Heat shock protein-90 (Hsp90) acts as a repressor of peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARbeta activity

The nuclear receptor (NR) peroxisome proliferator-activated receptor-alpha (PPARalpha) mediates the effects of several hypolipidemic drugs, endogenous fatty acids, and peroxisome proliferators. Despite belonging to a class of NR not known to interact with cytosolic chaperone complexes, we have recently shown that PPARalpha interacts with heat shock protein 90 (Hsp90), although the biological consequence of this association was unknown. In the present study, PPARalpha directly associated with Hsp90 in vitro to a much greater extent than either PPARbeta or PPARgamma. This interaction is similar to other NR-Hsp90 complexes with association occurring between the middle of Hsp90 and the hinge (D) and ligand binding domain (EF) of PPARalpha. Using several different approaches to disrupt Hsp90 complexes within the cell, we demonstrate that Hsp90 is a repressor of both PPARalpha and PPARbeta activity. Treatment with geldanamycin (GA) increased the activity of PPARalpha and in the presence of ligand in transient transfection assays. PPARalpha-response element (PPRE)-reporter assays in a stable cell line treated with GA resulted in enhanced expression of a known target gene, acyl-CoA oxidase. Similarly, overexpression of the tetratricopeptide repeat (TPR) of protein phosphatase 5 (PP5) increased PPARalpha or PPARbeta activity in a PPRE-reporter assay and decreased the interaction between PPARalpha or PPARbeta and Hsp90 in a mammalian two-hybrid assay. Finally, cotransfection with the C-terminal hsp-interacting protein (CHIP) construct, a TPR-containing ubiquitin ligase that interacts with hsp90, increased PPARalpha's and decreased PPARbeta's ability to regulate PPRE-reporter activity upon ligand activation. All three methods to disrupt Hsp90 function (GA, PP5-TPR, CHIP) resulted in an alteration in PPARalpha or PPARbeta activity to a much greater extent than PPARgamma. While FKBP52 had no effect on PPARalpha activity, p23 greatly enhanced constitutive and Wy14 643 induced PPRE-reporter activity. Thus, we describe the chaperone complex as being a regulator of PPARalpha and PPARbeta activity and have identified a novel, subtype-specific, inhibitory role for Hsp90.     

Involvement of heat shock protein (Hsp)90 beta but not Hsp90 alpha in antiapoptotic effect of CpG-B oligodeoxynucleotide

Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune responses in a TLR9-dependent manner. In this study, we found that stimulation of mouse macrophages and dendritic cells with B-type CpG ODN (CpG-B ODN) increased the cellular level of heat shock protein (Hsp) 90beta but not Hsp90alpha and prevented apoptosis induced by serum starvation or staurosporine treatment. The CpG-B ODN-induced Hsp90beta expression depended on TLR9, MyD88, and PI3K. Inhibition of Hsp90beta level by expressing small-interfering RNA suppressed not only Hsp90beta expression but also PI3K-dependent phosphorylation of Akt and CpG-B ODN-mediated antiapoptosis. Additional studies demonstrated that as described by other group in mast cells, Hsp90beta but not Hsp90alpha was associated with Bcl-2. Inhibition of Hsp90beta suppressed the CpG-B ODN-induced association of Hsp90beta with Bcl-2 and impaired the inhibitory effect of CpG-B ODN in the release of cytochrome c and activation of caspase-3. This study thus reveals the involvement of Hsp90beta but not Hsp90alpha in CpG-B ODN-mediated antiapoptotic response and that Hsp90beta is distinct from Hsp90alpha in regulation of the cellular function of immune cells.     

Hsp90 (heat shock protein 90) inhibitor occupancy is a direct determinant of client protein degradation and tumor growth arrest in vivo

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.     

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.     

Characterization of the nucleotide binding properties of the 90 kDa heat shock protein (Hsp90)

The recent crystallization and structural analysis of the ATP(ADP)-complex of the N-terminal domain of the 90 kDa heat shock protein (Hsp90) confirmed our earlier findings on the ATP-binding properties of Hsp90. Here we further characterize the nucleotide binding of Hsp90 by demonstrating that surface plasmon resonance measurements also indicate a low-affinity binding of ATP to Hsp90 and that [α-³²P]ATP seems to have an equal preference for monomers, dimers and oligomers of Hsp90 on native polyacrylamide gels. Finally we discuss some of our results which raise the possibility that Hsp90 has two nucleotide binding sites (one in its N-terminal and another in the C-terminal domain) and that the nucleotide binding to Hsp90 dimers may display a positive cooperativity under some special conditions. The submillimolar binding affinity of ATP to Hsp90 allows the regulation of some Hsp90-related functions just in the range of ATP-level fluctuations during stress or during the cell cycle.     

