Academic Appointments


Honors & Awards


  • Excellence in Teaching Award, Stanford School of Medicine (2014)
  • Senior Scholar Award in Aging, Ellison Medical Foundation (2010-2014)
  • Nobel Laureate Signature Award for Graduate Education, American Chemical Society (2009)
  • CAREER Award, NSF (2000-2004)
  • Alfred P. Sloan Research Fellow, Sloan Foundation (2000)
  • Camille Dreyfus Teacher-Scholar, Dreyfus Foundation (2000)
  • Dean's Award for Distinguished Teaching, Stanford University (1999)
  • Beckman Foundation Young Investigator, Arnold & Mabel Beckman Foundation (1998-2000)
  • Phi Beta Kappa Undergraduate Teaching Prize, Stanford Phi Beta Kappa (1998)

Professional Education


  • Ph.D., Harvard University, Chemistry (1993)
  • B.S., Trinity University, Biochemistry (1988)

Current Research and Scholarly Interests


Overview – The lab concentrates on the invention of molecules and techniques that enable better studies of biological processes. In short, we invent tools for biology and we are motivated by approaches that enable entirely new experiments with unprecedented control. These new techniques may also provide a window into mechanisms that cells use to maintain protein homeostasis. Protein quality control is a particular interest at present.

New Approaches for Conditional Control of Protein Function – We have developed an experimental system in which the stability of a specific protein depends on the presence or absence of a cell-permeable molecule. We started with a well-studied protein-ligand pair: the FKBP12 protein and a high-affinity ligand we synthesized called Shield-1. We screened a library of FKBP sequences to identify mutants that are unstable in the absence of Shield-1 and are stabilized by Shield-1. Further characterization of these mutants revealed that the most destabilizing mutants caused a 50-fold to 100-fold reduction in the expression levels of the proteins to which they were fused. Importantly, this instability is transmitted to any fused partner protein, allowing us to regulate the stability of any protein-of-interest using Shield-1.

These reagents are called destabilizing domains (DDs), and we have developed a broad portfolio of these reagents based on several distinct protein-ligand combinations. Most of these reagents are available through Addgene. The systems work well in cultured mammalian cells, a variety of model organisms (e.g., flies, worms, frogs) and in living mice and rats. This technique allows rapid and reversible elimination of a specific protein in a variety of biological contexts. We have also engineered a DD system that functions in the opposite sense. The fusion protein is stable in the absence of the ligand, and administration of the ligand causes the fusion protein to be rapidly degraded. Similarly, we have engineered a conditional stability system where light is used to regulate protein stability. Most of these reagents have been reviewed recently (Rakhit et al., Chemistry & Biology 2014, 21, 1238).

Protein Quality Control in Cells – The DDs can be thought of as model substrates that have the potential to help us understand how cells detect and deal with misfolded or unfolded proteins. The ability to conditionally regulate the folding state of these domains using high-affinity ligands allows us to correlate specific biophysical properties with biological stability. One focus of the lab is understanding how these technologies work in cells. Using purified proteins we have shown that the DD proteins are either unfolded or significantly populate the unfolded state in the absence of the stabilizing ligand (Egeler et al., JBC 2011, 286, 31328). We have also used a focused RNAi strategy to identify proteins involved in the cellular response to the unfolded DDs (Chu et al., JBC 2013, 288, 34575).

More recently, we are using the DDs as conditionally folded proteins to create an acute unfolded protein stress by withdrawing the stabilizing ligand from the cell culture media. This approach allowed us to identify a novel coordinated transcriptional response that mammalian cells trigger when unfolded protein appears (Miyazaki et al., eLIFE 2015). Interestingly, there is little overlap between this new cellular response and the heat shock response, which conventional wisdom holds as the "cytosolic unfolded protein response". Additionally, creating unfolded DD in either the cytosol or nucleus elicits distinct responses, suggesting that mammalian cells maintain different protein quality control surveillance environments in these compartments. Understanding the molecular mechanisms behind these new cellular responses as well as how they help to maintain protein homeostasis is a major focus of the lab.

2024-25 Courses


Stanford Advisees


All Publications


  • p53 engagement is a hallmark of an unfolded protein response in the nucleus of mammalian cells. bioRxiv : the preprint server for biology Park, J. H., Wandless, T. J. 2024

    Abstract

    Exposure to exogenous and endogenous stress is associated with the intracellular accumulation of aberrant unfolded and misfolded proteins. In eukaryotic cells, protein homeostasis within membrane-bound organelles is regulated by specialized signaling pathways, with the unfolded protein response in the endoplasmic reticulum serving as a foundational example. Yet, it is unclear if a similar surveillance mechanism exists in the nucleus. Here we leveraged engineered proteins called destabilizing domains to acutely expose mammalian cells to nuclear- or cytosolic- localized unfolded protein. We show that the appearance of unfolded protein in either compartment engages a common transcriptional response associated with the transcription factors Nrf1 and Nrf2. Uniquely, only in the nucleus does unfolded protein activate a robust p53-driven transcriptional response and a transient p53-independent cell cycle delay. These studies highlight the distinct effects of localized protein folding stress and the unique protein quality control environment of the nucleus.

    View details for DOI 10.1101/2024.11.08.622663

    View details for PubMedID 39574672

    View details for PubMedCentralID PMC11581032

  • Ubiquitin-Derived Fragment as a Peptide Linker for the Efficient Cleavage of a Target Protein from a Degron. ACS chemical biology Utsugi, Y., Nishimura, K., Yamanaka, S., Nishino, K., Kosako, H., Sawasaki, T., Shigemori, H., Wandless, T. J., Miyamae, Y. 2024

    Abstract

    The chemogenetic control of cellular protein stability using degron tags is a powerful experimental strategy in biomedical research. However, this technique requires permanent fusion of the degron to a target protein, which may interfere with the proper function of the protein. Here, we report a peptide fragment from the carboxyl terminus of ubiquitin as a cleavable linker that exhibits the slow but efficient cleavage of a degron tag via cellular deubiquitinating enzymes (DUBs). We designed a fusion protein consisting of a cleavable linker and a destabilizing domain (DD), which conditionally controls the expression and release of a target protein in a ligand-induced state, allowing the free unmodified protein to perform its function. Insertion of an AGIA epitope at the carboxyl terminus of the linker made space for the DUBs to access the site to assist the cleavage reaction when the amino terminus of the target protein caused steric hindrance. The developed system, termed a cleavable degron using ubiquitin-derived linkers (c-DUB), provides robust and tunable regulation of target proteins in their native forms. The c-DUB system is a useful tool for the regulation of proteins that have terminal sites that are essential for the proper localization and function. In addition, a mechanistic investigation using proximity labeling showed that DUBs associate with the refolded DD to reverse ubiquitination, suggesting a cellular surveillance system for distinguishing the refolded DD from misfolded proteins. The c-DUB method may benefit from this machinery so that DUBs subsequently cleave the neighboring linker.

