Search

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.

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.

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.

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.

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.

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.

Src homology 2 (SH2) domains are phosphotyrosine binding modules found within many cytoplasmic proteins. A major function of SH2 domains is to bring about the physical assembly of signaling complexes. We now show that, in addition, simultaneous occupancy of both SH2 domains of the phosphotyrosine phosphatase SH-PTP2 (Syp, PTP 1D, PTP-2C) by a tethered peptide with two IRS-1-derived phosphorylation sites potently stimulates phosphatase activity. The concentration required for activation by the tethered peptide is 80-160-fold lower than either corresponding monophosphorylated peptide. Moreover, the diphosphorylated peptide stimulates catalytic activity 37-fold, compared with 9-16-fold for the monophosphorylated peptides. Mutational analyses of the SH2 domains of SH-PTP2 confirm that both SH2 domains participate in this effect. Binding studies with a tandem construct comprising the N- plus C-terminal SH2 domains show that the diphosphorylated peptide binds with 60-90-fold higher affinity than either monophosphorylated sequence. These results demonstrate that SH-PTP2 activity can be potently regulated by interacting via both of its SH2 domains with phosphoproteins having two cognate phosphorylation sites.

The cytoplasmic insulin receptor substrate-1 (IRS-1), which is multiply phosphorylated in vivo on tyrosine residues, is a known binding protein for the tandem src homology 2 (SH2) domain-containing protein tyrosine phosphatase, SH-PTP2. Eleven phosphotyrosyl (pY) peptides from IRS-1 were screened for allosteric activation of SH-PTP2 phosphatase activity toward phosphorylated, reduced, carboxyamidomethylated, and maleylated-lysozyme. Peptides IRS-1pY895, IRS-1pY1172, and IRS-1pY1222 showed up to 50-fold acceleration of dephosphorylation. Analyses of Arg to Lys mutants in either or both SH2 domains indicate that both the N-terminal (N-SH2) and C-terminal (C-SH2) domains function in allosteric activation. Direct determination by surface plasmon resonance of the dissociation constants between pY peptides and glutathione S-transferase fusions to N-SH2 and C-SH2 domains reveals a 240-fold preference of the N-SH2 domain (compared with the C-SH2 domain) for IRS-1pY1172. The N-SH2 domain prefers IRS-1pY1172 > IRS-1pY895 > IRS-1pY1222, whereas C-SH2 domain prefers IRS-1pY1222 > IRS-1pY895 > IRS-1pY1172. These data suggest that each SH2 domain can bind to a distinct pY sequence of multiply phosphorylated protein substrates such as IRS-1, while activating hydrolysis at a third pY sequence bound in the SH-PTP2 active site. In addition, proteolysis and truncation studies reveal an autoregulatory function for the C-terminal region of SH-PTP2. Limited tryptic cleavage within the C-terminus results in 27-fold activation of protein tyrosine phosphatase activity. The activated tryptic fragment cannot be further activated by pY peptide binding to the SH2 domains indicating that autoregulatory functions of the SH2 domains are dependent on the C-terminal region. These data suggest that multiple levels for control of SH-PTP2 enzymatic activity may exist in vitro and in vivo.