Regulation of the Hsp90 system

Hsp90 is a highly conserved and abundant chaperone. It participates in essential cellular activities by supporting the maturation process of its client proteins, many of which are protein kinases and steroid receptors. Client processing is achieved via extensive conformational changes within the dimeric chaperone. This requires an ATP hydrolysis activity that is controlled by auto-inhibitory mechanisms and several structurally diverse cofactors. Especially the client-specificity of Hsp90 depends on client-specific cofactors, which can adapt Hsp90's activities to the client requirements at different conditions and in different cell types. Additionally, post-translational modifications can influence almost every aspect of Hsp90's interactions and activities. In this review, we present these regulatory principles, discuss the factors that have an impact on Hsp90's function and elaborate the mechanisms that are responsible for regulating the Hsp90 machinery.     

Evidence for chaperone heterocomplexes containing both Hsp90 and VCP

With assistance from co-chaperone partner proteins, Hsp90 plays an essential positive role in supporting the structure and function of numerous client proteins in vivo. Hsp90's co-chaperone partnerships are believed to regulate and/or target its function. Here we describe associations between Hsp90 chaperone machinery and another chaperone, the 97-kDa valosin-containing protein VCP. Coimmunoadsorption assays indicate that VCP occurs in one or more native heterocomplexes containing Hsp90 and the Hsp90 partner proteins Cdc37, FKBP52, and p23. Functional characterizations indicate that VCP is not an Hsp90 substrate, but rather demonstrate the biochemical hallmarks of an Hsp90 co-chaperone. Potential roles for a collaboration between for Hsp90 and VCP are discussed.     

Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90-cochaperone-client interactions

Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Recently, novobiocin has been shown to bind to a second nucleotide binding site located within the C-terminal domain of Hsp90. In this report, we have examined the effect of novobiocin on Hsp90 function in reticulocyte lysate. Novobiocin specifically inhibited the maturation of the heme-regulated eIF2alpha kinase (HRI) in a concentration-dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 cochaperones p23, FKBP52, and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by 10-fold. The recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/cochaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's autokinase activity. The data suggest that binding of novobiocin to the C-terminal nucleotide binding site of Hsp90 induces a change in Hsp90's conformation leading to the dissociation of bound kinase. The unique structure and properties of novobocin-bound Hsp90 suggest that it may represent the "client-release" conformation of the Hsp90 machine.     

The 'active life' of Hsp90 complexes

Hsp90 forms a variety of complexes differing both in clientele and co-chaperones. Central to the role of co-chaperones in the formation of Hsp90 complexes is the delivery of client proteins and the regulation of the ATPase activity of Hsp90. Determining the mechanisms by which co-chaperones regulate Hsp90 is essential in understanding the assembly of these complexes and the activation and maturation of Hsp90's clientele. Mechanistically, co-chaperones alter the kinetics of the ATP-coupled conformational changes of Hsp90. The structural changes leading to the formation of a catalytically active unit involve all regions of the Hsp90 dimer. Their complexity has allowed different orthologues of Hsp90 to evolve kinetically in slightly different ways. The interaction of the cytosolic Hsp90 with a variety of co-chaperones lends itself to a complex set of different regulatory mechanisms that modulate Hsp90's conformation and ATPase activity. It also appears that the conformational switches of Hsp90 are not necessarily coupled under all circumstances. Here, I described different co-chaperone complexes and then discuss in detail the mechanisms and role that specific co-chaperones play in this. I will also discuss emerging evidence that post-translational modifications also affect the ATPase activity of Hsp90, and thus complex formation. Finally, I will present evidence showing how Hsp90's active site, although being highly conserved, can be altered to show resistance to drug binding, but still maintain ATP binding and ATPase activity. Such changes are therefore unlikely to significantly alter Hsp90's interactions with client proteins and co-chaperones. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

