practical setting. Structural and combinatorial chemistry approaches are powerful techniques that will bring us closer to these goals.

  View details for Web of Science ID 000176957700008

  View details for PubMedID 12083006

• Chemical approaches for functionally probing the proteome MOLECULAR & CELLULAR PROTEOMICS Greenbaum, D., Baruch, A., Hayrapetian, L., Darula, Z., Burlingame, A., Medzihradszky, K. F., Bogyo, M. 2002; 1 (1): 60-68

  Abstract

  With the availability of complete genome sequences, emphasis has shifted toward the understanding of protein function. We have developed a functional proteomic methodology that makes use of chemically reactive fluorescent probes to profile and identify enzymes in complex mixtures by virtue of their catalytic activity. This methodology allows a comparison of changes in activity of multiple enzymes under a variety of conditions using a single two-dimensional separation. The probes can also be used to localize active enzymes in intact cells using fluorescence microscopy. Furthermore, the probes enable screens for selective small molecule inhibitors of each enzyme family member within crude lysates or intact cells. Ultimately, this technology allows the rapid identification of potential drug targets and small molecule lead compounds targeted to them.

  View details for DOI 10.1074/mcp.T100003-MCP200

  View details for Web of Science ID 000181445400007

  View details for PubMedID 12096141

• Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense MOLECULAR AND BIOCHEMICAL PARASITOLOGY Caffrey, C. R., Hansell, E., Lucas, K. D., Brinen, L. S., Hernandez, A. A., Cheng, J. N., Gwaltney, S. L., Roush, W. R., Stierhof, Y. D., Bogyo, M., STEVERDING, D., McKerrow, J. H. 2001; 118 (1): 61-73

  Abstract

  Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.

  View details for Web of Science ID 000172641700007

  View details for PubMedID 11704274

• Defining a link between gap junction communication, proteolysis, and cataract formation JOURNAL OF BIOLOGICAL CHEMISTRY Baruch, A., Greenbaum, D., Levy, E. T., Nielsen, P. A., GILULA, N. B., KUMAR, N. M., Bogyo, M. 2001; 276 (31): 28999-29006

  Abstract

  Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.

  View details for Web of Science ID 000170346000050

  View details for PubMedID 11395508

• Lysine 188 substitutions convert the pattern of proteasome activation by REG gamma to that of REGs alpha and beta EMBO JOURNAL Li, J., Gao, X. L., Ortega, J. Q., Nazif, T., Joss, L., Bogyo, M., STEVEN, A. C., Rechsteiner, M. 2001; 20 (13): 3359-3369

  Abstract

  11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGalpha activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGgamma only activates cleavage after basic residues. We have isolated REGgamma mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome beta subunits. The most robust REGgamma specificity mutants involve substitution of Glu or Asp for Lys188. REGgamma(K188E/D) variants are virtually identical to REGalpha in proteasome activation but assemble into less stable heptamers/hexamers. Based on the REGalpha crystal structure, Lys188 of REGgamma faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin-like subunits by 125I-YL3-VS demonstrates that REGgamma(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGgamma(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGgamma molecule.

  View details for Web of Science ID 000169803700009

  View details for PubMedID 11432824

• Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nazif, T., Bogyo, M. 2001; 98 (6): 2967-2972

  Abstract

  The proteasome is a large protease complex consisting of multiple catalytic subunits that function simultaneously to digest protein substrates. This complexity has made deciphering the role each subunit plays in the generation of specific protein fragments difficult. Positional scanning libraries of peptide vinyl sulfones were generated in which the amino acid located directly at the site of hydrolysis (P1 residue) was held constant and sequences distal to that residue (P2, P3, and P4 positions) were varied across all natural amino acids (except cysteine and methionine). Binding information for each of the individual catalytic subunits was obtained for each library under a variety of different conditions. The resulting specificity profiles indicated that substrate positions distal to P1 are critical for directing substrates to active subunits in the complex. Furthermore, specificity profiles of IFN-gamma-regulated subunits closely matched those of their noninducible counterparts, suggesting that subunit swapping may modulate substrate processing by a mechanism that does require a change in the primary sequence specificity of individual catalytic subunits in the complex. Finally, specificity profiles were used to design specific inhibitors of a single active site in the complex. These reagents can be used to further establish the role of each subunit in substrate processing by the proteasome.