Functional characterization of orchardgrass endoplasmic reticulum-resident Hsp90 (DgHsp90) as a chaperone and an ATPase

Hsp90 proteins are essential molecular chaperones regulating multiple cellular processes in distinct subcellular organelles. In this study, we report the functional characterization of a cDNA encoding endoplasmic reticulum (ER)-resident Hsp90 from orchardgrass (DgHsp90). DgHsp90 is a 2742 bp cDNA with an open reading frame predicted to encode an 808 amino acid protein. DgHsp90 has a well conserved N-terminal ATPase domain and a C-terminal Hsp90 domain and ER-retention motif. Expression of DgHsp90 increased during heat stress at 35 °C or H₂O₂ treatment. DgHsp90 also functions as a chaperone protein by preventing thermal aggregation of malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 2.3.3.1). The intrinsic ATPase activity of DgHsp90 was inhibited by geldanamycin, an Hsp90 inhibitor, and the inhibition reduced the chaperone activity of DgHsp90. Yeast cells overexpressing DgHsp90 exhibited enhanced thermotolerance.     

Independent regulation of Hsp70 and Hsp90 chaperones by Hsp70/Hsp90-organizing protein Sti1 (Hop1)

Hsp70 and Hsp90 protein chaperones cooperate in a protein-folding pathway required by many "client" proteins. The co-chaperone Sti1p coordinates functions of Hsp70 and Hsp90 in this pathway. Sti1p has three tetratricopeptide repeat (TPR) domains. TPR1 binds Hsp70, TPR2a binds Hsp90, and the ligand for TPR2b is unknown. Although Sti1p is thought to be dedicated to the client folding pathway, we earlier showed that Sti1p regulated Hsp70, independently of Hsp90, in a way that impairs yeast [PSI+] prion propagation. Using this prion system to monitor Sti1p regulation of Hsp70 and an Hsp90-inhibiting compound to monitor Hsp90 regulation, we identified Sti1p mutations that separately affect Hsp70 and Hsp90. TPR1 mutations impaired Sti1p regulation of Hsp70, but deletion of TPR2a and TPR2b did not. Conversely, TPR2a and TPR2b mutations impaired Sti1p regulation of Hsp90, but deletion of TPR1 did not. All Sti1p mutations variously impaired the client folding pathway, which requires both Hsp70 and Hsp90. Thus, Sti1p regulated Hsp70 and Hsp90 separately, Hsp90 is implicated as a TPR2b ligand, and mutations separately affecting regulation of either chaperone impair a pathway that is dependent upon both. We further demonstrate that client folding depended upon bridging of Hsp70 and Hsp90 by Sti1p and find conservation of the independent regulation of Hsp70 and Hsp90 by human Hop1.     

Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers

Hsp90 is an ATP dependent molecular chaperone protein which integrates multiple oncogenic pathways. As such, Hsp90 inhibition is a promising anti-cancer strategy. Several inhibitors that act on Hsp90 by binding to its N-terminal ATP pocket have entered clinical evaluation. Robust pre-clinical data suggested anti-tumor activity in multiple cancer types. Clinically, encouraging results have been demonstrated in melanoma, acute myeloid leukemia, castrate refractory prostate cancer, non-small cell lung carcinoma and multiple myeloma. In breast cancer, proof-of-concept was demonstrated by first generation Hsp90 inhibitors in combination with trastuzumab mainly in human epidermal growth factor receptor 2 (HER2)+metastatic breast cancer. There are a multitude of second generation Hsp90 inhibitors currently under investigation. To date, however, there is no FDA approved Hsp90 inhibitor nor standardized assay to ascertain Hsp90 inhibition. This review summarizes the current status of both first and second generation Hsp90 inhibitors based on their chemical classification and stage of clinical development. It also discusses the pharmacodynamic assays currently implemented in clinic as well as other novel strategies aimed at enhancing the effectiveness of Hsp90 inhibitors. Ultimately, these efforts will aid in maximizing the full potential of this class of agents. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Discovery of aminoquinolines as a new class of potent inhibitors of heat shock protein 90 (Hsp90): Synthesis, biology, and molecular modeling

The molecular chaperone Hsp90 plays important roles in maintaining malignant phenotypes. Recent studies suggest that Hsp90 exerts high-affinity interactions with multiple oncoproteins, which are essential for the growth of tumor cells. As a result, research has focused on finding Hsp90 probes as potential and selective anticancer agents. In a high-throughput screening exercise, we identified quinoline 7 as a moderate inhibitor of Hsp90. Further hit identification, SAR studies, and biological investigation revealed several synthetic analogs in this series with micromolar activities in both fluorescent polarization (FP) assay and a cell-based Western blot (WB) assay. These compounds represent a new class of Hsp90 inhibitors with simple chemical structures.     

