Mercury stimulates rat liver glucocorticoid receptor association with Hsp90 and Hsp70

The subject of the present study is the influence of mercury on association of rat liver glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70. The glucocorticoid receptor heterocomplexes with Hsp90 and Hsp70 were immunopurified from the liver cytosol of rats administered with different doses of mercury. The amounts of co-immunopurified apo-receptor, Hsp90 and Hsp70 were then determined by quantitative Western blotting. The ratio between the amount of heat shock protein Hsp90 or Hsp70 and the amount of apo-receptor within immunopurified heterocomplexes was found to increase in response to mercury administration. On the other hand, the levels of Hsp90 and Hsp70 in hepatic cytosol remained unaltered. The finding that mercury stimulates association of the two heat shock proteins with the glucocorticoid receptor, rendering the cytosolic heat shock protein levels unchanged, suggests that mercury affects the mechanisms controlling the assembly of the receptor heterocomplexes.     

Hsp70 Cochaperones HspBP1 and BAG-1M Differentially Regulate Steroid Hormone Receptor Function

Hsp70 binding protein 1 (HspBP1) and Bcl2-associated athanogene 1 (BAG-1), the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70) chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the androgen receptor (AR). BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.     

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.     

Phosphorylation of the Hsp90 Co-Chaperone Hop Changes its Conformational Dynamics and Biological Function

The molecular chaperones Hsp90 and Hsp70 and their regulatory co-chaperone Hop play a key role at the crossroads of the folding pathways of numerous client proteins by forming fine-tuned multiprotein complexes. Alterations of the biomolecules involved may functionally impact the chaperone machinery: here, we integrate simulations and experiments to unveil how Hop conformational fitness and interactions can be controlled by the perturbation of just one residue. Specifically, we unveil how mechanisms mediated by Hop residue Y354 control Hop open and closed states, which affect binding of Hsp70/Hsp90. Phosphorylation or mutation of Hop-Y354 are shown to favor structural ensembles that are indeed not optimal for stable interactions with Hsp90 and Hsp70. This disfavors cellular accumulation of the stringent Hsp90 clients glucocorticoid receptor and the viral tyrosine kinase v-Src, with detrimental effects on v-Src activity. Our results show how the post-translational modification of a specific residue in Hop provides a regulation mechanism for the larger chaperone complex of which it is part. In this framework, the effects of one single alteration are amplified at the cellular level through the perturbation of protein-interaction networks.     

Cyclophilin 40: An Hsp90-cochaperone associated with apo-steroid receptors

Cyclophilin 40, a divergent loop cyclophilin first identified in association with the estrogen receptor α, contains a C-terminal tetratricopeptide repeat domain through which it shares structural identity with FK506-binding protein 52 (FKBP52) and other partner cochaperones in steroid receptor-heat shock protein 90 (Hsp90) complexes. By dynamically competing for Hsp90 interaction, the cochaperones allow the receptors to establish distinct Hsp90-chaperone complexes, with the potential to exert tissue-specific control over receptor activity. Cyclophilin 40 regulates Hsp90 ATPase activity during receptor-Hsp90 assembly. Functional deletion of the cyclophilin 40 yeast homologue, Cpr7, adversely affected estrogen receptor α and glucocorticoid receptor activity that could be fully restored, either with wild type Cpr7 or Cpr7 with a cyclophilin domain lacking isomerase activity. We draw parallels with the mechanism already established for FKBP52 and propose that the cyclophilin 40 divergent loop interfaces with a contact surface on the steroid receptor ligand-binding domain to achieve an optimal orientation for receptor activity.     

Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones

Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1–3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398–719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1–550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310–529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.     

Hsp90: Still a viable target in prostate cancer

Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the maturation, activation and stability of critical signaling proteins that drive the development and progression of prostate cancer, including the androgen receptor. Despite robust preclinical data demonstrating anti-tumor activity of first-generation Hsp90 inhibitors in prostate cancer, poor clinical responses initially cast doubt over the clinical utility of this class of agent. Recent advances in compound design and development, use of novel preclinical models and further biological insights into Hsp90 structure and function have now stimulated a resurgence in enthusiasm for these drugs as a therapeutic option. This review highlights how the development of new-generation Hsp90 inhibitors with improved physical and pharmacological properties is unfolding, and discusses the potential contexts for their use either as single agents or in combination, for men with metastatic prostate cancer.     