    View details for DOI 10.1021/acschembio.3c00670

    View details for PubMedID 38270585

  • A cAMP Sensor Based on Ligand-Dependent Protein Stabilization. ACS chemical biology Sidoli, M., Chen, L., Lu, A. J., Wandless, T. J., Talbot, W. S. 2022

    Abstract

    cAMP is a ubiquitous second messenger with many functions in diverse organisms. Current cAMP sensors, including Foster resonance energy transfer (FRET)-based and single-wavelength-based sensors, allow for real time visualization of this small molecule in cultured cells and in some cases in vivo. Nonetheless the observation of cAMP in living animals is still difficult, typically requiring specialized microscopes and ex vivo tissue processing. Here we used ligand-dependent protein stabilization to create a new cAMP sensor. This sensor allows specific and sensitive detection of cAMP in living zebrafish embryos, which may enable new understanding of the functions of cAMP in living vertebrates.

    View details for DOI 10.1021/acschembio.2c00333

    View details for PubMedID 35839076

  • Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science (New York, N.Y.) Weber, E. W., Parker, K. R., Sotillo, E., Lynn, R. C., Anbunathan, H., Lattin, J., Good, Z., Belk, J. A., Daniel, B., Klysz, D., Malipatlolla, M., Xu, P., Bashti, M., Heitzeneder, S., Labanieh, L., Vandris, P., Majzner, R. G., Qi, Y., Sandor, K., Chen, L., Prabhu, S., Gentles, A. J., Wandless, T. J., Satpathy, A. T., Chang, H. Y., Mackall, C. L. 2021; 372 (6537)

    Abstract

    T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.

    View details for DOI 10.1126/science.aba1786

    View details for PubMedID 33795428

  • A method to rapidly create protein aggregates in living cells NATURE COMMUNICATIONS Miyazaki, Y., Mizumoto, K., Dey, G., Kudo, T., Perrino, J., Chen, L., Meyer, T., Wandless, T. J. 2016; 7

    Abstract

    The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates.

    View details for DOI 10.1038/ncomms11689

    View details for Web of Science ID 000376669800001

    View details for PubMedID 27229621

    View details for PubMedCentralID PMC4894968

  • A Method for Conditional Regulation of Protein Stability in Native or Near-Native Form. Cell chemical biology Miyamae, Y. n., Chen, L. C., Utsugi, Y. n., Farrants, H. n., Wandless, T. J. 2020

    Abstract

    Here, we report a method to regulate cellular protein levels by introducing a ubiquitin variant between a destabilizing domain (DD) and the regulated protein. When produced in the absence of a stabilizing ligand the DD dominates and the entire fusion protein is processively degraded by the proteasome. In the presence of the stabilizing ligand the fusion protein is metabolically stable and becomes a substrate for abundant ubiquitin-specific proteases, liberating a native, or a near-native protein-of-interest. This technique is thus particularly useful for the study of proteins whose free N terminus is required for proper function. In addition, removal of the DD in the presence of stabilizing ligand leads to higher expression levels of regulated protein when cells experience transient exposure to a stabilizing ligand, such as in a living animal receiving a single dose of a pharmacological agent as the stabilizing ligand.

    View details for DOI 10.1016/j.chembiol.2020.09.004

    View details for PubMedID 33007216

  • Titratable and Reversible Regulation of Therapeutic Proteins in Cell and Gene Therapies Using FDA Approved Drugs and a Modular Protein Stabilization Platform Suri, V., Inniss, M., Shamah, S., Wandless, T. CELL PRESS. 2019: 40–41
  • Methods to enable regulated gene therapy Wandless, T. AMER CHEMICAL SOC. 2018
  • A Novel Destabilizing Domain Based on a Small-Molecule Dependent Fluorophore ACS CHEMICAL BIOLOGY Navarro, R., Chen, L., Rakhit, R., Wandless, T. J. 2016; 11 (8): 2101-2104

    Abstract

    Tools that can directly regulate the activity of any protein-of-interest are valuable in the study of complex biological processes. Herein, we describe the development of a novel protein domain that exhibits small molecule-dependent stability and fluorescence based on the bilirubin-inducible fluorescent protein, UnaG. When genetically fused to any protein-of-interest, this fluorescent destabilizing domain (FDD) confers its instability to the entire fusion protein, facilitating the rapid degradation of the fusion. In the presence of its cognate ligand bilirubin (BR), the FDD fusion becomes stable and fluorescent. This new chemical genetic tool allows for rapid, reversible, and tunable control over the stability and fluorescence of a wide range of protein targets.

    View details for DOI 10.1021/acschembio.6b00234

    View details for Web of Science ID 000381847700003

    View details for PubMedID 27243964

  • Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells ELIFE Miyazaki, Y., Chen, L., Chu, B. W., Swigut, T., Wandless, T. J. 2015; 4

    Abstract

    Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called 'destabilizing domains', whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies.

    View details for DOI 10.7554/eLife.07687

    View details for Web of Science ID 000373822800001

    View details for PubMedID 26314864

    View details for PubMedCentralID PMC4566031

  • Using light to shape chemical gradients for parallel and automated analysis of chemotaxis. Molecular systems biology Collins, S. R., Yang, H. W., Bonger, K. M., Guignet, E. G., Wandless, T. J., Meyer, T. 2015; 11 (4): 804-?

    Abstract

    Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy.

    View details for DOI 10.15252/msb.20156027

    View details for PubMedID 25908733

    View details for PubMedCentralID PMC4422560

  • Chemical Biology Strategies for Posttranslational Control of Protein Function CHEMISTRY & BIOLOGY Rakhit, R., Navarro, R., Wandless, T. J. 2014; 21 (9): 1238-1252

    Abstract

    A common strategy to understand a biological system is to selectively perturb it and observe its response. Although technologies now exist to manipulate cellular systems at the genetic and transcript level, the direct manipulation of functions at the protein level can offer significant advantages in precision, speed, and reversibility. Combining the specificity of genetic manipulation and the spatiotemporal resolution of light- and small molecule-based approaches now allows exquisite control over biological systems to subtly perturb a system of interest in vitro and in vivo. Conditional perturbation mechanisms may be broadly characterized by change in intracellular localization, intramolecular activation, or degradation of a protein-of-interest. Here we review recent advances in technologies for conditional regulation of protein function and suggest further areas of potential development.

    View details for DOI 10.1016/j.chembiol.2014.08.011

    View details for Web of Science ID 000342626700018

    View details for PubMedCentralID PMC4174368

  • Chemical biology strategies for posttranslational control of protein function. Chemistry & biology Rakhit, R., Navarro, R., Wandless, T. J. 2014; 21 (9): 1238-1252

    Abstract

    A common strategy to understand a biological system is to selectively perturb it and observe its response. Although technologies now exist to manipulate cellular systems at the genetic and transcript level, the direct manipulation of functions at the protein level can offer significant advantages in precision, speed, and reversibility. Combining the specificity of genetic manipulation and the spatiotemporal resolution of light- and small molecule-based approaches now allows exquisite control over biological systems to subtly perturb a system of interest in vitro and in vivo. Conditional perturbation mechanisms may be broadly characterized by change in intracellular localization, intramolecular activation, or degradation of a protein-of-interest. Here we review recent advances in technologies for conditional regulation of protein function and suggest further areas of potential development.

    View details for DOI 10.1016/j.chembiol.2014.08.011

    View details for PubMedID 25237866

  • General method for regulating protein stability with light. ACS chemical biology Bonger, K. M., Rakhit, R., Payumo, A. Y., Chen, J. K., Wandless, T. J. 2014; 9 (1): 111-115

    Abstract

    Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.

