The chaperone Hsp90 and PPIases of the cyclophilin and FKBP families facilitate membrane translocation of Photorhabdus luminescens ADP‐ribosyltransferases

TccC3 and TccC5 from Photorhabdus luminescens are ADP‐ribosyltransferases, which modify actin and Rho GTPases, respectively, thereby inducing polymerization and clustering of actin. The bacterial proteins are components of the Photorhabdus toxin complexes, consisting of the binding and translocation component TcdA1, a proposed linker component TcdB2 and the enzymatic component TccC3/5. While the action of the toxins on target proteins is clearly defined, uptake and translocation of the toxins into the cytosol of target cells are not well understood. Here we show by using pharmacological inhibitors that heat shock protein 90 (Hsp90) and peptidyl prolyl cis/trans isomerases (PPIases) including cyclophilins and FK506‐binding proteins (FKBPs) facilitate the uptake of the ADP‐ribosylating toxins into the host cell cytosol. Inhibition of Hsp90 and/or PPIases resulted in decreased intoxication of target cells by Photorhabdus toxin complexes determined by cell rounding and reduction of transepithelial electrical resistance of cell monolayers. ADP‐ribosyltransferase activity of toxins and toxin‐induced pore formation were notimpaired by the inhibitors of Hsp90 and PPIases. The Photorhabdus toxins interacted with Hsp90, FKBP51, Cyp40 and CypA, suggesting a role of these host cell factors in translocation and/or refolding of the ADP‐ribosyltransferases.     

Hsp90 Activation and Cell Cycle Regulation

The widely-expressed molecular chaperone heat shock protein 90 (Hsp90) regulates several important cellular processes via its’ repertoire of ‘client’ proteins. Signal transduction pathways controlled by Hsp90 contribute to all major components of the malignant phenotype, so Hsp90 inhibitors are under investigation as anticancer agents. Since Hsp90 is also expressed at high levels in many normal tissues, it was unclear why Hsp90 inhibitors such as 17-allylamino-geldanamycin (17-AAG) have selective antitumor activity in animals and are well tolerated clinically. Recent findings indicate that Hsp90 is largely latent in unstressed normal cells, but tumor Hsp90 becomes completely utilized during malignant progression, resulting in an activation-dependent conformational shift that radically increases 17-AAG binding affinity in cancer cells. In this article, the implications of this discovery are discussed, with particular reference to cell cycle regulation in normal and malignant cells, and the consequences of inducing cell cycle arrest with Hsp90 inhibitors.     

The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock

Human heat shock 27-kDa protein 1 (HSPB1)/heat shock protein (Hsp) 27 is a small heat shock protein which is thought to have several roles within the cell. One of these roles includes regulating actin filament dynamics in cell movement, since Hsp27 has previously been found to inhibit actin polymerization in vitro. In this study, the role of Hsp27 in regulating actin filament dynamics is further investigated. Hsp27 protein levels were reduced using siRNA in SW480 cells, a human colon cancer cell line. An in vitro wound closure assay showed that cells with knocked down Hsp27 levels were unable to close wounds, indicating that this protein is involved in regulating cell motility. Immunoprecipitation pull down assays were done, to observe if and when Hsp27 and actin are in the same complex within the cell, before and after heat shock. At all time points tested, Hsp27 and actin were present in the same cell lysate fraction. Lastly, indirect immunostaining was done before and after heat shock to evaluate Hsp27 and actin interaction in cells. Hsp27 and actin showed colocalization before heat shock, little association 3 h after heat shock, and increased association 24 h after heat shock. Cytoprotection was observed as early as 3 h after heat shock, yet cells were still able to move. These results show that Hsp27 and actin are in the same complex in cells and that Hsp27 is important for cell motility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hsp90 regulates O-linked β-N-acetylglucosamine transferase: a novel mechanism of modulation of protein O-linked β-N-acetylglucosamine modification in endothelial cells