Intracellular localization of the 90 kDA heat shock protein (HSP90alpha) determined by expression of a EGFP-HSP90alpha-fusion protein in unstressed and heat stressed 3T3 cells

Heat shock protein 90 (Hsp90) is an abundant protein and essential for all eukaryotic cells. The expression of Hsp90 is further enhanced after exposure to stress factors, e.g. a heat shock. Many proteins interacting with Hsp90 as well as the various functions for Hsp90 have been described. In this study, an Hsp90alpha fusion protein along with the enhanced green fluorescence protein (EGFP) was expressed under the control of the human cytomegalovirus immediate early promoter. EGFP-Hsp90alpha was mainly localized in the cytoplasm, with only minor amounts inside the nuclei. No EGFP-Hsp90alpha could be detected inside the nucleoli. Following exposure to elevated temperatures, higher amounts of EGFP-Hsp90alpha are inside the nucleus, but not within the nucleoli. As the most remarkable finding under these conditions, an association of EGFP-Hsp90alpha with the nuclear membrane became visible.     

Functioning of the Hsp90 machine in chaperoning checkpoint kinase I (Chk1) and the progesterone receptor (PR)

Hsp90 is an abundant and highly conserved chaperone that functions at later stages of protein folding to maintain and regulate the activity of client proteins. Using a recently described in vitro system to fold a functional model kinase Chk1, we performed a side-by-side comparison of the Hsp90-dependent chaperoning of Chk1 to that of the progesterone receptor (PR) and show that these distinct types of clients have different chaperoning requirements. The less stable PR required more total chaperone protein(s) and p23, whereas Chk1 folding was critically dependent on Cdc37. When the 2 clients were reconstituted under identical conditions, each client folding was dose dependent for Hsp90 protein levels and was inhibited by geldanamycin. Using this tractable system, we found that Chk1 kinase folding was more effective if we used a type II Hsp40 cochaperone, whereas PR is chaperoned equally well with a type I or type II Hsp40. Additional dissection of Chk1-chaperone complexes and the resulting kinase activity suggests that kinase folding, like that previously shown for PR, is a dynamic, multistep process. Importantly, the cochaperones Hop and Cdc37 cooperate as the kinase transitions from immature Hsp70- to mature Hsp90-predominant complexes.     

Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion

NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.     

Evidence that the novobiocin-sensitive ATP-binding site of the heat shock protein 90 (hsp90) is necessary for its autophosphorylation

The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced. It is assumed, that under these conditions Hsp90 is required to protect other proteins from aggregation. One property of Hsp90 is its ability to undergo autophosphorylation. The N-terminal domain of Hsp90 has been shown to contain an unusual ATP-binding site. A well-known inhibitor of Hsp90 function is geldanamycin binding to the N-terminal ATP-binding site with high affinity. Recently it was shown that Hsp90 possesses a second ATP-binding site in the C-terminal region, which can be competed with novobiocin. Autophosphorylation of Hsp90 was analysed by incubation with gamma(32)P-ATP. Addition of geldanamycin did not interfere with the capability for autophosphorylation, while novobiocin indeed did. These results suggest that the C-terminal ATP-binding site is required for autophosphorylation of Hsp90.     

Secreted heat shock protein-90 (Hsp90) in wound healing and cancer

Extracellular Hsp90 proteins, including "membrane-bound", "released" and "secreted", were first reported more than two decades ago. Only studies of the past 7years have begun to reveal a picture for when, how and why Hsp90 gets exported by both normal and tumor cells. Normal cells secrete Hsp90 in response to tissue injury. Tumor cells have managed to constitutively secrete Hsp90 for tissue invasion. In either case, sufficient supply of the extracellular Hsp90 can be guaranteed by its unusually abundant storage inside the cells. A well-characterized function of secreted Hsp90α is to promote cell motility, a crucial event for both wound healing and cancer. The reported targets for extracellular Hsp90α include MMP2, LRP-1, tyrosine kinase receptors and possibly more. The pro-motility activity of secreted Hsp90α resides within a fragment, called 'F-5', at the boundary between linker region and middle domain. Inhibition of its secretion, neutralization of its extracellular action or interruption of its signaling through LRP-1 block wound healing and tumor invasion in vitro and in vivo. In normal tissue, topical application of F-5 promotes acute and diabetic wound healing far more effectively than US FDA-approved conventional growth factor therapy in mice. In cancer, drugs that selectively target the F-5 region of secreted Hsp90 by cancer cells may be more effective and less toxic than those that target the ATPase of the intracellular Hsp90. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase （IKK）

