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Sample records for hsp90 atpase activity

  1. Low Resolution Structural Studies Indicate that the Activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis Has an Elongated Shape Which Allows Its Interaction with Both N- and M-Domains of Hsp90

    PubMed Central

    Seraphim, Thiago V.; Alves, Marina M.; Silva, Indjara M.; Gomes, Francisco E. R.; Silva, Kelly P.; Murta, Silvane M. F.; Barbosa, Leandro R. S.; Borges, Júlio C.

    2013-01-01

    The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins’ interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90. PMID:23826147

  2. Low resolution structural studies indicate that the activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis has an elongated shape which allows its interaction with both N- and M-domains of Hsp90.

    PubMed

    Seraphim, Thiago V; Alves, Marina M; Silva, Indjara M; Gomes, Francisco E R; Silva, Kelly P; Murta, Silvane M F; Barbosa, Leandro R S; Borges, Júlio C

    2013-01-01

    The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins' interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90. PMID:23826147

  3. The Mechanism of Hsp90 ATPase Stimulation by Aha1.

    PubMed

    Wolmarans, Annemarie; Lee, Brian; Spyracopoulos, Leo; LaPointe, Paul

    2016-01-01

    Hsp90 is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called 'clients'. Numerous co-chaperone proteins regulate progression through the ATP-dependent client activation cycle. The most potent stimulator of the Hsp90 ATPase activity is the co-chaperone Aha1p. Only one molecule of Aha1p is required to fully stimulate the Hsp90 dimer despite the existence of two, presumably identical, binding sites for this regulator. Using ATPase assays with Hsp90 heterodimers, we find that Aha1p stimulates ATPase activity by a three-step mechanism via the catalytic loop in the middle domain of Hsp90. Binding of the Aha1p N domain to the Hsp90 middle domain exerts a small stimulatory effect but also drives a separate conformational rearrangement in the Hsp90 N domains. This second event drives a rearrangement in the N domain of the opposite subunit and is required for the stimulatory action of the Aha1p C domain. Furthermore, the second event can be blocked by a mutation in one subunit of the Hsp90 dimer but not the other. This work provides a foundation for understanding how post-translational modifications regulate co-chaperone engagement with the Hsp90 dimer. PMID:27615124

  4. ATPase Activity and ATP-dependent Conformational Change in the Co-chaperone HSP70/HSP90-organizing Protein (HOP)*

    PubMed Central

    Yamamoto, Soh; Subedi, Ganesh Prasad; Hanashima, Shinya; Satoh, Tadashi; Otaka, Michiro; Wakui, Hideki; Sawada, Ken-ichi; Yokota, Shin-ichi; Yamaguchi, Yoshiki; Kubota, Hiroshi; Itoh, Hideaki

    2014-01-01

    Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change. PMID:24535459

  5. Review: The HSP90 molecular chaperone—an enigmatic ATPase

    PubMed Central

    2016-01-01

    ABSTRACT The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of ‘client’ proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co‐chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally‐coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594–607, 2016. PMID:26991466

  6. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    PubMed

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. PMID:26991466

  7. Threonine 22 phosphorylation attenuates Hsp90 interaction with co-chaperones and affects its chaperone activity

    PubMed Central

    Mollapour, Mehdi; Tsutsumi, Shinji; Truman, Andrew W.; Xu, Wanping; Vaughan, Cara K.; Beebe, Kristin; Konstantinova, Anna; Vourganti, Srinivas; Panaretou, Barry; Piper, Peter W.; Trepel, Jane B.; Prodromou, Chrisostomos; Pearl, Laurence H.; Neckers, Len

    2011-01-01

    SUMMARY Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by co-chaperones but also by distinct post-translational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α-helix 1 of the yeast Hsp90 N-domain both in vitro and in vivo. This α-helix participates in a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase competent state. Phospho-mimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function, and impacts interaction with the co-chaperones Aha1 and Cdc37. Over-expression of Aha1 stimulates the ATPase activity, restores co-chaperone interactions, and compensates for the functional defects of these Hsp90 mutants. PMID:21419342

  8. Asymmetric activation of the hsp90 dimer by its cochaperone aha1.

    PubMed

    Retzlaff, Marco; Hagn, Franz; Mitschke, Lars; Hessling, Martin; Gugel, Frederik; Kessler, Horst; Richter, Klaus; Buchner, Johannes

    2010-02-12

    The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing. PMID:20159554

  9. Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism.

