Academic Appointments


Honors & Awards


  • Investigator in Pathogenesis of Infectious Disease, Burroughs Wellcome (May 2005)
  • Terman Fellow, Stanford University (2003)
  • Searle Scholar, Searle Scholars Program (2004)

Professional Education


  • B.S., Bates College, Chemistry (1993)
  • Ph.D., MIT, Biochemistry (1997)

Current Research and Scholarly Interests


Our lab uses chemical, biochemical, and cell biological methods to study protease function in human disease. Projects include:

1) Design and synthesis of novel chemical probes for each of the primary protease families.

2) Understanding the role of proteolysis in the life cycle of the human parasites, Plasmodium falciparum and Toxoplasma gondii.

3) Defining the specific functional roles of proteases during the process of tumorogenesis.

4) In vivo imaging of protease activity

Journal Articles


  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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 Web of Science ID 000313087300007

    View details for PubMedID 23142757

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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 Web of Science ID 000300404200009

    View details for PubMedID 22243962

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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 ?-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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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 BIOLOGICAL CHEMISTRY 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. 2008; 389 (8): 1067-1074

    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

  • 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

  • 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

  • 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

  • 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 Web of Science ID 000248027400024

    View details for PubMedID 17507477

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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
  • 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
  • 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

  • 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

  • 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

  • Growth phase-dependent production of a cell wall-associated elastinolytic cysteine proteinase by Staphylococcus epidermidis BIOLOGICAL CHEMISTRY Oleksy, A., Golonka, E., Banbula, A., Szmyd, G., Moon, J., Kubica, M., Greenbaum, D., Bogyo, M., Foster, T. J., Travis, J., Potempa, J. 2004; 385 (6): 525-535

    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 BIOLOGICAL CHEMISTRY Hook, V., Yasothornsrikul, S., Greenbaum, D., Medzihradszky, K. F., Troutner, K., Toneff, T., Bundey, R., Logrinova, A., Reinheckel, T., Peters, C., Bogyo, M. 2004; 385 (6): 473-480

    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

  • Peptide vinyl sulfones: Inhibitors and active site probes for the study of proteasome function in vivo PEPTIDES: FRONTIERS OF PEPTIDES SCIENCE Bogyo, M., McMaster, J. S., Glas, R., Gaczynska, M., Tortorella, D., Ploegh, H. L. 1999: 686-687
  • 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

Conference Proceedings


  • 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: 240-240
  • Intracellular inhibitors of cysteine cathepsins in activated macrophages Maher, K., Konjar, S., Ceru, S., Bogyo, M., Turk, B., Kopitar-Jerala, N. ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. 2009: 24-24
  • Selective targeting of papain-like proteases by double-headed epoxysuccinyl probes Verhelst, S. H., Sadaghiani, A. M., Arastu-Kapur, S., Bogyo, M. JOHN WILEY & SONS INC. 2005: 603-603
  • Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target 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: 11015-11022

    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