The co-chaperone p23 arrests the Hsp90 ATPase cycle to trap client proteins

The action of the molecular chaperone Hsp90 is essential for the activation and assembly of an increasing number of client proteins. This function of Hsp90 has been proposed to be governed by conformational changes driven by ATP binding and hydrolysis. Association of co-chaperones and client proteins regulate the ATPase activity of Hsp90. Here, we have examined the inhibition of the ATPase activity of human Hsp90beta by one such co-chaperone, human p23. We demonstrate that human p23 interacts with Hsp90 in both the absence and presence of nucleotide with a higher affinity in the presence of the ATP analogue AMP-PNP. This is consistent with an analysis of the effect of p23 on the steady-state kinetics that revealed a mixed mechanism of inhibition. Mass spectrometry of the intact Hsp90.p23 complex determined the stoichiometry of binding to be one p23 to each subunit of the Hsp90 dimer. p23 was also shown to interact with a monomeric, truncated fragment of Hsp90, lacking the C-terminal homodimerisation domain, indicating dimerisation of Hsp90 is not a prerequisite for association with p23. Complex formation between Hsp90 and p23 increased the apparent affinity of Hsp90 for AMP-PNP and completely inhibited the ATPase activity. We propose a model where the role of p23 is to lock individual subunits of Hsp90 in an ATP-dependent conformational state that has a high affinity for client proteins.     

Functional dissection of cdc37: characterization of domain structure and amino acid residues critical for protein kinase binding

Hsp90 and its co-chaperone Cdc37 facilitate the folding and activation of numerous protein kinases. In this report, we examine the structure-function relationships that regulate the interaction of Cdc37 with Hsp90 and with an Hsp90-dependent kinase, the heme-regulated eIF2alpha kinase (HRI). Limited proteolysis of native and recombinant Cdc37, in conjunction with MALDI-TOF mass spectrometry analysis of peptide fragments and peptide microsequencing, indicates that Cdc37 is comprised of three discrete domains. The N-terminal domain (residues 1-126) interacts with client HRI molecules. Cdc37's middle domain (residues 128-282) interacts with Hsp90, but does not bind to HRI. The C-terminal domain of Cdc37 (residues 283-378) does not bind Hsp90 or kinase, and no functions were ascribable to this domain. Functional assays did, however, suggest that residues S127-G163 of Cdc37 serve as an interdomain switch that modulates the ability of Cdc37 to sense Hsp90's conformation and thereby mediate Hsp90's regulation of Cdc37's kinase-binding activity. Additionally, scanning alanine mutagenesis identified four amino acid residues at the N-terminus of Cdc37 that are critical for high-affinity binding of Cdc37 to client HRI molecules. One mutation, Cdc37/W7A, also implicated this region as an interpreter of Hsp90's conformation. Results illuminate the specific Cdc37 motifs underlying the allosteric interactions that regulate binding of Hsp90-Cdc37 to immature kinase molecules.     

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.     

Hsp90 structure and function studied by NMR spectroscopy

The molecular chaperone Hsp90 plays a crucial role in folding and maturation of regulatory proteins. Key aspects of Hsp90's molecular mechanism and its adenosine-5'-triphosphate (ATP)-controlled active cycle remain elusive. In particular the role of conformational changes during the ATPase cycle and the molecular basis of the interactions with substrate proteins are poorly understood. The dynamic nature of the Hsp90 machine designates nuclear magnetic resonance (NMR) spectroscopy as an attractive method to unravel both the chaperoning mechanism and interaction with partner proteins. NMR is particularly suitable to provide a dynamic picture of protein-protein interactions at atomic resolution. Hsp90 is rather a challenging protein for NMR studies, due to its high molecular weight and its structural flexibility. The recent technologic advances allowed overcoming many of the traditional obstacles. Here, we describe the different approaches that allowed the investigation of Hsp90 using state-of-the-art NMR methods and the results that were obtained. NMR spectroscopy contributed to understanding Hsp90's interaction with the co-chaperones p23, Aha1 and Cdc37. A particular exciting prospect of NMR, however, is the analysis of Hsp90 interaction with substrate proteins. Here, the ability of this method to contribute to the structural characterization of not fully folded proteins becomes crucial. Especially the interaction of Hsp90 with one of its natural clients, the tumour suppressor p53, has been intensively studied by NMR spectroscopy. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

An acetylation site in the middle domain of Hsp90 regulates chaperone function

Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.     

Structure and in vivo function of Hsp90

Until recently, Hsp90 was one of the least well understood of the molecular chaperones, but considerable progress is now being made in unravelling its biochemistry. Hsp90 has now been shown to possess an inherent ATPase that is essential for the activation of authentic 'client' proteins in vivo and in vitro. The molecular detail of Hsp90's interactions with co-chaperones is also becoming clearer and the identification of key roles in assembling regulatory and signalling pathways has made it a target for anticancer drug development. Despite this, a clear understanding of how Hsp90 contributes to the folding and/or activation of its client proteins remains some way off.     

