Ph.D., Harvard University, Chemistry (1993)
B.S., Trinity University, Biochemistry (1988)
See http://wandless.stanford.edu/research.html for a full description.
Overview - We employ an interdisciplinary approach to studies of biological systems, combining a bit of synthetic chemistry with biochemistry, cell biology, and structural biology. More specifically, 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 new experiments with unprecedented control. These new techniques may also provide a window into mechanisms involved in maintaining cellular homeostasis. Protein quality control is a particular interest at present.
New Approaches for Conditional Control of Protein Function - We recently developed a new 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, synthetic ligand called Shield-1. We screened a library of FKBP sequences to identify mutants that are unstable in the absence of Shield-1. Additional screening enriched for fusion proteins that are stabilized by Shield-1, and 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. The system works in cultured mammalian cells and in living mice. Banaszynski et al (Cell 2006 and Nature Medicine 2008) describe the system in detail. Building on these early successes, we have expanded this technology is several useful ways. First, we have engineered additional DD systems using orthogonal protein- ligand combinations. Second, we have 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. Third, we have engineered an experimental system that works in yeast.
Protein Quality Control in Mammalian Cells - The DDs can be thought of as model substrates that have the potential to bridge our understanding of the physical basis of protein stability in vitro with intracellular stability. The ability to conditionally regulate the structures of these domains using high-affinity ligands allows us to quantitatively correlate specific biophysical properties with biological stability. There are two main questions that we would like to understand in this area. First, we focus on the DD proteins themselves. What property or properties of these DDs leads them to be recognized and degraded by the cellular quality control machinery? Second, we would like to have a more complete picture of the proteins that are involved in these quality control surveillance pathways. Efforts to address both of these questions are currently underway.
Protein-Protein Interactions & Bifunctional Molecules - One of the interests of the Wandless lab has been the study of synthetic molecules that are capable of binding to two different proteins. These molecules are typically comprised of two ligands for their respective proteins, and the ligands are linked by a covalent tether. In cases where the tether between the ligands is relatively long, both proteins can simultaneously bind to the bifunctional molecule to form a trimeric complex. The formation of a trimeric complex creates an environment wherein interactions between the two proteins are possible. These nascent protein-protein interactions may contribute either favorably or unfavorably to the overall stability of the ternary complex, and we have shown that these protein-protein interactions may endow the bifunctional molecules with biophysical and biological properties that are significantly different from the monomeric ligands that comprise them.
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 Web of Science ID 000330098800012
View details for PubMedID 24180414
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
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 Web of Science ID 000320328700060
View details for PubMedID 23650381
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
View details for Web of Science ID 000165730300009
View details for Web of Science ID 000083036800062
View details for Web of Science ID 000081216800043
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 Web of Science ID 000077498400079