O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins is involved in many important cellular processes. Increased O-GlcNAc has been implicated in major diseases, such as diabetes and its complications and cardiovascular and neurodegenerative diseases. Recently, we reported that O-GlcNAc modification occurs in the proteasome and serves to inhibit proteasome function by blocking the ATPase activity in the 19S regulatory cap, explaining, at least in part, the adverse effects of O-GlcNAc modification and suggesting that downregulating O-GlcNAc might be important in the treatment of human diseases. In this study, we report on a novel mechanism to modulate cellular O-GlcNAc modification, namely through heat shock protein 90 (Hsp90) inhibition. We observed that O-linked β-N-acetylglucosamine transferase (OGT) interacts with the tetratricopeptide repeat binding site of Hsp90. Inhibition of Hsp90 by its specific inhibitors, radicicol or 17-N-allylamino-17-demethoxygeldanamycin, destabilized OGT in primary endothelial cell cultures and enhanced its degradation by the proteasome. Furthermore, Hsp90 inhibition downregulated O-GlcNAc protein modifications and attenuated the high glucose-induced increase in O-GlcNAc protein modification, including high glucose-induced increase in endothelial or type 3 isoform of nitric oxide synthase (eNOS) O-GlcNAcylation. These results suggest that Hsp90 is involved in the regulation of OGT and O-GlcNAc modification and that Hsp90 inhibitors might be used to modulate O-GlcNAc modification and reverse its adverse effects in human diseases.     

Stabilization of integrin‐linked kinase by the Hsp90‐CHIP axis impacts cellular force generation,migration and the fibrotic response

Integrin‐linked kinase (ILK) is an adaptor protein required to establish and maintain the connection between integrins and the actin cytoskeleton. This linkage is essential for generating force between the extracellular matrix (ECM) and the cell during migration and matrix remodelling. The mechanisms by which ILK stability and turnover are regulated are unknown. Here we report that the E3 ligase CHIP–heat shock protein 90 (Hsp90) axis regulates ILK turnover in fibroblasts. The chaperone Hsp90 stabilizes ILK and facilitates the interaction of ILK with α‐parvin. When Hsp90 activity is blocked, ILK is ubiquitinated by CHIP and degraded by the proteasome, resulting in impaired fibroblast migration and a dramatic reduction in the fibrotic response to bleomycin in mice. Together, our results uncover how Hsp90 regulates ILK stability and identify a potential therapeutic strategy to alleviate fibrotic diseases.     

热休克蛋白90三磷酸腺苷酶的调控机制研究

热休克蛋白90（heat shock protein90,Hsp90）是一类在生物进化中高度保守的蛋白,与细胞凋亡密切相关,作为抗癌新靶标已经得到了广泛的关注。相关研究表明,Hsp90可以通过多种方式调控ATPase的活性,如自身构象改变、与辅伴侣分子形成复合物以及转录后修饰等。在Hsp90基本构象改变的基础上,综述了不同因素对ATPase的调控作用,着重阐述近几年的研究进展,为进一步研究Hsp90调控ATPase的机制提供一定的参考。     

Synthesis and biological activities of novel 17-aminogeldanamycin derivatives

Geldanamycin interferes with the action of heat shock protein 90 (Hsp90) by binding to the N-terminal ATP binding site and inhibiting an essential ATPase activity. In a program directed toward finding potent, water soluble inhibitors of Hsp90, we prepared a library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogs, and compared their affinity for Hsp90, ability to inhibit growth of SKBr3 mammalian cells, and in selected cases, water solubility. Over 20 analogs showed cell growth inhibition potencies similar to that of 17-allylamino-17-demethoxygeldanamycin (17-AAG), the front-runner geldanamycin analog that is currently in multiple clinical trials. Many of these analogs showed water solubility properties that were desirable for formulation. One of the most potent and water-soluble analogs in the series was 17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG), which was independently prepared by the NCI and will soon enter clinical trials. Importantly, the binding affinity of these analogs to the molecular target Hsp90 does not correlate well with their cytotoxicity in SKBr3 cells.     

Histone deacetylase 6 regulates growth factor-induced actin remodeling and endocytosis

Histone deacetylase 6 (HDAC6) is a cytoplasmic deacetylase that uniquely catalyzes α-tubulin deacetylation and promotes cell motility. However, the mechanism underlying HDAC6-dependent cell migration and the role for microtubule acetylation in motility are not known. Here we show that HDAC6-induced global microtubule deacetylation was not sufficient to stimulate cell migration. Unexpectedly, in response to growth factor stimulation, HDAC6 underwent rapid translocation to actin-enriched membrane ruffles and subsequently became associated with macropinosomes, the vesicles for fluid-phase endocytosis. Supporting the importance of these associations, membrane ruffle formation, macropinocytosis, and cell migration were all impaired in HDAC6-deficient cells. Conversely, elevated HDAC6 levels promoted membrane ruffle formation with a concomitant increase in macropinocytosis and motility. In search for an HDAC6 target, we found that heat shock protein 90 (Hsp90), another prominent substrate of HDAC6, was also recruited to membrane ruffles and macropinosomes. Significantly, inhibition of Hsp90 activity suppressed membrane ruffling and cell migration, while expression of an acetylation-resistant Hsp90 mutant promoted ruffle formation. Our results uncover a surprising role for HDAC6 in actin remodeling-dependent processes and identify the actin cytoskeleton as an important target of HDAC6-regulated protein deacetylation.     

