Hsp90's secrets unfold: new insights from structural and functional studies.

Hsp90 is a molecular chaperone associated with the folding of signal-transducing proteins, such as steroid hormone receptors and protein kinases. Results from recent studies have shed light on the structure of Hsp90 and have demonstrated that it can bind to and hydrolyse ATP. Hsp90 forms several discrete subcomplexes, each containing distinct groups of co-chaperones that function in folding pathways. Although Hsp90 is not generally involved in the folding of nascent polypeptide chains, there is a growing list of proteins whose activity depends on its function, including heat-shock factor. This review addresses recent developments in our understanding of the structure and function of Hsp90.     

Hsp90伴侣复合体装配及对类固醇受体调节

真核细胞中近100种蛋白质都受Hsp90的调节。这些蛋白质多与信号转导作用有关,它们与Hsp90一起进入一个以Hsp90/Hsp70为主的伴侣复合体,在复合体内完成信号转导作用。Hsp90除了和蛋白质的伴侣位点结合以外,还在其他位点与辅助因子连接,这是Hsp90能与蛋白质及辅助因子组装成复合体,并进而调节其信号作用的结构基础。类固醇受体等蛋白质的信号转导作用是在Hsp70、Hsp90为基础的5种蛋白质(Hsp90,Hsp70,Hop,Hsp40和p23)组成的复合体中进行的。这个系统可以帮助理解在真核细胞中,Hsp70和Hsp90怎样联合作用,改变底物蛋白构象,以及怎样应答信号作用。     

Genetic and Biochemical Analysis of p23 and Ansamycin Antibiotics in the Function of Hsp90-Dependent Signaling Proteins

The ubiquitous molecular chaperone Hsp90 acts in concert with a cohort of associated proteins to facilitate the functional maturation of a number of cellular signaling proteins, such as steroid hormone receptors and oncogene tyrosine kinases. The Hsp90-associated protein p23 is required for the assembly of functional steroid aporeceptor complexes in cell lysates, and Hsp90-binding ansamycin antibiotics disrupt the activity of Hsp90-dependent signaling proteins in cultured mammalian cells and prevent the association of p23 with Hsp90-receptor heterocomplexes; these observations have led to the hypotheses that p23 is required for the maturation of Hsp90 target proteins and that ansamycin antibiotics abrogate the activity of such proteins by disrupting the interaction of p23 with Hsp90. In this study, I demonstrate that ansamycin antibiotics disrupt the function of Hsp90 target proteins expressed in yeast cells; prevent the assembly of Sba1, a yeast p23-like protein, into steroid receptor-Hsp90 complexes; and result in the assembly of receptor-Hsp90 complexes that are defective for ligand binding. To assess the role of p23 in Hsp90 target protein function, I show that the activity of Hsp90 target proteins is unaffected by deletion of SBA1. Interestingly, steroid receptor activity in cells lacking Sba1 displays increased sensitivity to ansamycin antibiotics, and this phenotype is rescued by the expression of human p23 in yeast cells. These findings indicate that Hsp90-dependent signaling proteins can achieve a functional conformation in vivo in the absence of p23. Furthermore, while the presence of p23 decreases the sensitivity of Hsp90-dependent processes to ansamycin treatment, ansamycin antibiotics disrupt signaling through some mechanism other than altering the Hsp90-p23 interaction.     

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.     

High-yield expression and purification of the Hsp90-associated p23, FKBP52, HOP and SGTα proteins

Hsp90 is a ubiquitous molecular chaperone that plays a key role in the malignant development of hormone-dependent pathologies such as cancer. An important role for Hsp90 is to facilitate the stable binding of steroid hormones to their respective receptors enabling the ligand-based signal to be carried to the nucleus and ultimately resulting in the up-regulation of gene expression. Along with Hsp90, this dynamic and transient process also involves the recruitment of additional proteins and co-chaperones that add further stability to the mature receptor–chaperone complex. In the work presented here, we describe four new protocols for the bacterial over-expression and column chromatographic purification of the human p23, FKBP52, HOP and SGTα proteins. Each of these proteins plays a distinct role in the steroid hormone receptor regulatory cycle. Affinity, ion-exchange and size-exclusion techniques were used to produce target yields greater than 50 mg/L of cultured media, with each purified sample reaching near absolute sample homogeneity. These results reveal a reliable system for the production of p23, FKBP52, HOP and SGTα substrate proteins for use in the investigation of the Hsp90-associated protein interactions of the steroid hormone receptor cycle.     

Hsp90: a chaperone for protein folding and gene regulation.