  View details for Web of Science ID 000167521300009

  View details for PubMedID 11248015

• Release of signal peptide fragments into the cytosol requires cleavage in the transmembrane region by a protease activity that is specifically blocked by a novel cysteine protease inhibitor JOURNAL OF BIOLOGICAL CHEMISTRY Weihofen, A., Lemberg, M. K., Ploegh, H. L., Bogyo, M., Martoglio, B. 2000; 275 (40): 30951-30956

  Abstract

  Signal peptides of secretory and membrane proteins are generated by proteolytic processing of precursor proteins after insertion into the endoplasmic reticulum membrane. Liberated signal peptides can be further processed, and the resulting N-terminal fragments are released toward the cytosol, where they may interact with target proteins like calmodulin. We show here that the processing of signal peptides requires a protease activity distinct from signal peptidase. This activity is inhibited specifically with a newly developed cysteine protease inhibitor, 1, 3-di-(N-carboxybenzoyl-l-leucyl-l-leucyl)amino acetone ((Z-LL)(2) ketone). Inhibitor studies revealed that the final, (Z-LL)(2) ketone-sensitive cleavage event occurs within the hydrophobic transmembrane region of the signal peptide, thus promoting the release of an N-terminal fragment into the cytosol.

  View details for Web of Science ID 000089762700032

  View details for PubMedID 10921927

• Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, E. W., Kessler, B. M., Borodovsky, A., Cravatt, B. F., Bogyo, M., Ploegh, H. L., Glas, R. 2000; 97 (18): 9990-9995

  Abstract

  Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.

  View details for Web of Science ID 000089067500034

  View details for PubMedID 10954757

• Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools CHEMISTRY & BIOLOGY Greenbaum, D., Medzihradszky, K. F., Burlingame, A., Bogyo, M. 2000; 7 (8): 569-581

  Abstract

  Analysis of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymatic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly determining activities of an entire class of enzymes on a genome-wide scale would be of great utility.We have engineered chemical probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes containing a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivatives was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular extracts. Finally, the affinity tag of DCG-04 allowed purification of modified proteases and identification by mass spectrometry.We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixtures. These probes were used to determine relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purification/identification of targets.

  View details for Web of Science ID 000089866200003

  View details for PubMedID 11048948

• Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris FEBS LETTERS Caffrey, C. R., Mathieu, M. A., Gaffney, A. M., Salter, J. P., Sajid, M., Lucas, K. D., Franklin, C., Bogyo, M., McKerrow, J. H. 2000; 466 (2-3): 244-248

  Abstract

  Asparaginyl endopeptidases, or legumains, are a recently identified family of cysteine-class endopeptidases. A single gene encoding a Schistosoma mansoni asparaginyl endopeptidase (a.k.a. Sm32 or schistosome legumain) has been reported, but by sequence homology it would be expected to yield an inactive product as the active site C197 had been replaced by N. We now describe a new S. mansoni gene in which C197 is present. Both gene products were expressed in Pichia pastoris. Autocatalytic processing to fully active C197 Sm32 occurred at acid pH. In contrast, N197 Sm32 was not processed and this is consistent with the hypothesis that C197 is essential for catalysis. This was confirmed by mutation of N197 to C and re-expression in Pichia. The availability of recombinant active Sm32 allows detailed analysis of its catalytic mechanism and its function(s) in the biology of this important human parasite.

  View details for Web of Science ID 000085122200008

  View details for PubMedID 10682836

• Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs CHEMISTRY & BIOLOGY Bogyo, M., Verhelst, S., Bellingard-Dubouchaud, V., Toba, S., Greenbaum, D. 2000; 7 (1): 27-38

  Abstract

  The lysosomal cysteine proteases of the papain family are some of the best studied proteolytic enzymes. Small-molecule inhibitors and fluorogenic substrate mimics have been used to probe the physiological roles of these proteases. A high degree of homology between family members and overlap in substrate specificity have made elucidating individual protease function, expression and activity difficult.Using peptide vinyl sulfones and epoxide as templates, we have generated probes that can be tagged with radioactive iodine. The resulting compounds covalently label various cathepsins and several unidentified polypeptides likely to be proteases. MB-074 was found to be a highly selective probe of cathepsin B activity. Probes that labeled several cathepsins were used to examine the specificity and cell permeability of the CA-074 family of inhibitors. Although CA-074 reportedly acts in vivo, we find it is unable to penetrate cells. Esterifying CA-074 resulted in a cell-permeable inhibitor with dramatically reduced activity and specificity for cathepsin B. The probes were also used to monitor protease activity in primary human tumor tissue and cells derived from human placenta.We have generated a highly selective cathepsin B probe and several less specific reagents for the study of cathepsin biology. The reagents have several advantages over commonly used fluorogenic substrates, allowing inhibitor targets to be identified in a pool of total cellular enzymes. We have used the probes to show that cathepsin activity is regulated in tumor tissues and during differentiation of placental-derived cytotrophoblasts to invasive cells required for establishing blood circulation in a developing embryo.