Importance of the C-terminal domain of Harc for binding to Hsp70 and Hop as well as its response to heat shock

Hsp90 is a molecular chaperone that acts in concert with Hsp70 to mediate the folding of many important regulatory proteins (e.g., protein kinases) into functional conformations. The chaperone activity of Hsp90 is primarily regulated by its cochaperones. For example, the Hsp90 cochaperone Cdc37 recruits Hsp90 to protein kinases as well as inhibiting its ATPase activity to promote the binding of Hsp90 to protein kinases. Harc is a structurally related Hsp90 cochaperone with a three-domain structure in which the middle domain binds Hsp90. In contrast to Cdc37 though, Harc also binds to Hsp70 and Hop (Hsp70/Hsp90 organizing protein). Here we demonstrate that deletion of the C-terminal domain of Harc abolished the binding of Hsp70 and Hop and reduced the affinity of Hsp90 binding to Harc. Significantly, the C-terminal domain of Harc bound Hsp70, but it did not bind Hop or Hsp90. Size exclusion chromatography of cell lysates revealed that Hop only formed a complex with Harc in the presence of Hsp90 and Hsp70, consistent with a model in which the interaction of Hop with Harc is mediated via the binding of Hop to Harc-bound Hsp90 and Hsp70. Notably, heat shock resulted in a marked decrease in the solubility of Harc, a response that was further augmented by the deletion of the C-terminal domain of Harc. This latter finding is especially interesting given that bioinformatics analysis indicated that cells may express splice variants of Harc that encode C-terminally truncated Harc isoforms. Together, these findings indicate that the C-terminal domain of Harc is a key determinant of its cochaperone functions.     

Heat shock protein synthesis over time in infective Trichinella spiralis larvae raised in suboptimal culture conditions

Changes in the viability, infectivity and heat shock protein (Hsp) levels are reported in Trichinella spiralis first stage larvae (L1) stored in 199 medium for up to seven days at 37 degrees C. These conditions induce stress that the larvae, eventually, cannot overcome. After three days of storage, the infectivity and viability were unchanged, although higher Hsp70 levels were observed. After this time, larvae gradually lost viability and infectivity, coinciding with a decrease in Hsp70 and Hsp90 and an increase in actin (a housekeeping protein). In addition, a possibly inducible heat shock protein, Hsp90i, appeared as constitutive Hsp90 disappeared. No significant changes in Hsp60 levels were detected at any time. These results suggest that heat shock proteins initially try to maintain homeostasis, but on failing, may be involved in cell death.     

Dihydroquinone ansamycins: toward resolving the conflict between low in vitro affinity and high cellular potency of geldanamycin derivatives

Heat shock protein 90 (Hsp90) is critical for the maturation of numerous client proteins, many of which are involved in cellular transformation and oncogenesis. The ansamycins, geldanamycin (GA) and its derivative, 17-allylaminogeldanamycin (17-AAG), inhibit Hsp90. As such, the prototypical Hsp90 inhibitor, 17-AAG, has advanced into clinical oncology trials. GA and 17-AAG potently inhibit tumor cell proliferation and survival but have been reported to bind weakly to Hsp90 in vitro. Recent studies have suggested that the in vitro potency of ansamycins against Hsp90 may be enhanced in the presence of cochaperones. Here, we present evidence of an alternative explanation. Ansamycins reduced to their dihydroquinones in the presence of common reducing agents in vitro have approximately 40-fold greater affinity than the corresponding oxidized quinones. The dihydroquinone of 17-AAG is not generated in an aqueous environment in the absence of reducing agents but is produced in both tumor and normal quiescent epithelial cells. The reduced form of 17-AAG is differentiated from its oxidized form not only by the higher affinity for Hsp90 but also by a protracted K(off) rate. Therefore, the in vivo accumulation of the high-affinity dihydroquinone ansamycins in tumor cells contributes to the antitumor activity of these compounds and alters our understanding of the active species driving the efficacy of this class of compounds.     

Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin

Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.     