    View details for DOI 10.1021/cb400755b

    View details for PubMedID 24180414

    View details for PubMedCentralID PMC3906921

  • The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates. journal of biological chemistry Chu, B. W., Kovary, K. M., Guillaume, J., Chen, L., Teruel, M. N., Wandless, T. J. 2013; 288 (48): 34575-34587

    Abstract

    To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.

    View details for DOI 10.1074/jbc.M113.499350

    View details for PubMedID 24158444

    View details for PubMedCentralID PMC3843071

  • Inducible control of gene expression with destabilized Cre NATURE METHODS Sando, R., Baumgaertel, K., Pieraut, S., Torabi-Rander, N., Wandless, T. J., Mayford, M., Maximov, A. 2013; 10 (11)

    Abstract

    Acute manipulation of gene and protein function in the brain is essential for understanding the mechanisms of nervous system development, plasticity and information processing. Here we describe a technique based on a destabilized Cre recombinase (DD-Cre) whose activity is controlled by the antibiotic trimethoprim (TMP). We show that DD-Cre triggers rapid TMP-dependent recombination of loxP-flanked ('floxed') alleles in mouse neurons in vivo and validate the use of this system for neurobehavioral research.

    View details for DOI 10.1038/NMETH.2640

    View details for Web of Science ID 000326507600021

    View details for PubMedID 24056874

  • FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension. journal of clinical investigation Spiekerkoetter, E., Tian, X., Cai, J., Hopper, R. K., Sudheendra, D., Li, C. G., El-Bizri, N., Sawada, H., Haghighat, R., Chan, R., Haghighat, L., de Jesus Perez, V., Wang, L., Reddy, S., Zhao, M., Bernstein, D., Solow-Cordero, D. E., Beachy, P. A., Wandless, T. J., ten Dijke, P., Rabinovitch, M. 2013; 123 (8): 3600-3613

    Abstract

    Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

    View details for DOI 10.1172/JCI65592

    View details for PubMedID 23867624

  • Par-4 Downregulation Promotes Breast Cancer Recurrence by Preventing Multinucleation following Targeted Therapy CANCER CELL Alvarez, J. V., Pan, T., Ruth, J., Feng, Y., Zhou, A., Pant, D., Grimley, J. S., Wandless, T. J., DeMichele, A., Chodosh, L. A. 2013; 24 (1): 30-44

    Abstract

    Most deaths from breast cancer result from tumor recurrence, but mechanisms underlying tumor relapse are largely unknown. We now report that Par-4 is downregulated during tumor recurrence and that Par-4 downregulation is necessary and sufficient to promote recurrence. Tumor cells with low Par-4 expression survive therapy by evading a program of Par-4-dependent multinucleation and apoptosis that is otherwise engaged following treatment. Low Par-4 expression is associated with poor response to neoadjuvant chemotherapy and an increased risk of relapse in patients with breast cancer, and Par-4 is downregulated in residual tumor cells that survive neoadjuvant chemotherapy. Our findings identify Par-4-induced multinucleation as a mechanism of cell death in oncogene-addicted cells and establish Par-4 as a negative regulator of breast cancer recurrence.

    View details for DOI 10.1016/j.ccr.2013.05.007

    View details for Web of Science ID 000321604000008

    View details for PubMedID 23770012

  • Visualizing cellular interactions with a generalized proximity reporter. Proceedings of the National Academy of Sciences of the United States of America Sellmyer, M. A., Bronsart, L., Imoto, H., Contag, C. H., Wandless, T. J., Prescher, J. A. 2013; 110 (21): 8567-8572

    Abstract

    Interactions among neighboring cells underpin many physiological processes ranging from early development to immune responses. When these interactions do not function properly, numerous pathologies, including infection and cancer, can result. Molecular imaging technologies, especially optical imaging, are uniquely suited to illuminate complex cellular interactions within the context of living tissues in the body. However, no tools yet exist that allow the detection of microscopic events, such as two cells coming into close proximity, on a global, whole-animal scale. We report here a broadly applicable, longitudinal strategy for probing interactions among cells in living subjects. This approach relies on the generation of bioluminescent light when two distinct cell populations come into close proximity, with the intensity of the optical signal correlating with relative cellular location. We demonstrate the ability of this reporter strategy to gauge cell-cell proximity in culture models in vitro and then evaluate this approach for imaging tumor-immune cell interactions using a murine breast cancer model. In these studies, our imaging strategy enabled the facile visualization of features that are otherwise difficult to observe with conventional imaging techniques, including detection of micrometastatic lesions and potential sites of tumor immunosurveillance. This proximity reporter will facilitate probing of numerous types of cell-cell interactions and will stimulate the development of similar techniques to detect rare events and pathological processes in live animals.

    View details for DOI 10.1073/pnas.1218336110

    View details for PubMedID 23650381

    View details for PubMedCentralID PMC3666728

  • Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule. PloS one Cho, U., Zimmerman, S. M., Chen, L., Owen, E., Kim, J. V., Kim, S. K., Wandless, T. J. 2013; 8 (8)

    Abstract

    Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

    View details for DOI 10.1371/journal.pone.0072393

    View details for PubMedID 23991108

    View details for PubMedCentralID PMC3750007

  • Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule. PloS one Cho, U., Zimmerman, S. M., Chen, L., Owen, E., Kim, J. V., Kim, S. K., Wandless, T. J. 2013; 8 (8)

    View details for DOI 10.1371/journal.pone.0072393

    View details for PubMedID 23991108

  • Networks of Polarized Actin Filaments in the Axon Initial Segment Provide a Mechanism for Sorting Axonal and Dendritic Proteins CELL REPORTS Watanabe, K., Al-Bassam, S., Miyazaki, Y., Wandless, T. J., Webster, P., Arnold, D. B. 2012; 2 (6): 1546-1553

    Abstract

    Trafficking of proteins specifically to the axonal or somatodendritic membrane allows neurons to establish and maintain polarized compartments with distinct morphology and function. Diverse evidence suggests that an actin-dependent vesicle filter within the axon initial segment (AIS) plays a critical role in polarized trafficking; however, no distinctive actin-based structures capable of comprising such a filter have been found within the AIS. Here, using correlative light and scanning electron microscopy, we visualized networks of actin filaments several microns wide within the AIS of cortical neurons in culture. Individual filaments within these patches are predominantly oriented with their plus ends facing toward the cell body, consistent with models of filter selectivity. Vesicles carrying dendritic proteins are much more likely to stop in regions occupied by the actin patches than in other regions, indicating that the patches likely prevent movement of dendritic proteins to the axon and thereby act as a vesicle filter.

    View details for DOI 10.1016/j.celrep.2012.11.015

    View details for Web of Science ID 000314459200011

    View details for PubMedID 23246006

    View details for PubMedCentralID PMC3663144

  • Intracellular Context Affects Levels of a Chemically Dependent Destabilizing Domain PLOS ONE Sellmyer, M. A., Chen, L., Egeler, E. L., Rakhit, R., Wandless, T. J. 2012; 7 (9)

    Abstract

    The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.