    PubMed

    Schulze, Andrea; Beliu, Gerti; Helmerich, Dominic A; Schubert, Jonathan; Pearl, Laurence H; Prodromou, Chrisostomos; Neuweiler, Hannes

    2016-08-01

    The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle. PMID:27322067

  10. Hsp90 Activity Modulation by Plant Secondary Metabolites.

    PubMed

    Dal Piaz, Fabrizio; Terracciano, Stefania; De Tommasi, Nunziatina; Braca, Alessandra

    2015-09-01

    Hsp90 is an evolutionarily conserved adenosine triphosphate-dependent molecular chaperone and is one of the most abundant proteins in the cells (1-3 %). Hsp90 is induced when a cell undergoes various types of environmental stresses such as heat, cold, or oxygen deprivation. It is involved in the turnover, trafficking, and activity of client proteins, including apoptotic factors, protein kinases, transcription factors, signaling proteins, and a number of oncoproteins. Most of the Hsp90 client proteins are involved in cell growth, differentiation, and survival, and include kinases, nuclear hormone receptors, transcription factors, and other proteins associated with almost all the hallmarks of cancer. Consistent with these diverse activities, genetic and biochemical studies have demonstrated the implication of Hsp90 in a range of diseases, including cancer, making this chaperone an interesting target for drug research.During the last few decades, plant secondary metabolites have been studied as a major source for lead compounds in drug discovery. Recently, several plant-derived small molecules have been discovered exhibiting inhibitory activity towards Hsp90, such as epigallocatechin gallate, gedunin, lentiginosine, celastrol, and deguelin. In this work, an overview of plant secondary metabolites interfering with Hsp90 activities is provided. PMID:26227505

  11. HSP90 inhibitors enhance differentiation and MITF (microphthalmia transcription factor) activity in osteoclast progenitors.

    PubMed

    van der Kraan, A Gabrielle J; Chai, Ryan C C; Singh, Preetinder P; Lang, Benjamin J; Xu, Jiake; Gillespie, Matthew T; Price, John T; Quinn, Julian M W

    2013-04-15

    The HSP90 (heat-shock protein 90) inhibitor 17-AAG (17-allylamino-demethoxygeldanamycin) increases osteoclast formation both in vitro and in vivo, an action that can enhance cancer invasion and growth in the bone microenvironment. The cellular mechanisms through which 17-AAG exerts this action are not understood. Thus we sought to clarify the actions of 17-AAG on osteoclasts and determine whether other HSP90 inhibitors had similar properties. We determined that 17-AAG and the structurally unrelated HSP90 inhibitors CCT018159 and NVP-AUY922 dose-dependently increased RANKL [receptor activator of NF-κB (nuclear factor κB) ligand]-stimulated osteoclastogenesis in mouse bone marrow and pre-osteoclastic RAW264.7 cell cultures. Moreover, 17-AAG also enhanced RANKL- and TNF (tumour necrosis factor)-elicited osteoclastogenesis, but did not affect RANKL-induced osteoclast survival, suggesting that only differentiation mechanisms are targeted. 17-AAG affected the later stages of progenitor maturation (after 3 days of incubation), whereas the osteoclast formation enhancer TGFβ (transforming growth factor β) acted prior to this, suggesting different mechanisms of action. In studies of RANKL-elicited intracellular signalling, 17-AAG treatment did not increase c-Fos or NFAT (nuclear factor of activated T-cells) c1 protein levels nor did 17-AAG increase activity in luciferase-based NF-κB- and NFAT-response assays. In contrast, 17-AAG treatment (and RANKL treatment) increased both MITF (microphthalmia-associated transcription factor) protein levels and MITF-dependent vATPase-d2 (V-type proton ATPase subunit d2) gene promoter activity. These results indicate that HSP90 inhibitors enhance osteoclast differentiation in an NFATc1-independent manner that involves elevated MITF levels and activity. PMID:23379601