  View details for Web of Science ID 000087225200008

  View details for PubMedID 10662686

• How an inhibitor of the HIV-I protease modulates proteasome activity JOURNAL OF BIOLOGICAL CHEMISTRY Schmidtke, G., Holzhutter, H. G., Bogyo, M., Kairies, N., Groll, M., De Giuli, R., Emch, S., Groettrup, M. 1999; 274 (50): 35734-35740

  Abstract

  The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.

  View details for Web of Science ID 000084187900066

  View details for PubMedID 10585454

• Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target National Academy of Sciences Colloquium on Proteolytic Processing and Physiological Regulation Selzer, P. M., Pingel, S., Hsieh, I., Ugele, B., Chan, V. J., Engel, J. C., Bogyo, M., Russell, D. G., Sakanari, J. A., McKerrow, J. H. NATL ACAD SCIENCES. 1999: 11015–22

  Abstract

  Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  View details for Web of Science ID 000082868500015

  View details for PubMedID 10500116

• Peptide vinyl sulfones: Inhibitors and active site probes for the study of proteasome function in vivo 15th American Peptide Symposium Bogyo, M., McMaster, J. S., Glas, R., Gaczynska, M., Tortorella, D., Ploegh, H. L. SPRINGER. 1999: 686–687

  View details for Web of Science ID 000085202200301

• Antigen presentation - A protease draws first blood NATURE Bogyo, M., Ploegh, H. L. 1998; 396 (6712): 625-?

  View details for Web of Science ID 000077694200026

  View details for PubMedID 9872306

• Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes CHEMISTRY & BIOLOGY Bogyo, M., Shin, S., McMaster, J. S., Ploegh, H. L. 1998; 5 (6): 307-320

  Abstract

  The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active beta subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 125I-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of beta-subunit modification.The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual beta subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual beta subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.

  View details for Web of Science ID 000074359900004

  View details for PubMedID 9653549

• A proteolytic system that compensates for loss of proteasome function NATURE Glas, R., Bogyo, M., McMaster, J. S., Gaczynska, M., Ploegh, H. L. 1998; 392 (6676): 618-622

  Abstract

  Proteolysis is essential for the execution of many cellular functions. These include removal of incorrectly folded or damaged proteins, the activation of transcription factors, the ordered degradation of proteins involved in cell cycle control, and the generation of peptides destined for presentation by class I molecules of the major histocompatibility complex. A multisubunit protease complex, the proteasome, accomplishes these tasks. Here we show that in mammalian cells inactivation of the proteasome by covalent inhibitors allows the outgrowth of inhibitor-resistant cells. The growth of such adapted cells is apparently maintained by the induction of other proteolytic systems that compensate for the loss of proteasomal activity.

  View details for Web of Science ID 000072987200064

  View details for PubMedID 9560160

• Proteasome function is dispensable under normal but not under heat shock conditions in Thermoplasma acidophilum FEBS LETTERS Ruepp, A., Eckerskorn, C., Bogyo, M., Baumeister, W. 1998; 425 (1): 87-90

  Abstract

  Hitherto the biology of proteolysis in prokaryotes, particularly in archaea, is only poorly understood. We have used the tri-peptide vinyl sulfone inhibitor carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) to study the in vivo function of proteasomes in Thermoplasma acidophilum. Z-L3VS is a potent inhibitor of the Thermoplasma proteasome and is capable of modifying 75 to 80% of the proteasomal beta-subunits in cell cultures. Inhibition of proteasomes has only marginal effects under normal growth conditions. Under heat shock conditions, however, the effects of proteasome inhibition are much more severe, to the extent of complete cell growth arrest. These data suggest that other proteolytic systems may exist that can compensate for the loss of proteasome function in T. acidophilum.

