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.     

Heat shock protein 90 stabilization of ErbB2 expression is disrupted by ATP depletion in myocytes

Heat shock protein (Hsp) 90 is a ubiquitously expressed chaperone that stabilizes expression of multiple signaling kinases involved in growth regulation, including ErbB2, Raf-1, and Akt. The chaperone activity of Hsp90 requires ATP, which binds with approximately 10-fold lower affinity than ADP. This suggests that Hsp90 may be a physiological ATP sensor, regulating the stability of growth signaling cascades in relation to cellular energy charge. Here we show that lowering ATP concentration by inhibiting glycolysis or mitochondrial respiration in isolated myocytes triggers rapid dissociation of Hsp90 from ErbB2 and degradation of ErbB2 along with other client proteins. The effect of disrupting Hsp90 chaperone activity by ATP depletion was similar to the effect of the pharmacological Hsp90 inhibitor geldanamycin. ATP depletion-induced disruption of Hsp90 chaperone activity was associated with cellular resistance to growth factor activation of intracellular signaling. ErbB2 degradation was also induced by the physiological stress of beta-adrenergic receptor stimulation in electrically stimulated cells. These results support a role for Hsp90 as an ATP sensor that modulates tissue growth factor responsiveness under metabolically stressed conditions and provide a novel mechanism by which cellular responsiveness to growth factor stimulation is modulated by cellular energy charge.     

Hsp90 recognizes a common surface on client kinases

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.     

Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation

Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.     

Aha1 Can Act as an Autonomous Chaperone to Prevent Aggregation of Stressed Proteins

Aha1 (activator of Hsp90 ATPase) stimulates the ATPase activity of the molecular chaperone Hsp90 to accelerate the conformational cycle during which client proteins attain their final shape. Thereby, Aha1 promotes effective folding of Hsp90-dependent clients such as steroid receptors and many kinases involved in cellular signaling. In our current study, we find that Aha1 plays a novel, additional role beyond regulating the Hsp90 ATP hydrolysis rate. We propose a new concept suggesting that Aha1 acts as an autonomous chaperone and associates with stress-denatured proteins to prevent them from aggregation similar to the chaperonin GroEL. Our study reveals that an N-terminal sequence of 22 amino acids, present in human but absent from yeast Aha1, is critical for this capability. However, in lieu of fostering their refolding, Aha1 allows ubiquitination of bound clients by the E3 ubiquitin ligase CHIP. Accordingly, Aha1 may promote disposal of folding defective proteins by the cellular protein quality control.     

The cellular chaperone heat shock protein 90 facilitates Flock House virus RNA replication in Drosophila cells

The assembly of viral RNA replication complexes on intracellular membranes represents a critical step in the life cycle of positive-strand RNA viruses. We investigated the role of the cellular chaperone heat shock protein 90 (Hsp90) in viral RNA replication complex assembly and function using Flock House virus (FHV), an alphanodavirus whose RNA-dependent RNA polymerase, protein A, is essential for viral RNA replication complex assembly on mitochondrial outer membranes. The Hsp90 chaperone complex transports cellular mitochondrial proteins to the outer mitochondrial membrane import receptors, and thus we hypothesized that Hsp90 may also facilitate FHV RNA replication complex assembly or function. Treatment of FHV-infected Drosophila S2 cells with the Hsp90-specific inhibitor geldanamycin or radicicol potently suppressed the production of infectious virions and the accumulation of protein A and genomic, subgenomic, and template viral RNA. In contrast, geldanamycin did not inhibit the activity of preformed FHV RNA replication complexes. Hsp90 inhibitors also suppressed viral RNA and protein A accumulation in S2 cells expressing an FHV RNA replicon. Furthermore, Hsp90 inhibition with either geldanamycin or RNAi-mediated chaperone downregulation suppressed protein A accumulation in the absence of viral RNA replication. These results identify Hsp90 as a host factor involved in FHV RNA replication and suggest that FHV uses established cellular chaperone pathways to assemble its RNA replication complexes on intracellular membranes.     

Hsp90 and the Fanconi Anemia Pathway: A Molecular Link Between Protein Quality Control and the DNA Damage Response

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an essential role in cell growth and survival. The chaperone exerts these functions by regulating key signaling proteins involved in cell growth/survival and protecting cells from proteotoxic stress. Importantly, Hsp90 inhibitors including geldanamycin analogues show anti-tumor effects. We recently found that Hsp90 promotes stabilization and nuclear localization of the Fanconi anemia (FA) protein FANCA, which is required for activation of the FA pathway. The FA pathway is a multiprotein biochemical pathway involved in genotoxic signaling, defects in which cause genomic instability, hematopoietic stem cell failure and tumor development. Inhibition of Hsp90 impairs the intracellular homeostasis of FANCA, resulting in disruption of the FA pathway. These findings have important implications for rational cancer chemotherapy using Hsp90 inhibitors. We also discuss the possible functions of Hsp90 in FA pathophysiology and stem cell/cancer biology. Based on our findings and other data, we propose that Hsp90 functions as “a guardian of the genome” through the control of DNA repair proteins.     

The role of Hsp90 in protein complex assembly

Hsp90 is a ubiquitous and essential molecular chaperone that plays central roles in many signaling and other cellular pathways. The in vivo and in vitro activity of Hsp90 depends on its association with a wide variety of cochaperones and cofactors, which form large multi-protein complexes involved in folding client proteins. Based on our proteomic work mapping the molecular chaperone interaction networks in yeast, especially that of Hsp90, as well as, on experiments and results presented in the published literature, one major role of Hsp90 appears to be the promotion and maintenance of proper assembly of protein complexes. To highlight this role of Hsp90, the effect of the chaperone on the assembly of the following seven complexes is discussed in this review: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome. For some complexes, it is observed that Hsp90 mediates complex assembly by stabilizing an unstable protein subunit and facilitating its incorporation into the complex; for other complexes, Hsp90 promotes change in the composition of that complex. In all cases, Hsp90 does not appear to be part of the final assembled complex. This article is part of a Special Issue entitled:Heat Shock Protein 90 (HSP90).     