    View details for DOI 10.1371/journal.pone.0043297

    View details for Web of Science ID 000308738500009

    View details for PubMedID 22984418

    View details for PubMedCentralID PMC3440426

  • Differential Trafficking of Transport Vesicles Contributes to the Localization of Dendritic Proteins CELL REPORTS Al-Bassam, S., Xu, M., Wandless, T. J., Arnold, D. B. 2012; 2 (1): 89-100

    Abstract

    In neurons, transmembrane proteins are targeted to dendrites in vesicles that traffic solely within the somatodendritic compartment. How these vesicles are retained within the somatodendritic domain is unknown. Here, we use a novel pulse-chase system, which allows synchronous release of exogenous transmembrane proteins from the endoplasmic reticulum to follow movements of post-Golgi transport vesicles. Surprisingly, we found that post-Golgi vesicles carrying dendritic proteins were equally likely to enter axons and dendrites. However, once such vesicles entered the axon, they very rarely moved beyond the axon initial segment but instead either halted or reversed direction in an actin and Myosin Va-dependent manner. In contrast, vesicles carrying either an axonal or a nonspecifically localized protein only rarely halted or reversed and instead generally proceeded to the distal axon. Thus, our results are consistent with the axon initial segment behaving as a vesicle filter that mediates the differential trafficking of transport vesicles.

    View details for DOI 10.1016/j.celrep.2012.05.018

    View details for Web of Science ID 000309713900011

    View details for PubMedID 22840400

    View details for PubMedCentralID PMC3408588

  • Destabilizing Domains Derived from the Human Estrogen Receptor JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Miyazaki, Y., Imoto, H., Chen, L., Wandless, T. J. 2012; 134 (9): 3942-3945

    Abstract

    Methods to rapidly and reversibly perturb the functions of specific proteins are desirable tools for studies of complex biological processes. We have demonstrated an experimental strategy to regulate the intracellular concentration of any protein of interest by using an engineered destabilizing protein domain and a cell-permeable small molecule. Destabilizing domains have general utility to confer instability to a wide range of proteins including integral transmembrane proteins. This study reports a destabilizing domain system based on the ligand binding domain of the estrogen receptor that can be regulated by one of two synthetic ligands, CMP8 or 4-hydroxytamoxifen.

    View details for DOI 10.1021/ja209933r

    View details for Web of Science ID 000301550800003

    View details for PubMedID 22332638

    View details for PubMedCentralID PMC3296833

  • Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo PLOS ONE Leong, H. S., Lizardo, M. M., Ablack, A., McPherson, V. A., Wandless, T. J., Chambers, A. F., Lewis, J. D. 2012; 7 (1)

    Abstract

    The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters.

    View details for DOI 10.1371/journal.pone.0030177

    View details for Web of Science ID 000301457200040

    View details for PubMedID 22276156

    View details for PubMedCentralID PMC3261888

  • Ligand-switchable Substrates for a Ubiquitin-Proteasome System JOURNAL OF BIOLOGICAL CHEMISTRY Egeler, E. L., Urner, L. M., Rakhit, R., Liu, C. W., Wandless, T. J. 2011; 286 (36): 31328-31336

    Abstract

    Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.

    View details for DOI 10.1074/jbc.M111.264101

    View details for Web of Science ID 000294487500028

    View details for PubMedID 21768107

    View details for PubMedCentralID PMC3173072

  • Evaluation of FKBP and DHFR based destabilizing domains in Saccharomyces cerevisiae BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Rakhit, R., Edwards, S. R., Iwamoto, M., Wandless, T. J. 2011; 21 (17): 4965-4968

    Abstract

    Two orthogonal destabilizing domains have been developed based on mutants of human FKBP12 as well as bacterial DHFR and these engineered domains have been used to control protein concentration in a variety of contexts in vitro and in vivo. FKBP12 based destabilizing domains cannot be rescued in the yeast Saccharomyces cerevisiae; ecDHFR based destabilizing domains are not degraded as efficiently in S. cerevisiae as in mammalian cells or Plasmodium, but provide a starting point for the development of domains with increased signal-to-noise in S. cerevisiae.

    View details for DOI 10.1016/j.bmcl.2011.06.006

    View details for Web of Science ID 000293884100006

    View details for PubMedID 21741238

    View details for PubMedCentralID PMC3156383

  • Small-molecule displacement of a cryptic degron causes conditional protein degradation NATURE CHEMICAL BIOLOGY Bonger, K. M., Chen, L., Liu, C. W., Wandless, T. J. 2011; 7 (8): 531-537

    Abstract

    The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.

    View details for DOI 10.1038/NCHEMBIO.598

    View details for Web of Science ID 000292825400014

    View details for PubMedID 21725303

    View details for PubMedCentralID PMC3139708

  • Chemical Control of FGF-2 Release for Promoting Calvarial Healing with Adipose Stem Cells JOURNAL OF BIOLOGICAL CHEMISTRY Kwan, M. D., Sellmyer, M. A., Quarto, N., Ho, A. M., Wandless, T. J., Longaker, M. T. 2011; 286 (13): 11307-11313

    Abstract

    Chemical control of protein secretion using a small molecule approach provides a powerful tool to optimize tissue engineering strategies by regulating the spatial and temporal dimensions that are exposed to a specific protein. We placed fibroblast growth factor 2 (FGF-2) under conditional control of a small molecule and demonstrated greater than 50-fold regulation of FGF-2 release as well as tunability, reversibility, and functionality in vitro. We then applied conditional control of FGF-2 secretion to a cell-based, skeletal tissue engineering construct consisting of adipose stem cells (ASCs) on a biomimetic scaffold to promote bone formation in a murine critical-sized calvarial defect model. ASCs are an easily harvested and abundant source of postnatal multipotent cells and have previously been demonstrated to regenerate bone in critical-sized defects. These results suggest that chemically controlled FGF-2 secretion can significantly increase bone formation by ASCs in vivo. This study represents a novel approach toward refining protein delivery for tissue engineering applications.

    View details for DOI 10.1074/jbc.M110.180042

    View details for Web of Science ID 000288797100043

    View details for PubMedID 21262969

    View details for PubMedCentralID PMC3064187

  • Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Muralidharan, V., Oksman, A., Iwamoto, M., Wandless, T. J., Goldberg, D. E. 2011; 108 (11): 4411-4416

    Abstract

    One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.

    View details for DOI 10.1073/pnas.1018449108

    View details for Web of Science ID 000288450900033

    View details for PubMedID 21368162

    View details for PubMedCentralID PMC3060247

  • A General Chemical Method to Regulate Protein Stability in the Mammalian Central Nervous System CHEMISTRY & BIOLOGY Iwamoto, M., Bjorklund, T., Lundberg, C., Kirik, D., Wandless, T. J. 2010; 17 (9): 981-988

    Abstract

    The ability to make specific perturbations to biological molecules in a cell or organism is a central experimental strategy in modern research biology. We have developed a general technique in which the stability of a specific protein is regulated by a cell-permeable small molecule. Mutants of the Escherichia coli dihydrofolate reductase (ecDHFR) were engineered to be degraded, and, when this destabilizing domain is fused to a protein of interest, its instability is conferred to the fused protein resulting in rapid degradation of the entire fusion protein. A small-molecule ligand trimethoprim (TMP) stabilizes the destabilizing domain in a rapid, reversible, and dose-dependent manner, and protein levels in the absence of TMP are barely detectable. The ability of TMP to cross the blood-brain barrier enables the tunable regulation of proteins expressed in the mammalian central nervous system.