  12. Structural and functional studies of Leishmania braziliensis Hsp90.

    PubMed

    Silva, K P; Seraphim, T V; Borges, J C

    2013-01-01

    The ubiquitous Hsp90 is critical for protein homeostasis in the cells, stabilizing "client" proteins in a functional state. Hsp90 activity depends on its ability to bind and hydrolyze ATP, involving various conformational changes that are regulated by co-chaperones, posttranslational modifications and small molecules. Compounds like geldanamycin (GA) and radicicol inhibit the Hsp90 ATPase activity by occupying the ATP binding site, which can lead client protein to degradation and also inhibit cell growth and differentiation in protozoan parasites. Our goal was to produce the recombinant Hsp90 of Leishmania braziliensis (LbHsp90) and construct of its N-terminal (LbHsp90N) and N-domain and middle-domain (LbHsp90NM), which lacks the C-terminal dimerization domain, in order to understand how Hsp90 works in protozoa. The recombinant proteins were produced folded as attested by spectroscopy experiments. Hydrodynamic experiments revealed that LbHsp90N and LbHsp90NM behaved as elongated monomers while LbHsp90 is an elongated dimer. All proteins prevented the in vitro citrate synthase and malate dehydrogenase aggregation, attesting that they have chaperone activity, and interacted with adenosine ligands with similar dissociation constants. The LbHsp90 has low ATPase activity (k(cat)=0.320min(-1)) in agreement with Hsp90 orthologs, whereas the LbHsp90NM has negligible activity, suggesting the importance of the dimeric protein for this activity. The GA interacts with LbHsp90 and with its domain constructions with different affinities and also inhibits the LbHsp90 ATPase activity with an IC(50) of 0.7μM. All these results shed light on the LbHsp90 activity and are the first step to understanding the Hsp90 molecular chaperone system in L. braziliensis. PMID:22910377

  13. A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase activity requires interaction with Hsp90.

    PubMed Central

    Aligue, R; Akhavan-Niak, H; Russell, P

    1994-01-01

    Wee1 protein kinase regulates the length of G2 phase by carrying out the inhibitory tyrosyl phosphorylation of Cdc2-cyclin B kinase. Mutations were isolated that suppressed the G2 cell cycle arrest caused by overproduction of Wee1. One class of swo (suppressor of wee1 overproduction) mutation, exemplified by swo1-26, also caused a temperature sensitive lethal phenotype in a wee1+ background. The swo1+ gene encodes a member of the Hsp90 family of stress proteins. Swo1 is essential for viability at all temperatures. Swo1 coimmunoprecipitates with Wee1, showing that the two proteins interact. The swo1-26 mutant undergoes premature mitosis when grown at a semi-permissive temperature. These data strongly indicate that formation of active Wee1 tyrosine kinase requires interaction with Swo1, perhaps in a manner analogous to the previously demonstrated interaction between Hsp90 and v-src tyrosine kinase. These observations demonstrate a unexpected role for Hsp90 in cell cycle control. Images PMID:7813446

  14. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    PubMed Central

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  15. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    NASA Astrophysics Data System (ADS)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  16. Hsp90 Oligomers Interacting with the Aha1 Cochaperone: An Outlook for the Hsp90 Chaperone Machineries.

    PubMed

    Lepvrier, Eléonore; Moullintraffort, Laura; Nigen, Michaël; Goude, Renan; Allegro, Diane; Barbier, Pascale; Peyrot, Vincent; Thomas, Daniel; Nazabal, Alexis; Garnier, Cyrille

    2015-07-21

    The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer able to self-associate in the presence of divalent cations or under heat shock. This study investigated the relationship between Hsp90 oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase). The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated by ultracentrifugation, size-exclusion chromatography coupled to multiangle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Hsp90 dimer was able to bind up to four Aha1 molecules, and Hsp90 oligomers are also able to interact with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization process. Except for Hsp90 dimer, the stoichiometry of the interaction remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to bind the Aha1 ATPase activator reinforces their role within the Hsp90 chaperone machineries. PMID:26076190

  17. Both the Charged Linker Region and ATPase Domain of Hsp90 Are Essential for Rad51-Dependent DNA Repair

    PubMed Central

    Suhane, Tanvi; Laskar, Shyamasree; Advani, Siddheshwari; Roy, Nabamita; Varunan, Shalu; Bhattacharyya, Dibyendu

    2014-01-01

    The inhibition of Hsp90 in cancerous cells has been correlated with the reduction in double-strand break (DSB repair) activity. However, the precise effect of Hsp90 on the DSB repair pathway in normal cells has remained enigmatic. Our results show that the Hsp82 chaperone, the ortholog of mammalian Hsp90, is indispensable for homologous-recombination (HR)-mediated DNA repair in the budding yeast Saccharomyces cerevisiae. A considerable reduction in cell viability is observed in an Hsp82-inactivated mutant upon methyl methanesulfonate (MMS) treatment as well as upon UV treatment. The loss of Hsp82 function results in a dramatic decrease in gene-targeting efficiency and a marked decrease in the endogenous levels of the key recombination proteins Rad51 and Rad52 without any notable change in the levels of RAD51 or RAD52 transcripts. Our results establish Rad51 as a client of Hsp82, since they interact physically in vivo, and also show that when Hsp82 is inhibited by 17-AAG, Rad51 undergoes proteasomal degradation. By analyzing a number of point mutants with mutations in different domains of Hsp82, we observe a strong association between the sensitivity of an ATPase mutant of Hsp82 to DNA damage and the decreases in the amounts of Rad51 and Rad52 proteins. The most significant observations include the dramatic abrogation of HR activity and the marked decrease in Rad51 focus formation in the charged linker deletion mutant of Hsp82 upon MMS treatment. The charged linker region of Hsp82 is evolutionarily conserved in all eukaryotes, but until now, no biological significance has been assigned to it. Our findings elucidate the importance of this region in DNA repair for the first time. PMID:25380755

