The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization

Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.     

The Molecular Chaperone Hsp90 Modulates Intermediate Steps of Amyloid Assembly of the Parkinson-related Protein ��-Synuclein

α-Synuclein is an intrinsically unstructured protein that binds to membranes, forms fibrils, and is involved in neurodegeneration. We used a reconstituted in vitro system to show that the molecular chaperone Hsp90 influenced α-synuclein vesicle binding and amyloid fibril formation, two processes that are tightly coupled to α-synuclein folding. Binding of Hsp90 to monomeric α-synuclein occurred in the low micromolar range, involving regions of α-synuclein that are critical for vesicle binding and amyloidogenesis. As a consequence, both processes were affected. In the absence of ATP, the accumulation of non-amyloid α-synuclein oligomers prevailed over fibril formation, whereas ATP favored fibril growth. This suggests that Hsp90 modulates the assembly of α-synuclein in an ATP-dependent manner. We propose that Hsp90 affects these folding processes by restricting conformational fluctuations of α-synuclein.     

In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH₂-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH₂-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis–dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.     

Force Distribution Reveals Signal Transduction in E.?coli Hsp90

Heat-shock protein 90 (Hsp90) is an ubiquitous chaperone that is essential for cell function in that it promotes client-protein folding and stabilization. Its function is tightly controlled by an ATP-dependent large conformational transition between the open and closed states of the Hsp90 dimer. The underlying allosteric pathway has remained largely unknown, but it is revealed here in atomistic detail for the Escherichia coli homolog HtpG. Using force-distribution analysis based on molecular-dynamics simulations (>1 μs in total), we identify an internal signaling pathway that spans from the nucleotide-binding site to an ∼2.3-nm-distant region in the HtpG middle domain, that serves as a dynamic hinge region, and to a putative client-protein-binding site in the middle domain. The force transmission is triggered by ATP capturing a magnesium ion and thereby rotating and bending a proximal long α-helix, which represents the major force channel into the middle domain. This allosteric mechanism is, with statistical significance, distinct from the dynamics in the ADP and apo states. Tracking the distribution of forces is likely to be a promising tool for understanding and guiding experiments of complex allosteric proteins in general.     

Hsp90 chaperones PPARγ and regulates differentiation and survival of 3T3-L1 adipocytes

Adipose tissue dysregulation has a major role in various human diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) is a key regulator of adipocyte differentiation and function, as well as a target of insulin-sensitizing drugs. The Hsp90 chaperone stabilizes a diverse set of signaling ‘client'' proteins, thereby regulates various biological processes. Here we report a novel role for Hsp90 in controlling PPARγ stability and cellular differentiation. Specifically, we show that the Hsp90 inhibitors geldanamycin and novobiocin efficiently impede the differentiation of murine 3T3-L1 preadipocytes. Geldanamycin at higher concentrations also inhibits the survival of both developing and mature adipocytes, respectively. Further, Hsp90 inhibition disrupts an Hsp90-PPARγ complex, leads to the destabilization and proteasomal degradation of PPARγ, and inhibits the expression of PPARγ target genes, identifying PPARγ as an Hsp90 client. A similar destabilization of PPARγ and a halt of adipogenesis also occur in response to protein denaturing stresses caused by a single transient heat-shock or proteasome inhibition. Recovery from stress restores PPARγ stability and adipocyte differentiation. Thus, our findings reveal Hsp90 as a critical stress-responsive regulator of adipocyte biology and offer a potential therapeutic target in obesity and the metabolic syndrome.     

Folding or holding?—Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease

The toxic accumulation of misfolded proteins as inclusions, fibrils, or aggregates is a hallmark of many neurodegenerative diseases. However, how molecular chaperones, such as heat shock protein 70 kDa (Hsp70) and heat shock protein 90 kDa (Hsp90), defend cells against the accumulation of misfolded proteins remains unclear. The ATP-dependent foldase function of both Hsp70 and Hsp90 actively transitions misfolded proteins back to their native conformation. By contrast, the ATP-independent holdase function of Hsp70 and Hsp90 prevents the accumulation of misfolded proteins. Foldase and holdase functions can protect against the toxicity associated with protein misfolding, yet we are only beginning to understand the mechanisms through which they modulate neurodegeneration. This review compares recent structural findings regarding the binding of Hsp90 to misfolded and intrinsically disordered proteins, such as tau, α-synuclein, and Tar DNA-binding protein 43. We propose that Hsp90 and Hsp70 interact with these proteins through an extended and dynamic interface that spans the surface of multiple domains of the chaperone proteins. This contrasts with many other Hsp90–client protein interactions for which only a single bound conformation of Hsp90 is proposed. The dynamic nature of these multidomain interactions allows for polymorphic binding of multiple conformations to vast regions of Hsp90. The holdase functions of Hsp70 and Hsp90 may thus allow neuronal cells to modulate misfolded proteins more efficiently by reducing the long-term ATP running costs of the chaperone budget. However, it remains unclear whether holdase functions protect cells by preventing aggregate formation or can increase neurotoxicity by inadvertently stabilizing deleterious oligomers.     