    View details for DOI 10.1016/j.chembiol.2010.07.009

    View details for Web of Science ID 000283283200012

    View details for PubMedID 20851347

    View details for PubMedCentralID PMC2943492

  • A Plant-Like Kinase in Plasmodium falciparum Regulates Parasite Egress from Erythrocytes SCIENCE Dvorin, J. D., Martyn, D. C., Patel, S. D., Grimley, J. S., Collins, C. R., Hopp, C. S., Bright, A. T., Westenberger, S., Winzeler, E., Blackman, M. J., Baker, D. A., Wandless, T. J., Duraisingh, M. T. 2010; 328 (5980): 910-912

    Abstract

    Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate-dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

    View details for DOI 10.1126/science.1188191

    View details for Web of Science ID 000277618800054

    View details for PubMedID 20466936

    View details for PubMedCentralID PMC3109083

  • Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae YEAST Edwards, S. R., Wandless, T. J. 2010; 27 (4): 229-236

    Abstract

    Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co-expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5'-gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast-enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast-enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon-optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest.

    View details for DOI 10.1002/yea.1744

    View details for Web of Science ID 000276932700005

    View details for PubMedID 20017217

    View details for PubMedCentralID PMC3210078

  • General Methods to Conditionally Regulate Protein Function Wandless, T. J. FEDERATION AMER SOC EXP BIOL. 2010
  • A general method to rapidly and reversibly regulate protein function using synthetic small molecules Banaszynski, L. A., Wandless, T. J. AMER CHEMICAL SOC. 2009: 590
  • A general method for conditional regulation of protein stability in living animals. Cold Spring Harbor protocols Sellmyer, M. A., Thorne, S. H., Banaszynski, L. A., Contag, C. H., Wandless, T. J. 2009; 2009 (3): pdb prot5173-?

    View details for DOI 10.1101/pdb.prot5173

    View details for PubMedID 20147108

    View details for PubMedCentralID PMC3215584

  • Regulating protein stability in mammalian cells using small molecules. Cold Spring Harbor protocols Hagan, E. L., Banaszynski, L. A., Chen, L., Maynard-Smith, L. A., Wandless, T. J. 2009; 2009 (3): pdb prot5172-?

    View details for DOI 10.1101/pdb.prot5172

    View details for PubMedID 20147107

    View details for PubMedCentralID PMC3213858

  • The proteasome makes sense of mixed signals NATURE CHEMICAL BIOLOGY Wandless, T. J. 2009; 5 (1): 3-4

    View details for DOI 10.1038/nchembio0109-3

    View details for Web of Science ID 000261935500003

    View details for PubMedID 19088709

    View details for PubMedCentralID PMC2654574

  • Recent progress with FKBP-derived destabilizing domains BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Chu, B. W., Banaszynski, L. A., Chen, L., Wandless, T. J. 2008; 18 (22): 5941-5944

    Abstract

    The FKBP-derived destabilizing domains are increasingly being used to confer small molecule-dependent stability to many different proteins. The L106P domain confers instability to yellow fluorescent protein when it is fused to the N-terminus, the C-terminus, or spliced into the middle of yellow fluorescent protein, however multiple copies of L106P do not confer greater instability. These engineered destabilizing domains are not dominant to endogenous degrons that regulate protein stability.

    View details for DOI 10.1016/j.bmcl.2008.09.043

    View details for Web of Science ID 000260966800027

    View details for PubMedID 18815033

    View details for PubMedCentralID PMC2593907

  • Chemical control of protein stability and function in living mice NATURE MEDICINE Banaszynski, L. A., Sellmyer, M. A., Contag, C. H., Wandless, T. J., Thorne, S. H. 2008; 14 (10): 1123-1127

    Abstract

    Conditional control of protein function in vivo offers great potential for deconvoluting the roles of individual proteins in complicated systems. We recently developed a method in which a small protein domain, termed a destabilizing domain, confers instability to fusion protein partners in cultured cells. Instability is reversed when a cell-permeable small molecule binds this domain. Here we describe the use of this system to regulate protein function in living mammals. We show regulation of secreted proteins and their biological activity with conditional secretion of an immunomodulatory cytokine, resulting in tumor burden reduction in mouse models. Additionally, we use this approach to control the function of a specific protein after systemic delivery of the gene that encodes it to a tumor, suggesting uses for enhancing the specificity and efficacy of targeted gene-based therapies. This method represents a new strategy to regulate protein function in living organisms with a high level of control.

    View details for DOI 10.1038/nm.1754

    View details for Web of Science ID 000259892300044

    View details for PubMedID 18836461

    View details for PubMedCentralID PMC2605277

  • Tunable control of FGF-2 secretion for skeletal tissue engineering Kwan, M. D., Sellmyer, M. A., Quarto, N., Wandless, T. J., Longaker, M. T. ELSEVIER SCIENCE INC. 2008: S63–S64
  • Synthesis and analysis of stabilizing ligands for FKBP-derived destabilizing domains BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Grimley, J. S., Chen, D. A., Banaszynski, L. A., Wandless, T. J. 2008; 18 (2): 759-761

    Abstract

    We recently identified mutants of the human FKBP12 protein that are unstable and rapidly degraded when expressed in mammalian cells. We call these FKBP mutants destabilizing domains (DDs), because their instability is conferred to any protein fused to the DDs. A cell-permeable ligand binds tightly to the DDs and prevents their degradation, thus providing small molecule control over intracellular protein levels. We now report the synthesis and functional characterization of a stabilizing ligand called Shield-2. The synthesis of Shield-2 is efficient, and this ligand binds to the FKBP(F36V) protein with a dissociation constant of 29 nM.

    View details for DOI 10.1016/j.bmcl.2007.11.044

    View details for Web of Science ID 000253410100060

    View details for PubMedID 18039574

    View details for PubMedCentralID PMC2245802

  • Rapid control of protein level in the apicomplexan Toxoplasma gondii NATURE METHODS Herm-Goetz, A., Agop-Nersesian, C., Muenter, S., Grimley, J. S., Wandless, T. J., Frischknecht, F., Meissner, M. 2007; 4 (12): 1003-U8

    Abstract

    Analysis of gene function in apicomplexan parasites is limited by the absence of reverse genetic tools that allow easy and rapid modulation of protein levels. The fusion of a ligand-controlled destabilization domain (ddFKBP) to a protein of interest enables rapid and reversible protein stabilization in T. gondii. This allows an efficient functional analysis of proteins that have a dual role during host cell invasion and/or intracellular growth of the parasite.

    View details for DOI 10.1038/nmeth1134

    View details for Web of Science ID 000251282600011

    View details for PubMedID 17994029

    View details for PubMedCentralID PMC2601725

  • A directed approach for engineering conditional protein stability using biologically silent small molecules JOURNAL OF BIOLOGICAL CHEMISTRY Maynard-Smith, L. A., Chen, L., Banaszynski, L. A., Ooi, A. G., Wandless, T. J. 2007; 282 (34): 24866-24872

    Abstract

    The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506- and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (DeltaDeltaG(U-F)) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.

    View details for Web of Science ID 000248933000043

    View details for PubMedID 17603093

  • Rescue of degradation-prone mutants of the FK506-rapamycin binding (FRB) protein with chemical ligands CHEMBIOCHEM Stankunas, K., Bayle, J. H., Havranek, J. J., Wandless, T. J., Baker, D., Crabtree, G. R., Gestwicki, J. E. 2007; 8 (10): 1162-1169

    Abstract

    We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell, 2003, 12, 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Delta G) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilized by up to 6 kcal mol(-1) relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Delta G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Delta G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.