Single-point mutation in a conserved TPR domain of Hip disrupts enhancement of glucocorticoid receptor signaling

The Hsp70-interacting protein Hip has been identified as a transient participant in the assembly of both glucocorticoid (GR) and progesterone receptor complexes. Although it has been difficult to identify a physiological role for Hip, it is believed to have intrinsic chaperoning properties and has been identified as a potential anti-apoptotic target of Granzyme B. In vitro assays have provided evidence that Hip may interact with GR complexes in an Hsp70 independent manner and can enhance the function of GR in hormone based reporter assays. In this study, a cDNA for human Hip was used in mutational analysis to map Hip function to critical structural elements. A single amino acid substitution (L211S) resulted in a loss of Hip function. This mutation also appears to disrupt the interaction of Hip with Hsp70 in vitro. Failure to recover Hip-L211S constructs in co-immunoprecipitation assays with an Hsp70 monoclonal antibody suggests that the mutation is unlikely to result in a misfolded substrate.     

Folding and stability of the ligand-binding domain of the glucocorticoid receptor

A complex pathway involving many molecular chaperones has been proposed for the folding, assembly, and maintenance of a high-affinity ligand-binding form of steroid receptors in vivo, including the glucocorticoid receptor. To better understand this intricate folding and assembly process, we studied the folding of the ligand-binding domain of the glucocorticoid receptor in vitro. We found that this domain can be refolded into a compact, highly structured state in vitro in the absence of chaperones. However, the presence of zwitterionic detergent is required to maintain the domain in a soluble form. In this state, the protein is dimeric and has considerable helical structure as shown by far-UV circular dichroism. Further investigation of the properties of this in vitro refolded state show that it is stable and resistant to denaturation by heat or low concentrations of chemical denaturants. A detailed analysis of the unfolding equilibria using three different structural probes demonstrated that this state unfolds via a highly populated dimeric intermediate state. Together, these data clearly show that the ligand-binding domain of the glucocorticoid receptor does not require chaperones for folding per se. However, this in vitro refolded state binds the ligand dexamethasone only weakly (K(d) = 45 microM) compared to the in vivo assembled receptor (K(d) = 3.4 nM). We suggest that the role of Hsp90 and associated chaperones is to bind to, and stabilize, a specific conformational state of the receptor which binds ligand with high affinity.     

Molecular cloning of human FKBP51 and comparisons of immunophilin interactions with Hsp90 and progesterone receptor.

A cDNA for human FKBP51 has been cloned and sequenced, and protein products have been expressed in both in vitro and bacterial systems. The deduced amino acid sequence for human FKBP51 is 90% identical to sequences of recently described murine proteins and is 55% identical to the sequence of human FKBP52. Human FKBP51 mRNA is expressed in a wide range of tissues, and the protein has peptidylprolyl isomerase activity that is inhibited by FK506 but not cyclosporine. FKBP51 is the same as a previously described progesterone receptor-associated immunophilin that, similar to FKBP52 and cyclophilin 40, is an Hsp90-binding protein and appears in functionally mature steroid receptor complexes along with Hsp90 and p23. Each of the three receptor-associated immunophilins displays interactions with progesterone receptor that are more dynamic than Hsp90-receptor interactions. Whereas FKBP52 and FKBP51 compete about equally well for binding to Hsp90 in a purified system, FKBP51 accumulates preferentially in progesterone receptor complexes assembled in a cell-free system. This observation provides a precedent for differential interactions between Hsp90-associated immunophilins and target proteins such as steroid receptors.     

The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.     

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

Human Hsp90 cochaperones: perspectives on tissue-specific expression and identification of cochaperones with similar in vivo functions

The Hsp90 molecular chaperone is required for the function of hundreds of different cellular proteins. Hsp90 and a cohort of interacting proteins called cochaperones interact with clients in an ATP-dependent cycle. Cochaperone functions include targeting clients to Hsp90, regulating Hsp90 ATPase activity, and/or promoting Hsp90 conformational changes as it progresses through the cycle. Over the last 20 years, the list of cochaperones identified in human cells has grown from the initial six identified in complex with steroid hormone receptors and protein kinases to about fifty different cochaperones found in Hsp90-client complexes. These cochaperones may be placed into three groups based on shared Hsp90 interaction domains. Available evidence indicates that cochaperones vary in client specificity, abundance, and tissue distribution. Many of the cochaperones have critical roles in regulation of cancer and neurodegeneration. A more limited set of cochaperones have cellular functions that may be limited to tissues such as muscle and testis. It is likely that a small set of cochaperones are part of the core Hsp90 machinery required for the folding of a wide range of clients. The presence of more selective cochaperones may allow greater control of Hsp90 activities across different tissues or during development.Electronic supplementary materialThe online version of this article (10.1007/s12192-020-01167-0) contains supplementary material, which is available to authorized users.     

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.