Inhibition of apoptosome formation by suppression of Hsp90beta phosphorylation in tyrosine kinase-induced leukemias

Constitutively active tyrosine kinases promote leukemogenesis by increasing cell proliferation and inhibiting apoptosis. However, mechanisms underlying apoptotic inhibition have not been fully elucidated. In many settings, apoptosis occurs by mitochondrial cytochrome c release, which nucleates the Apaf-1/caspase-9 apoptosome. Here we report that the leukemogenic kinases, Bcr-Abl, FLT3/D835Y, and Tel-PDGFRβ, all can inhibit apoptosome function. In cells expressing these kinases, the previously reported apoptosome inhibitor, Hsp90β, bound strongly to Apaf-1, preventing cytochrome c-induced Apaf-1 oligomerization and caspase-9 recruitment. Hsp90β interacted weakly with the apoptosome in untransformed cells. While Hsp90β was phosphorylated at Ser 226/Ser 255 in untransformed cells, phosphorylation was absent in leukemic cells. Expression of mutant Hsp90β (S226A/S255A), which mimics the hypophosphorylated form in leukemic cells, conferred resistance to cytochrome c-induced apoptosome activation in normal cells, reflecting enhanced binding of nonphosphorylatable Hsp90β to Apaf-1. In Bcr-Abl-positive mouse bone marrow cells, nonphosphorylatable Hsp90β expression conferred imatinib (Gleevec) resistance. These data provide an explanation for apoptosome inhibition by activated leukemogenic tyrosine kinases and suggest that alterations in Hsp90β-apoptosome interactions may contribute to chemoresistance in leukemias.     

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H₂O₂, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins.     

Extracellular Heat Shock Protein 90 Signals through Subdomain II and the NPVY Motif of LRP-1 Receptor to Akt1 and Akt2: a Circuit Essential for Promoting Skin Cell Migration In Vitro and Wound Healing In Vivo

Normal cells secrete heat shock protein 90 alpha (Hsp90α) in response to tissue injury. Tumor cells have managed to constitutively secrete Hsp90α during invasion and metastasis. The sole function of extracellular Hsp90α (eHsp90α) is to promote cell motility, a critical event for both wound healing and tumor progression. The mechanism of promotility action by eHsp90α, however, has remained elusive. A key issue is whether eHsp90α still acts as a chaperone outside the cells or is a new and bona fide signaling molecule. Here, we have provided evidence that eHsp90α utilizes a unique transmembrane signaling mechanism to promote cell motility and wound healing. First, subdomain II in the extracellular part of low-density lipoprotein receptor-related protein 1 (LRP-1) receives the eHsp90α signal. Then, the NPVY but not the NPTY motif in the cytoplasmic tail of LRP-1 connects eHsp90α signaling to serine 473 but not threonine 308 phosphorylation in Akt kinases. Individual knockdown of Akt1, Akt2, or Akt3 revealed the importance of Akt1 and Akt2 in eHsp90α-induced cell motility. Akt gene rescue experiments suggest that Akt1 and Akt2 work in concert, rather than independently, to mediate eHsp90α promotility signaling. Finally, Akt1 and Akt2 knockout mice showed impaired wound healing that cannot be corrected by topical application with the eHsp90α protein.     

Coordinated ATP hydrolysis by the Hsp90 dimer.

The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrolysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determined to be 60 nm. Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form readily and exhibit dimerization constants similar to full-length Hsp90. However, the ATPase activity of these heterodimers was significantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activation of the ATPase activity. To further address the contribution of the N-terminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this mutant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. Thus, contact formation between the N-terminal domains might not depend on ATP bound to both domains. Together, these results suggest a mechanism for coupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone.     

Hsp90 Nuclear Accumulation in Quiescence Is Linked to Chaperone Function and Spore Development in Yeast

The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Δ cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the α/β importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.     

The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle

The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction. During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases. It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation. The specific role of the partner proteins in this process is unknown. Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1. Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range. Two Sba1 molecules were found to bind per Hsp90 dimer. Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP. Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1. Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.