    View details for DOI 10.1002/cbic.200700087

    View details for Web of Science ID 000248067100014

    View details for PubMedID 17525916

  • Engineering small molecule specificity in nearly identical cellular environments BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Sellmyer, M. A., Stankunas, K., Briesewitz, R., Crabtree, G. R., Wandless, T. J. 2007; 17 (10): 2703-2705

    Abstract

    Methotrexate (MTX), an inhibitor of dihydrofolate reductase, was tethered to an FKBP12 ligand (SLF), and the resulting bifunctional molecule (MTXSLF) potently inhibits either enzyme but not both simultaneously. MTXSLF is cytotoxic to fibroblasts derived from FKBP12-null mice but is detoxified 40-fold by FKBP12 in wild-type fibroblasts. These studies demonstrate that non-target proteins in an otherwise identical genetic background can be used to predictably regulate the biological activity of synthetic molecules.

    View details for DOI 10.1016/j.bmcl.2007.03.012

    View details for Web of Science ID 000246675700005

    View details for PubMedID 17383876

    View details for PubMedCentralID PMC1949043

  • The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain JOURNAL OF BIOLOGICAL CHEMISTRY Edwards, S. R., Wandless, T. J. 2007; 282 (18): 13395-13401

    Abstract

    Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.

    View details for DOI 10.1074/jbc.M700498200

    View details for Web of Science ID 000246060300028

    View details for PubMedID 17350953

  • SIK1 is a class IIHDAC kinase that promotes survival of skeletal myocytes NATURE MEDICINE Berdeaux, R., Goebel, N., Banaszynski, L., Takemori, H., Wandless, T., Shelton, G. D., Montminy, M. 2007; 13 (5): 597-603

    Abstract

    During physical exercise, increases in motor neuron activity stimulate the expression of muscle-specific genes through the myocyte enhancer factor 2 (MEF2) family of transcription factors. Elevations in intracellular calcium increase MEF2 activity via the phosphorylation-dependent inactivation of class II histone deacetylases (HDACs). In studies to determine the role of the cAMP responsive element binding protein (CREB) in skeletal muscle, we found that mice expressing a dominant-negative CREB transgene (M-ACREB mice) exhibited a dystrophic phenotype along with reduced MEF2 activity. Class II HDAC phosphorylation was decreased in M-ACREB myofibers due to a reduction in amounts of Snf1lk (encoding salt inducible kinase, SIK1), a CREB target gene that functions as a class II HDAC kinase. Inhibiting class II HDAC activity either by viral expression of Snf1lk or by the administration of a small molecule antagonist improved the dystrophic phenotype in M-ACREB mice, pointing to an important role for the SIK1-HDAC pathway in regulating muscle function.

    View details for DOI 10.1038/nm1573

    View details for Web of Science ID 000246302800027

    View details for PubMedID 17468767

  • The enantioselective synthesis of phomopsin b ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Grimley, J. S., Sawayama, A. M., Tanaka, H., Stohlmeyer, M. M., Woiwode, T. E., Wandless, T. J. 2007; 46 (43): 8157-8159

    View details for DOI 10.1002/anie.200702537

    View details for Web of Science ID 000250966100008

    View details for PubMedID 17886312

    View details for PubMedCentralID PMC3290521

  • PI(3,4,5)P-3 and PI(4,5)P-2 lipids target proteins with polybasic clusters to the plasma membrane SCIENCE Heo, W. D., Inoue, T., Park, W. S., Kim, M. L., Park, B. O., Wandless, T. J., Meyer, T. 2006; 314 (5804): 1458-1461

    Abstract

    Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P2. Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.

    View details for DOI 10.1126/science.1134389

    View details for Web of Science ID 000242406100043

    View details for PubMedID 17095657

    View details for PubMedCentralID PMC3579512

  • ORGN 735-Progress towards the total synthesis of celogentin C Grimley, J. S., Wandless, T. J. AMER CHEMICAL SOC. 2006
  • A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules CELL Banaszynski, L. A., Chen, L., Maynard-Smith, L. A., Ooi, A. G., Wandless, T. J. 2006; 126 (5): 995-1004

    Abstract

    Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.

    View details for DOI 10.1016/j.cell.2006.07.025

    View details for Web of Science ID 000240675000025

    View details for PubMedID 16959577

    View details for PubMedCentralID PMC3290523

  • Conditional mislocalization to regulate protein function Edwards, Wandless, T. J. FEDERATION AMER SOC EXP BIOL. 2006: A76
  • Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity CHEMISTRY & BIOLOGY Bayle, J. H., Grimley, J. S., Stankunas, K., Gestwicki, J. E., Wandless, T. J., Crabtree, G. R. 2006; 13 (1): 99-107

    Abstract

    Controlling protein dimerization with small molecules has broad application to the study of protein function. Rapamycin has two binding surfaces: one that binds to FKBP12 and the other to the Frb domain of mTor/FRAP, directing their dimerization. Rapamycin is a potent cell growth inhibitor, but chemical modification of the surface contacting Frb alleviates this effect. Productive interactions with Frb-fused proteins can be restored by mutation of Frb to accommodate the rapamycin analog (a rapalog). We have quantitatively assessed the interaction between rapalogs functionalized at C16 and C20 and a panel of Frb mutants. Several drug-Frb mutant combinations have different and nonoverlapping specificities. These Frb-rapalog partners permit the selective control of different Frb fusion proteins without crossreaction. The orthogonal control of multiple target proteins broadens the capabilities of chemical induction of dimerization to regulate biologic processes.

    View details for DOI 10.1016/j.chembiol.2005.10.017

    View details for Web of Science ID 000235090900014

    View details for PubMedID 16426976

  • Conditional control of protein function CHEMISTRY & BIOLOGY Banaszynski, L. A., Wandless, T. J. 2006; 13 (1): 11-21

    Abstract

    Deciphering the myriad ways in which proteins interact with each other to give rise to complex behaviors that define living systems is a significant challenge. Using perturbations of DNA, genetic analyses have provided many insights into the functions of proteins encoded by specific genes. However, it can be difficult to study essential genes using these approaches, and many biological processes occur on a fast timescale that precludes study using genetic methods. For these reasons and others, it is often desirable to target proteins directly rather than the genes that encode them. Over the past 20 years, several methods to regulate protein function have been developed. In this review, we discuss the genesis and use of these methods, with particular emphasis on the elements of specificity, speed, and reversibility.

    View details for DOI 10.1016/j.chembiol.2005.10.010

    View details for Web of Science ID 000235090900005

    View details for PubMedID 16426967

  • A cell-permeable, activity-based probe for protein and lipid kinases JOURNAL OF BIOLOGICAL CHEMISTRY Yee, M., Fas, S. C., Stohlmeyer, M. M., Wandless, T. J., Cimprich, K. A. 2005; 280 (32): 29053-29059

    Abstract

    Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

    View details for DOI 10.1074/jbc.M504730200

    View details for Web of Science ID 000231021300030

    View details for PubMedID 15975929

  • An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways NATURE METHODS Inoue, T., Do Heo, W., Grimley, J. S., Wandless, T. J., Meyer, T. 2005; 2 (6): 415-418

    Abstract

    We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase-mediated signaling processes.