Molecular chaperones are essential components of a quality control machinery present in the cell. They can either aid in the folding and maintenance of newly translated proteins, or they can lead to the degradation of misfolded and destabilized proteins. Hsp90 is a key member of this machinery. It is a ubiquitous molecular chaperone that is found in eubacteria and all branches of eukarya. It plays a central role in cellular signaling since it is essential for maintaining the activity of several signaling proteins, including steroid hormone receptors and protein kinases. Hsp90 is currently a novel anticancer drug target since it is overexpressed in some cancer cells. The chaperone typically functions as part of large complexes, which include other chaperones and essential cofactors that regulate its function. It is thought that different cofactors target Hsp90 to different sets of substrates. However, the mechanism of Hsp90 function remains poorly understood. As part of an effort to elucidate the Hsp90 chaperone network, we carried out a large-scale proteomics study to identify physical and genetic interactors of the chaperone. We identified 2 highly conserved novel Hsp90 cofactors, termed Tah1 and Pih1, that bind to the chaperone and that also associate physically and functionally with the essential DNA helicases Rvb1 and Rvb2. These helicases are key components of the chromatin remodeling complexes Ino80 and SWR-C. Tah1 and Pih1 seem to represent a novel class of Hsp90 cofactors that allow the chaperone to indirectly affect gene regulation in the cell in addition to its ability to directly promote protein folding. In this review, we provide an overview of Hsp90 structure and function, and we discuss the literature that links the chaperone activity to gene regulation.     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.     

Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.     

The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila.

The heat shock protein Hsp90 has been shown to associate with various cellular signalling proteins such as steroid hormone receptors, src-like kinases and the serine/threonine kinase Raf. While the interaction between steroid hormone receptors and Hsp90 appears to be essential for ligand binding and activation of the receptors, the role of Hsp90 in Raf activation is less clear. We have identified mutations in the hsp83 gene, the Drosophila homologue of hsp90, in a search for dominant mutations that attenuate signalling from Raf in the developing eye. The mutations result in single amino acid substitutions in the Hsp83 protein and cause a dominant-negative effect on the function of the wild-type protein. We show that both wild-type and mutant forms of Hsp83 bind to the activated Drosophila Raf but the mutant Hsp83 protein causes a reduction in the kinase activity of Raf. Our results indicate that Hsp83 is essential for Raf function in vivo.     

Chaperoning steroidal physiology: lessons from mouse genetic models of Hsp90 and its cochaperones

The molecular chaperone Hsp90 is abundant, ubiquitous, and catholic to biological processes in eukaryotes, controlling phosphorylation cascades, protein stability and turnover, client localization and trafficking, and ligand-receptor interactions. Not surprisingly, Hsp90 does not accomplish these activities alone. Instead, an ever-growing number of cochaperones have been identified, leading to an explosion of reports on their molecular and cellular effects on Hsp90 chaperoning of client substrates. Notable among these clients are many members of the steroid receptor family, such as glucocorticoid, androgen, estrogen and progesterone receptors. Cochaperones typically associated with the mature, hormone-competent states of these receptors include p23, the FK506-binding protein 52 (FKBP52), FKBP51, protein phosphatase 5 (PP5) and cyclophilin 40 (Cyp40). The ultimate relevance of these cochaperones to steroid receptor action depends on their physiological effects. In recent years, the first mouse genetic models of these cochaperones have been developed. This work will review the complex and intriguing phenotypes so far obtained in genetically-altered mice and compare them to the known molecular and cellular impacts of cochaperones on steroid receptors. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Solubility-promoting function of Hsp90 contributes to client maturation and robust cell growth

The Hsp90 chaperone is required for the maturation of signal transduction clients, including many kinases and nuclear steroid hormone receptors. The binding and hydrolysis of ATP by Hsp90 drive conformational rearrangements in three structure domains. Two intrinsically disordered regions of Hsp90 located between these domains and at the C terminus have traditionally been considered to impart flexibility. We discovered that the charged nature of these acid-rich disordered regions imparts a solubility-promoting function to Hsp90 that is important for its cellular activity in yeast. Both the solubility-promoting function and ATPase activity must occur in the same Hsp90 molecule in order to support robust growth, suggesting that the solubility-promoting function is required during the ATP-driven client maturation process. Expression of model clients together with Hsp90 variants indicated interdependent solubilities mediated by the aggregation propensities of both the client and Hsp90. We propose a model whereby the charge-rich disordered regions of Hsp90 serve a solubility-promoting function important for complexes with aggregation-prone clients. These findings demonstrate a novel biological function of the intrinsically disordered regions in Hsp90 and provide a compelling rationale for why their charged properties are conserved throughout eukaryotic evolution.     

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.     

Control of estrogen receptor ligand binding by Hsp90

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.