Molecular analysis of Plasmodium falciparum co-chaperone Aha1 supports its interaction with and regulation of Hsp90 in the malaria parasite

The recent recognition of Plasmodium falciparum Hsp90 (PfHsp90) as a promising anti-malaria drug target has sparked interest in identifying factors that regulate its function and drug-interaction. Co-chaperones are well-known regulators of Hsp90's chaperone function, and certain members have been implicated in conferring protection against lethal cellular effects of Hsp90-specific inhibitors. In this context, studies on PfHsp90's co-chaperones are imperative to gain insight into the regulation of the chaperone in the malaria parasite. In this study, a putative co-chaperone P. falciparum Aha1 (PfAha1) was identified and investigated for its interaction and regulation of PfHsp90. A previous genome-wide yeast two-hybrid study failed to identify PfAha1's association with PfHsp90, which prompted us to use a directed assay to investigate their interaction. PfAha1 was shown to interact with PfHsp90 via the in vivo split-ubiquitin assay and the association was confirmed in vitro by GST pull-down experiments. The GST pull-down assay further revealed PfAha1's interaction with PfHsp90 to be dependent on MgCl₂ and ATP, and was competed by co-chaperone Pfp23 that binds PfHsp90 under the same condition. In addition, the PfHsp90-PfAha1 complex was found to be sensitive to disruption by high salt, indicating a polar interaction between them. Using bio-computational modelling coupled with site-directed mutagenesis, the polar residue N108 in PfAha1 was found to be strategically located and essential for PfHsp90 interaction. The functional significance of PfAha1's interaction was clearly that of exerting a stimulatory effect on the ATPase activity of PfHsp90, likely to be essential for promoting the activation of PfHsp90's client proteins.     

Approaches for defining the Hsp90-dependent proteome

Hsp90 is the target of ongoing drug discovery studies seeking new compounds to treat cancer, neurodegenerative diseases, and protein folding disorders. To better understand Hsp90's roles in cellular pathologies and in normal cells, numerous studies have utilized proteomics assays and related high-throughput tools to characterize its physical and functional protein partnerships. This review surveys these studies, and summarizes the strengths and limitations of the individual attacks. We also include downloadable spreadsheets compiling all of the Hsp90-interacting proteins identified in more than 23 studies. These tools include cross-references among gene aliases, human homologues of yeast Hsp90-interacting proteins, hyperlinks to database entries, summaries of canonical pathways that are enriched in the Hsp90 interactome, and additional bioinformatic annotations. In addition to summarizing Hsp90 proteomics studies performed to date and the insights they have provided, we identify gaps in our current understanding of Hsp90-mediated proteostasis. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Geldanamycin anisimycins activate Rho and stimulate Rho- and ROCK-dependent actin stress fiber formation

Heat shock protein 90 (Hsp90) is a member of the heat shock family of molecular chaperones that regulate protein conformation and activity. Hsp90 regulates multiple cell signaling pathways by controlling the abundance and activity of several important protein kinases and cell cycle-related proteins. In this report, we show that inhibition of Hsp90 by geldanamycin or its derivative, 17-allylamino-17-desmethoxygeldamycin, leads to activation of the Rho GTPase and a dramatic increase in actin stress fiber formation in human tumor cell lines. Inactivation of Rho prevents geldanamycin-induced actin reorganization. Hsp90 inactivation does not alter the appearance of filopodia or lamellipodia and tubulin architecture is not visibly perturbed. Our observations suggest that Hsp90 has an important and specific role in regulating Rho activity and Rho-dependent actin cytoskeleton remodeling.     

Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.     