    View details for DOI 10.1038/NMETH763

    View details for Web of Science ID 000229424300011

    View details for PubMedID 15908919

    View details for PubMedCentralID PMC3579513

  • Characterization of the FKBP.rapamycin.FRB ternary complex. Journal of the American Chemical Society Banaszynski, L. A., Liu, C. W., Wandless, T. J. 2005; 127 (13): 4715-4721

    Abstract

    Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough analysis of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP.rapamycin.FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Analysis of the data shows that rapamycin binds to FRB with moderate affinity (K(d) = 26 +/- 0.8 microM). The FKBP12.rapamycin complex, however, binds to FRB 2000-fold more tightly (K(d) = 12 +/- 0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiological conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.

    View details for PubMedID 15796538

  • Characterization of the FKBP center dot Rapamycin center dot FRB ternary complex JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Banaszynski, L. A., Liu, C. W., Wandless, T. J. 2005; 127 (13): 4715-4721

    Abstract

    Rapamycin is an important immunosuppressant, a possible anticancer therapeutic, and a widely used research tool. Essential to its various functions is its ability to bind simultaneously to two different proteins, FKBP and mTOR. Despite its widespread use, a thorough analysis of the interactions between FKBP, rapamycin, and the rapamycin-binding domain of mTOR, FRB, is lacking. To probe the affinities involved in the formation of the FKBP.rapamycin.FRB complex, we used fluorescence polarization, surface plasmon resonance, and NMR spectroscopy. Analysis of the data shows that rapamycin binds to FRB with moderate affinity (K(d) = 26 +/- 0.8 microM). The FKBP12.rapamycin complex, however, binds to FRB 2000-fold more tightly (K(d) = 12 +/- 0.8 nM) than rapamycin alone. No interaction between FKBP and FRB was detected in the absence of rapamycin. These studies suggest that rapamycin's ability to bind to FRB, and by extension to mTOR, in the absence of FKBP is of little consequence under physiological conditions. Furthermore, protein-protein interactions at the FKBP12-FRB interface play a role in the stability of the ternary complex.

    View details for DOI 10.1021/ja043277y

    View details for Web of Science ID 000228089300041

  • Total synthesis of ustiloxin D and considerations on the origin of selectivity of the asymmetric allylic alkylation JOURNAL OF ORGANIC CHEMISTRY Sawayama, A. M., Tanaka, H., Wandless, T. J. 2004; 69 (25): 8810-8820

    Abstract

    As part of investigations into cell cycle checkpoint inhibitors, an asymmetric synthesis of the antimitotic natural product, ustiloxin D, has been completed. A salen-Al-catalyzed aldol reaction was employed to construct a chiral oxazoline 9 (99% yield, 98% ee) that served the dual purpose of installing the necessary 1,2-amino alcohol functionality as well as providing an efficient synthon for the requisite methylamino group at C9. The chiral aryl-alkyl ether was assembled using a Pd-catalyzed asymmetric allylic alkylation that notably delivered a product with stereochemistry opposite to that predicted by precedent. The linear tetrapeptide was subsequently cyclized to produce ustiloxin D. The mechanistic origin of the allylic alkylation selectivity was further investigated, and a working hypothesis for the origin of the observed stereoselectivity has been proposed.

    View details for DOI 10.1021/jo048854f

    View details for Web of Science ID 000225550300032

    View details for PubMedID 15575762

  • Quantitative analyses of bifunctional molecules BIOCHEMISTRY Braun, P. D., Wandless, T. J. 2004; 43 (18): 5406-5413

    Abstract

    Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications.

    View details for DOI 10.1021/bi035839g

    View details for Web of Science ID 000221343200030

    View details for PubMedID 15122906

  • Conditional protein alleles using knockin mice and a chemical inducer of dimerization MOLECULAR CELL Stankunas, K., Bayle, J. H., Gestwicki, J. E., Lin, Y. M., Wandless, T. J., Crabtree, G. R. 2003; 12 (6): 1615-1624

    Abstract

    We have developed a general method of making conditional alleles that allows the rapid and reversible regulation of specific proteins. A mouse line was produced in which proteins encoded by the endogenous glycogen synthase kinase-3 beta (GSK-3beta) gene are fused to an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3beta, producing a severe loss-of-function allele. In the presence of C20-MaRap, a highly specific, nontoxic, cell-permeable small molecule, GSK-3betaFRB* binds to the ubiquitously expressed FKBP12 protein. This interaction stabilizes GSK-3betaFRB* and restores both protein levels and activity. C20-MaRap-mediated stabilization is rapidly reversed by the addition of an FKBP12 binding competitor molecule. This technology may be applied to a wide range of FRB*-tagged mouse genes while retaining their native transcriptional control. Inducible stabilization could be valuable for many developmental and physiological studies and for drug target validation.

    View details for Web of Science ID 000187511600028

    View details for PubMedID 14690613

  • A bifunctional molecule that displays context-dependent cellular activity JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Braun, P. D., Barglow, K. T., Lin, Y. M., Akompong, T., Briesewitz, R., Ray, G. T., Haldar, K., Wandless, T. J. 2003; 125 (25): 7575-7580

    Abstract

    The cell-permeable dihydrofolate reductase inhibitor methotrexate was covalently linked to a ligand for the protein FKBP to create a bifunctional molecule called MTXSLF. The covalent tether between the two ligands was designed to be prohibitively short, so that unfavorable protein-protein interactions between DHFR and FKBP preclude formation of a trimeric complex. In vitro and in vivo experiments demonstrate that MTXSLF is an effective inhibitor of human DHFR, but that efficacy is decreased in the presence of human FKBP due to the high concentration of FKBP and its tight affinity for MTXSLF. MTXSLF also inhibits Plasmodium falciparum DHFR in vitro, but a low concentration of the weaker binding Plasmodium FKBP has no effect on the inhibitory potency of MTXSLF in vivo. These studies illustrate a potentially general strategy for modulating the biological activity of synthetic molecules that depends on the ligand-binding properties of a nontarget protein.

    View details for DOI 10.1021/ja035176q

    View details for Web of Science ID 000183646400033

    View details for PubMedID 12812497

  • Enantioselective total synthesis of ustiloxin D JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Tanaka, H., Sawayama, A. M., Wandless, T. J. 2003; 125 (23): 6864-6865

    Abstract

    Ustiloxin D and phomopsin A are potent antimitotic agents that bind to tubulin and interfere with cellular microtubule function. A synthetic strategy has been developed to allow access to both of the natural products as well as a variety of variants of the ustiloxin and phomopsin family members in order to provide sufficient quantities for biological studies. Herein we report the enantioselective total synthesis of ustiloxin D using a longest linear sequence of 20 steps. Four of the five stereocenters were set using catalytic asymmetric methodologies. In particular, Evans's new Al-catalyzed asymmetric aldol reaction facilitated access to both syn and anti products corresponding to the different benzylic stereochemistries found in ustiloxins and phomopsins. In addition, due to its high functional group tolerance, Trost's Pd-mediated etherification was used to construct the chiral tertiary alkyl-aryl ether. Taken together, these synthetic strategies allow us to use densely functionalized intermediates to realize an efficient synthesis of ustiloxin D.

    View details for DOI 10.1021/ja035429f

    View details for Web of Science ID 000183359300012

    View details for PubMedID 12783528

  • Calcineurin inhibitors and the generalization of the presenting protein strategy ADVANCES IN PROTEIN CHEMISTRY, VOL 56 Vogel, K. W., Briesewitz, R., Wandless, T. J., Crabtree, G. R. 2001; 56: 253-?