Heat shock protein 90 regulates the stability of MEKK3 in HEK293 cells

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the conformational maturation and function of certain signaling proteins. Hsp90 inhibitors cause the inactivation, destabilization and eventual degradation of Hsp90 client proteins through occupying the ATP/ADP binding pocket of Hsp90. In the present study, we found that Hsp90 interacted with MEKK3 in HEK293 cells. Hsp90 inhibitors reduced the level of endogenous MEKK3 in time- and dose-dependent manners, and this decrease was reversed by Hsp90 overexpression. In addition, Hsp90 RNAi destabilized MEKK3. A selective inhibitor of Hsp90, geldanamycin (GA), shortened MEKK3 half-life, and induced ubiquitination and proteasomal degradation of MEKK3. These results strongly suggested that Hsp90 could work as the molecular chaperone of MEKK3.     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

An interaction network predicted from public data as a discovery tool: application to the Hsp90 molecular chaperone machine

Understanding the functions of proteins requires information about their protein-protein interactions (PPI). The collective effort of the scientific community generates far more data on any given protein than individual experimental approaches. The latter are often too limited to reveal an interactome comprehensively. We developed a workflow for parallel mining of all major PPI databases, containing data from several model organisms, and to integrate data from the literature for a protein of interest. We applied this novel approach to build the PPI network of the human Hsp90 molecular chaperone machine (Hsp90Int) for which previous efforts have yielded limited and poorly overlapping sets of interactors. We demonstrate the power of the Hsp90Int database as a discovery tool by validating the prediction that the Hsp90 co-chaperone Aha1 is involved in nucleocytoplasmic transport. Thus, we both describe how to build a custom database and introduce a powerful new resource for the scientific community.     

Improved data normalization methods for reverse phase protein microarray analysis of complex biological samples

Members of the heat shock protein-90 (Hsp90) family are key regulators of biological processes through dynamic interaction with a multitude of protein partners. However, the transient nature of these interactions hinders the identification of Hsp90 interactors. Here we show that chemical cross-linking with ethylene glycolbis (succinimidylsuccinate), but not shorter cross-linkers, generated an abundant 240-kDa heteroconjugate of the molecular chaperone Hsp90 in different cell types. The combined use of pharmacological and genetic approaches allowed the characterization of the subunit composition and subcellular compartmentalization of the multimeric protein complex, termed p240. The in situ formation of p240 did not require the N-terminal domain or the ATPase activity of Hsp90. Utilizing subcellular fractionation techniques and a cell-impermeant cross-linker, subpopulations of p240 were found to be present in both the plasma membrane and the mitochondria. The Hsp90-interacting proteins, including Hsp70, p60Hop and the scaffolding protein filamin A, had no role in governing the formation of p240. Therefore, chemical cross-linking combined with proteomic methods has the potential to unravel the protein components of this p240 complex and, more importantly, may provide an approach to expand the range of tools available to the study of the Hsp90 interactome.     

The mammalian CHORD-containing protein melusin is a stress response protein interacting with Hsp90 and Sgt1

Melusin is a mammalian muscle specific CHORD containing protein capable of activating signal transduction pathways leading to cardiomyocytes hypertrophy in response to mechanical stress. To define melusin function we searched for molecular partners possibly involved in melusin dependent signal transduction. Here we show that melusin and heat shock proteins are co-regulated. Moreover, melusin directly binds to Hsp90, a ubiquitous chaperone involved in regulating several signaling pathways. In addition, melusin interacts with Sgt1, an Hsp90 binding molecule. Melusin does not behave as an Hsp90 substrate but rather as a chaperone capable to protect citrate synthase from heat induced aggregation. These results describe melusin as a new component of the Hsp90 chaperone machinery.     

Tom34: a cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import

Most mitochondrial membrane proteins are synthesized in the cytosol and must be delivered to the organelle in an unfolded, import competent form. In mammalian cells, the cytosolic chaperones Hsp90 and Hsp70 are part of a large cytosolic complex that deliver the membrane protein to the mitochondrion by docking with the import receptor Tom70. These two abundant chaperones have other functions in the cell suggesting that the specificity for the targeting of mitochondrial proteins requires the addition of specific factors within the targeting complex. We identify Tom34 as a cochaperone of Hsp70/Hsp90 in mitochondrial protein import. We show that Tom34 is an integral component with Hsp70 and Hsp90 in the large complex. We also demonstrate the role of Tom34 in the mitochondrial import process, as the addition of an excess of Tom34 prevents efficient mitochondrial translocation of precursor proteins that have requirements for Hsp70/Hsp90. Tom34 exhibits an affinity for mitochondrial preproteins of the Tom70 translocation pathway as demonstrated by binding assays using in vitro translated proteins as baits. In addition, we examined the specificity and the size of different complex cytosolic machines. Separation of different radiolabeled cell-free translated proteins on Native-PAGE showed the presence of a high molecular weight complex which binds hydrophobic proteins. Importantly we show that the formation of the chaperone cytosolic complex that mediates the targeting of proteins to the mitochondria contains Tom34 and assembles in the presence of a fully translated substrate protein.     

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.