Modular Control of Cross-oligomerization: ANALYSIS OF SUPERSTABILIZED Hsp90 HOMODIMERS IN VIVO*

Homo-oligomeric proteins fulfill numerous functions in all cells. The ability to co-express subunits of these proteins that preferentially self-assemble without cross-oligomerizing provides for controlled experiments to analyze the function of mutant homo-oligomers in vivo. Hsp90 is a dimeric chaperone involved in the maturation of many kinases and steroid hormone receptors. We observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered superstabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. Superstabilized Hsp90 dimers supported robust growth of S. cerevisiae, indicating that dissociation of Hsp90 dimers could be hindered without compromising essential function. We utilized superstabilized dimers to analyze the activity of ATPase mutant homodimers in a temperature-sensitive yeast background where elevated temperature inactivated all other Hsp90 species. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of glucocorticoid receptor and v-Src, confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo.     

Differential interactions of p23 and the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90 mutants

Hsp90 is required for the normal function of steroid receptors, but its binding to steroid receptors is mediated by Hsc70 and several hsp-associated accessory proteins. An assortment of Hsp90 mutants were tested for their abilities to interact with each of the following accessories: Hop, Cyp40, FKBP52, FKBP51, and p23. Of the 11 Hsp90 mutants tested, all were defective to some extent in associating with progestin (PR) complexes. In every case, however, reduced PR binding correlated with a defect in binding of one or more accessories. Co-precipitation of mutant Hsp90 forms with individual accessories was used to map Hsp90 sequences required for accessory protein interactions. Mutation of Hsp90's highly conserved C-terminal EEVD to AAVD resulted in diminished interactions with several accessory proteins, most particularly with Hop. Deletion of amino acids 661–677 resulted in loss of Hsp90 dimerization and also caused diminished interactions with all accessory proteins. Binding of p23 mapped most strongly to the N-terminal ATP-binding domain of Hsp90 while binding of TPR proteins mapped to the C-terminal half of Hsp90. These results and others further suggest that the N- and C-terminal regions of Hsp90 maintain important conformational links through intramolecular interactions and/or intermolecular influences in homodimers.     

Hsp90 chaperone code and the tumor suppressor VHL cooperatively regulate the mitotic checkpoint

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes that plays a vital role in protecting and maintaining the functional integrity of deregulated signaling proteins in tumors. We have previously reported that the stability and activity of the mitotic checkpoint kinase Mps1 depend on Hsp90. In turn, Mps1-mediated phosphorylation Hsp90 regulates its chaperone function and is essential for the mitotic arrest. Cdc14-assisted dephosphorylation of Hsp90 is vital for the mitotic exit. Post-translational regulation of Hsp90 function is also known as the Hsp90 “Chaperone Code.” Here, we demonstrate that only the active Mps1 is ubiquitinated on K86, K827, and K848 by the tumor suppressor von Hippel-Lindau (VHL) containing E3 enzyme, in a prolyl hydroxylation-independent manner and degraded in the proteasome. Furthermore, we show that this process regulates cell exit from the mitotic checkpoint. Collectively, our data demonstrates an interplay between the Hsp90 chaperone and VHL degradation machinery in regulating mitosis.     

High affinity binding of Hsp90 is triggered by multiple discrete segments of its kinase clients

The 90 kDa heat shock protein (Hsp90) cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, crystal structures of protein kinases were used to guide the dissection of two kinases [the Src-family tyrosine kinase, Lck, and the heme-regulated eIF2alpha kinase (HRI)], and the association of Hsp90 and Cdc37 with these constructs was assessed. Hsp90 interacted with both the N-terminal (NL) and C-terminal (CL) lobes of the kinases' catalytic domains. In contrast, Cdc37 interacted only with the NL. The Hsp90 antagonist molybdate was necessary to stabilize the interactions between isolated subdomains and Hsp90 or Cdc37, but the presence of both lobes of the kinases' catalytic domain generated a stable salt-resistant chaperone-client heterocomplex. The Hsp90 co-chaperones FKBP52 and p23 interacted with the catalytic domain and the NL of Lck, whereas protein phosphatase 5 demonstrated unique modes of kinase binding. Cyp40 was a salt labile component of Hsp90 complexes formed with the full-length, catalytic domains, and N-terminal catalytic lobes of Lck and HRI. Additionally, dissections identify a specific kinase motif that triggers Hsp90's conformational switching to a high-affinity client binding state. Results indicate that the Hsp90 machine acts as a versatile chaperone that recognizes multiple regions of non-native proteins, while Cdc37 binds to a more specific kinase segment, and that concomitant recognition of multiple client segments is communicated to generate or stabilize high-affinity chaperone-client heterocomplexes.