    View details for Web of Science ID 000168999900008

    View details for PubMedID 11329856

  • Synthesis of flavonol derivatives as probes of biological processes TETRAHEDRON LETTERS Tanaka, H., Stohlmeyer, M. M., Wandless, T. J., Taylor, L. P. 2000; 41 (50): 9735-9739
  • Mechanistic studies of affinity modulation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rosen, M. K., Amos, C. D., Wandless, T. J. 2000; 122 (48): 11979-11982

    View details for DOI 10.1021/ja002991m

    View details for Web of Science ID 000165696900029

  • A confederacy of bunches: Fundamentals and applications of a self-associating protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wandless, T. J. 2000; 97 (13): 6921-6923

    View details for Web of Science ID 000087811600001

    View details for PubMedID 10852948

    View details for PubMedCentralID PMC34359

  • Efficient synthesis of beta,gamma-dehydrovaline JOURNAL OF ORGANIC CHEMISTRY Woiwode, T. F., Wandless, T. J. 1999; 64 (20): 7670-7674
  • A stereospecific elimination to form dehydroamino acids: Synthesis of the phomopsin tripeptide side chain JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Stohlmeyer, M. M., Tanaka, H., Wandless, T. J. 1999; 121 (25): 6100-6101
  • Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Briesewitz, R., Ray, G. T., Wandless, T. J., Crabtree, G. R. 1999; 96 (5): 1953-1958

    Abstract

    A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein-protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds.

    View details for Web of Science ID 000078956600029

    View details for PubMedID 10051576

    View details for PubMedCentralID PMC26718

  • A simple and efficient method for the preparation of hindered alkyl-aryl ethers JOURNAL OF ORGANIC CHEMISTRY Woiwode, T. F., Rose, C., Wandless, T. J. 1998; 63 (25): 9594-9596
  • INHIBITION OF T-CELL SIGNALING BY IMMUNOPHILIN LIGAND COMPLEXES CORRELATES WITH LOSS OF CALCINEURIN PHOSPHATASE-ACTIVITY BIOCHEMISTRY Liu, J., Albers, M. W., Wandless, T. J., Luan, S., Alberg, D. G., Belshaw, P. J., Cohen, P., Mackintosh, C., Klee, C. B., Schreiber, S. L. 1992; 31 (16): 3896-3901

    Abstract

    Calcineurin, a Ca2+, calmodulin-dependent protein phosphatase, was recently found to bind with high affinity to two different immunosuppressant binding proteins (immunophilins) with absolute dependence on the presence of the immunosuppressants FK506 or cyclosporin A (CsA) [Liu et al. (1991) Cell 66, 807-815]. The binding affinities of the immunophilin-drug complexes toward calcineurin and the stoichiometry of the resultant multimeric complexes have now been determined, and structural elements of FK506, CsA, and calcineurin that are critical for mediating their interactions have been identified. Analogues of FK506 (FK520, FK523, 15-O-demethyl-FK520) and CsA (MeBm2t1-CsA and MeAla6-CsA) whose affinities for their cognate immunophilins do not correlate with their immunosuppressive activities have been prepared and evaluated in biochemical and cellular assays. We demonstrate a strong correlation between the ability of these analogues, when bound to their immunophilins, to inhibit the phosphatase activity of calcineurin and their ability to inhibit transcriptional activation by NF-AT, a T cell specific transcription factor that regulates IL-2 gene synthesis in human T cells. In addition, FKBP-FK506 and CyP-CsA do not inhibit members of the PP1, PP2A, and PP2C classes of serine/threonine phosphatases. These data suggest that calcineurin is the relevant cellular target of these immunosuppressive agents and is involved in Ca(2+)-dependent signal transduction pathways in, among others, T cells and mast cells.

    View details for Web of Science ID A1992HR18700002

    View details for PubMedID 1373650

  • MOLECULAR RECOGNITION OF IMMUNOPHILINS AND IMMUNOPHILIN-LIGAND COMPLEXES SYMP ON NEW DIRECTIONS IN ORGANIC SYNTHESIS, IN HONOR OF HARRY H WASSERMAN Schreiber, S. L., Liu, J., Albers, M. W., Rosen, M. K., Standaert, R. F., Wandless, T. J., Somers, P. K. PERGAMON-ELSEVIER SCIENCE LTD. 1992: 2545–58
  • IMMUNOPHILIN-LIGAND COMPLEXES AS PROBES OF INTRACELLULAR SIGNALING PATHWAYS TRANSPLANTATION PROCEEDINGS Schreiber, S. L., Liu, J., Albers, M. W., Karmacharya, R., Koh, E., Martin, P. K., Rosen, M. K., Standaert, R. F., Wandless, T. J. 1991; 23 (6): 2839-2844

    View details for Web of Science ID A1991GV17500041

    View details for PubMedID 1721293

  • SYNTHESIS AND ANALYSIS OF 506BD, A HIGH-AFFINITY LIGAND FOR THE IMMUNOPHILIN FKBP JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Somers, P. K., Wandless, T. J., Schreiber, S. L. 1991; 113 (21): 8045-8056
  • SOLUTION STRUCTURE OF FKBP, A ROTAMASE ENZYME AND RECEPTOR FOR FK506 AND RAPAMYCIN SCIENCE Michnick, S. W., Rosen, M. K., Wandless, T. J., Karplus, M., Schreiber, S. L. 1991; 252 (5007): 836-839

    Abstract

    Immunophilins, when complexed to immunosuppressive ligands, appear to inhibit signal transduction pathways that result in exocytosis and transcription. The solution structure of one of these, the human FK506 and rapamycin binding protein (FKBP), has been determined by nuclear magnetic resonance (NMR). FKBP has a previously unobserved antiparallel beta-sheet folding topology that results in a novel loop crossing and produces a large cavity lined by a conserved array of aromatic residues; this cavity serves as the rotamase active site and drug-binding pocket. There are other significant structural features (such as a protruding positively charged loop and an apparently flexible loop) that may be involved in the biological activity of FKBP.

    View details for Web of Science ID A1991FL12300044

    View details for PubMedID 1709301

  • PROBING IMMUNOSUPPRESSANT ACTION WITH A NONNATURAL IMMUNOPHILIN LIGAND SCIENCE Bierer, B. E., Somers, P. K., Wandless, T. J., Burakoff, S. J., Schreiber, S. L. 1990; 250 (4980): 556-559

    Abstract

    The immunosuppressants FK506 and rapamycin bind to the same immunophilin, FK506 binding protein (FKBP), and inhibit distinct signal transduction pathways in T lymphocytes. A nonnatural immunophilin ligand, 506BD, which contains only the common structural elements of FK506 and rapamycin, was synthesized and found to be a high-affinity ligand of FKBP and a potent inhibitor of FKBP rotamase activity. Whereas 506BD does not interfere with T cell activation, it does block the immunosuppressive effects of both FK506 and rapamycin. Thus, the common immunophilin binding element of these immunosuppressants, which is responsible for rotamase inhibition, is fused to different effector elements, resulting in the inhibition of different signaling pathways. Inhibition of rotamase activity is an insufficient requirement for mediating these effects.

    View details for Web of Science ID A1990EE66000037

    View details for PubMedID 1700475