Autophagic and proteasomal proteolysis are two major pathways for degradation of cellular constituents. Current models suggest that autophagy is responsible for the nonselective bulk degradation of long-lived proteins and organelles while the proteasome specifically degrades short-lived proteins including misfolded proteins caused by the absence of Hsp90 function. Here, we show that the IκB kinase （IKK）, an essential activator of NF-κB, is selectively degraded by autophagy when Hsp90 is inhibited by geldanamycin （GA）, a specific Hsp90 inhibitor showing highly effective anti-tumor activity. We find that in this case inactivation of ubiquitination or proteasome fails to block IKK degradation. However, inhibition of autophagy by an autophagy inhibitor or knockout of Atg5, a key component of the autophagy pathway, significantly rescues IKK from GA-induced degradation. These findings provide the first evidence that an Hsp90 client may be degraded by a mechanism different from the proteasome pathway and establish a molecular link among Hsp90, NF-κB and autophagy     

Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of growth and differentiation, whose aberrant activation causes a number of genetic diseases including achondroplasia and cancer. Hsp90 is a specialized molecular chaperone involved in stabilizing a select set of proteins termed clients. Here, we delineate the relationship of Hsp90 and co-chaperone Cdc37 with FGFR3 and the FGFR family. FGFR3 strongly associates with these chaperone complexes and depends on them for stability and function. Inhibition of Hsp90 function using the geldanamycin analog 17-AAG induces the ubiquitination and degradation of FGFR3 and reduces the signaling capacity of FGFR3. Other FGFRs weakly interact with these chaperones and are differentially influenced by Hsp90 inhibition. The Hsp90-related ubiquitin ligase CHIP is able to interact and destabilize FGFR3. Our results establish FGFR3 as a strong Hsp90 client and suggest that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease.     

Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90beta,a property that can be exploited in screens for new Hsp90 chaperone inhibitors

Heat shock protein 90 (Hsp90) is essential for activation of many of the most important regulatory proteins of eukaryotic cells. It is an extremely conserved protein, such that heterologous expressions of either human Hsp90beta or Caenorhabditis elegans Hsp90 will provide the essential Hsp90 function in yeast. The ability of these metazoan Hsp90s to provide this Hsp90 function to yeast cells requires Sti, a Hsp90 system cochaperone. Yeast that is expressing human Hsp90beta in place of the normal native yeast Hsp90 is selectively hypersensitised to Hsp90 inhibitor drugs. Hsp90 drugs are promising anticancer agents, their administration simultaneously destabilizing a number of the proteins critical to multistep carcinogenesis. Though one of these drugs (17-allylaminogeldanamycin, 17-AAG) is now progressing to Phase 2 clinical trials, there is a pressing need to identify selective Hsp90 inhibitors that are more soluble than 17-AAG. High-throughput screening for chemical agents that exert greater inhibitory effects against yeast expressing the human Hsp90beta relative to yeast expressing its native Hsp90 should therefore facilitate the search for new Hsp90 inhibitors.     

Delimitation of two regions in the 90-kDa heat shock protein (Hsp90) able to interact with the glucocorticosteroid receptor (GR)

The role of the 90-kDa heat shock protein (Hsp90) as a chaperone and its regulatory functions for cellular proteins such as the glucocorticosteroid receptor (GR) depends on the direct interaction of the Hsp90 with the corresponding protein as part of a multiprotein complex. The search for the amino acid sequence(s) in Hsp90 involved in interaction with the human GR has been carried out by mutational deletion analysis in whole cells, studying the effects of interaction on the nucleocytoplasmic distributions of transiently expressed Hsp90 and GR derivatives in COS-7 cells. Using a recently developed confocal microscopic immunofluorescence method that allows quantification of the nucleocytoplasmic ratios of the proteins in individual cells and statistical comparison of cell populations, two subregions of the Hsp90 molecule have been defined that allow interaction with GR (residues 206-291 and 446-581). The latter region may play a fundamental role in the interaction, while the former may merely stabilize the binding to GR of the intact Hsp90 molecule. Moreover, the dissection of the Hsp90 molecule allowed us to define two regions displaying nuclear localization activity (residues 1-206 and 381-581), followed by two regions having a predominantly cytoplasmic localization activity (residues 287-381 and 581-728) and counteracting the nuclear localization activities.