Heat shock protein (hsp90) interacts with smooth muscle calponin and affects calponin-binding to actin

Interaction of smooth muscle calponin with 90 kDa heat shock protein (hsp90) was analyzed by means of native gel electrophoresis and affinity chromatography. Under conditions used, calponin and hsp90 form a complex with an apparent dissociation constant in the micromolar range. The major hsp90-binding site is located in the N-terminal (residues 7-144) part of calponin. Addition of calponin to actin-tropomyosin complex results in formation of actin bundles. Hsp90 partially prevents bundle formation without affecting the molar ratio calponin/actin in single actin filaments or actin bundles. At low ionic strength, calponin induces polymerization of G-actin. Hsp90 decreases calponin-induced polymerization of G-actin. It is supposed that hsp90 may be involved in the assembly of actin filaments.     

Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation

Heat shock protein 90 (Hsp90), which chaperones multiple client proteins, has been shown to be implicated in HCV replication. Pharmacological inhibitors of Hsp90 display an anti-HCV activity. However, little is known about the mechanisms of regulation of HCV replication by Hsp90. Here, we show that Hsp90 inhibition by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) destabilizes phosphoinositide-dependent kinase-1 (PDK1), an upstream kinase of the protein kinase C-related kinase 2 (PRK2) responsible for phosphorylation of HCV RNA polymerase, through the proteosome pathway. Destabilization of PDK1 led to inhibition of phosphorylation of the viral RNA polymerase through a decrease in the abundance of active form PRK2 level. Consequently, Hsp90 inhibition resulted in suppression of HCV replication both in human hepatoma Huh7 cells harboring an HCV subgenomic replicon and in HCV-infected cells. 17-DMAG treatment did not interfere with HCV internal ribosome entry site-mediated translation and the cell cycle in Huh7 cells. Co-treatment of 17-DMAG with interferon-α or HA1077, an inhibitor of PRK2, enhanced the anti-HCV activity of 17-DMAG. Taken together, these findings suggest that Hsp90 plays a critical role in the regulation of HCV RNA polymerase phosphorylation via the PDK1-PRK2 signaling pathway.     

热休克蛋白90的N端与ATP类似物的晶体结构揭示其功能调控

分子伴侣热休克蛋白90(Hsp90)对于许多涉及细胞周期调控、信号转导以及细胞生长调控蛋白质的折叠、成熟及稳定是必需的．Hsp90的N端结构高度保守,包含一个ATP结合口袋并具有ATP酶活性,Hsp90的功能依赖于ATP与Hsp90结合后诱导的构象重排及之后的ATP水解．为了深入研究ATP与Hsp90结合后N端的结构及其功能状态,使用悬滴法共结晶了Hsp90的N端与ATP类似物AMPPNP及ATPγS的复合物,并利用分子置换法对其结构进行了解析．两个复合物晶体结构都捕获到了核苷酸的电子密度,尤其是γ-磷酸的电子密度,从而观察到γ-磷酸与蛋白质之间的相互作用．ATPγS中γ-磷酸的捕获证实了之前报道的结构中没有捕获到γ-磷酸是其处于无序状态而非被水解．单体状态下的人源Hsp90^N- AMPPNP与处于二聚体化的酵母Hsp90-AMPPNP结构对比可见S1和ATP lid的位置有明显区别,结构分析表明,E18-K100和N40-D127之间形成的氢键相互作用,在一定程度上阻碍了S1和ATP lid的摆动,很可能阻止了二聚体的形成．     

Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion

NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.     

Inhibition of Hsp90 function delays and impairs recovery from heat shock

The induction of the heat shock response as well as its termination is autoregulated by heat shock protein activities. In this study we have investigated whether Hsp90 functional protein levels influence the characteristics and duration of the heat shock response. Treatment of cells with several benzoquinone ansamycin inhibitors of Hsp90 (geldanamycin, herbimycin A) activated a heat shock response in the absence of heat shock, as reported previously. Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock. Concurrently, the rate of Hsp synthesis and accumulation was substantially increased and prolonged. The cessation of heat shock protein synthesis did not occur until the levels of Hsp70 were substantially elevated relative to its standard threshold for autoregulation. The elevated levels of HSPS 22-28 (the small HSPS) and Hsp70 are not able to promote thermotolerance when Hsp90 activity is repressed by ansamycins; rather a suppression of thermotolerance is observed. These results suggest that a multicomponent protein chaperone complex involving both Hsp90 and Hsp70 signals the cessation of heat shock protein synthesis, the restoration of normal translation, and likely the establishment of thermotolerance. Impaired function of either component is sufficient to alter the heat shock response.