  View details for Web of Science ID 000072783000017

  View details for PubMedID 9541012

• Covalent modification of the active site threonine of proteasomal beta subunits and the Escherichia coli homolog HslV by a new class of inhibitors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bogyo, M., McMaster, J. S., Gaczynska, M., Tortorella, D., Goldberg, A. L., Ploegh, H. 1997; 94 (13): 6629-6634

  Abstract

  The proteasome is a multicatalytic protease complex that plays a key role in diverse cellular functions. The peptide vinyl sulfone, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) covalently inhibits the trypsin-like, chymotrypsin-like and, unlike lactacystin, also the peptidylglutamyl peptidase activity in isolated proteasomes, and blocks their function in living cells. Although described as a class of mechanism-based inhibitors for cysteine proteases, the peptide vinyl sulfone Z-L3VS and a 125I-labeled nitrophenol derivative (125I-NIP-L3VS) covalently modify the active site threonine of the catalytic beta subunits of the proteasome. Modification of Thermoplasma proteasomes demonstrates the requirement for a hydroxyl amino acid (threonine, serine) as nucleophile at the beta subunit's NH2 terminus. 125I-NIP-L3VS covalently modifies the HslV subunit of the Escherichia coli protease complex HslV/HslU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.

  View details for Web of Science ID A1997XH03400010

  View details for PubMedID 9192616

• Proteasome inhibitors and antigen presentation BIOPOLYMERS Bogyo, M., Gaczynska, M., Ploegh, H. L. 1997; 43 (4): 269-280

  Abstract

  Protein degradation plays an important role in the control and regulation of many crucial biological functions, ranging from cell cycle progression to presentation of viral antigens for scrutiny by cells of the immune system. At the heart of many of these catabolic events is the multicatalytic proteinase complex known as the proteasome. This large barrel-shaped protein complex executes a remarkable set of functions ranging from the complete destruction of abnormal and misfolded proteins to the specific proteolytic activation of crucial signaling molecules. Inhibitors of this proteolytic complex have thus been extremely useful for perturbing its function and deciphering its role in these diverse biological processes. Inhibitors of the proteasome consist mainly of peptides that are modified at the predicted site of hydrolysis with a reactive functional group capable of modifying the attacking nucleophile, either reversibly or irreversibly. Many of these inhibitors can be used in living cells and have proved to be invaluable tools for the study of proteasome function.

  View details for Web of Science ID A1997XY12600002

  View details for PubMedID 9316392

• Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction NATURE Wiertz, E. J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., JONES, T. R., Rapoport, T. A., Ploegh, H. L. 1996; 384 (6608): 432-438

  Abstract

  The human cytomegalovirus genome encodes proteins that trigger destruction of newly synthesized major histocompatibility complex (MHC) class I molecules. The human cytomegalovirus gene US2 specifies a product capable of dislocating MHC class I molecules from the endoplasmic reticulum to the cytosol and delivering them to the proteasome. This process involves the Sec61 complex, in what appears to be a reversal of the reaction by which it translocates nascent chains into the endoplasmic reticulum.

  View details for Web of Science ID A1996VW68700053

  View details for PubMedID 8945469

• The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol CELL Wiertz, E. J., JONES, T. R., Sun, L., Bogyo, M., Geuze, H. J., Ploegh, H. L. 1996; 84 (5): 769-779

  Abstract

  Human cytomegalovirus (HCMV) down-regulates expression of MHC class I products by selective proteolysis. A single HCMV gene, US11, which encodes an endoplasmic reticulum (ER) resident type-I transmembrane glycoprotein, is sufficient to cause this effect. In US11+cells, MHC class I molecules are core-glycosylated and therefore inserted into the ER. They are degraded with a half-time of less than 1 min. A full length breakdown intermediate that has lost the single N-linked glycan in an N-glycanase-catalyzed reaction transiently accumulates in cells exposed to the protease inhibitors LLnL, Cbz-LLL, and lactacystin, identifying the proteasome as a key protease. Subcellular fractionation experiments show this intermediate to be cytosolic. Thus, US11 dislocates newly synthesized class I molecules from the ER to the cytosol, where they are acted upon by an N-glycanase and the proteasome.

  View details for Web of Science ID A1996TZ99000014

  View details for PubMedID 8625414