  18. Luteolin Induces Carcinoma Cell Apoptosis through Binding Hsp90 to Suppress Constitutive Activation of STAT3

    PubMed Central

    Zhao, Bo; Zhao, Zhihui; Zhou, Jiahong; Xu, Yimiao; Xin, Yinqiang; Liu, Chang; Luo, Lan; Yin, Zhimin

    2012-01-01

    Background Abnormal activity of STAT3 is associated with a number of human malignancies. Hsp90 plays a central role in stabilizing newly synthesized proteins and participates in maintaining the functional competency of a number of signaling transducers involved in cell growth, survival and oncogenesis, such as STAT3. Hsp90 interacts with STAT3 and stabilizes Tyr-phosphorylated STAT3. It has been reported that luteolin possesses anticancer activity through degradation of Tyr705-phosphorylated STAT3. Methodology/Principal Findings We found that overexpression of Hsp90 inhibited luteolin-induced degradation of Tyr705-phosphorylated STAT3 and luteolin also reduced the levels of some other Hsp90 interacting proteins. Results from co-immunoprecipitation and immunoblot analysis demonstrated that luteolin prevented the association between Hsp90 and STAT3 and induced both Tyr705- and Ser727-phosphorylated STAT3 degradation through proteasome-dependent pathway. The molecular modeling analysis with CHARMm–Discovery Studio 2.1(DS 2.1) indicated that luteolin could bind to the ATP-binding pocket of Hsp90. SPR technology-based binding assay confirmed the association between luteolin and Hsp90. ATP-sepharose binding assay displayed that luteolin inhibited Hsp90-ATP binding. Conclusions/Significance Luteolin promoted the degradation of Tyr705- and Ser727-phosphorylated STAT3 through interacting with Hsp90 and induced apoptosis of cancer cells. This study indicated that luteolin may act as a potent HSP90 inhibitor in antitumor strategies. PMID:23145121

  19. AIDing cancer treatment: Reducing AID activity via HSP90 inhibition.

    PubMed

    Rebhandl, Stefan; Geisberger, Roland

    2015-08-01

    The activation induced deaminase (AID) catalyses the two key events underlying humoral adaptive immunity: class switch recombination and somatic hypermutation of antibody genes in B lymphocytes. AID accomplishes this task by directly deaminating cytosines within the genomic immunoglobulin locus, thereby triggering a complex mutagenic process eventually leading to improved effector function of antibodies. However, it has long been noticed that AID can be aberrantly expressed in cancer and that its activity is not absolutely restricted to antibody genes, as substantial genome-wide off-target mutations have been observed, which contribute to tumorigenesis and clonal evolution of AID-expressing malignancies. In this issue of the European Journal of Immunology, Montamat-Sicotte et al. [Eur. J. Immunol. 2015. 45: 2365-2376] investigate the feasibility and efficacy of in vivo inhibition of AID with HSP90 inhibitors in a mouse model of B-cell leukemia and in vitro with a human breast cancer cell line, thereby demonstrating that cancer patients may benefit from preventing noncanonical AID functions. PMID:26151367

  20. HSP90 inhibitors decrease AID levels and activity in mice and in human cells

    PubMed Central

    Montamat-Sicotte, Damien; Liztler, Ludivine C; Abreu, Cecilia; Safavi, Shiva; Zahn, Astrid; Orthwein, Alexandre; Muschen, Markus; Oppezzo, Pablo; Muñoz, Denise P; Di Noia, Javier M

    2015-01-01

    Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert non-canonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications. PMID:25912253

  1. HSP90 inhibitors decrease AID levels and activity in mice and in human cells.

    PubMed

    Montamat-Sicotte, Damien; Litzler, Ludivine C; Abreu, Cecilia; Safavi, Shiva; Zahn, Astrid; Orthwein, Alexandre; Müschen, Markus; Oppezzo, Pablo; Muñoz, Denise P; Di Noia, Javier M

    2015-08-01

    Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert noncanonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications. PMID:25912253

  2. Effects of various salinities on Na(+)-K(+)-ATPase, Hsp70 and Hsp90 expression profiles in juvenile mitten crabs, Eriocheir sinensis.

    PubMed

    Sun, M; Jiang, K; Zhang, F; Zhang, D; Shen, A; Jiang, M; Shen, X; Ma, L

    2012-01-01

    Eriocheir sinensis is a euryhaline crab migrating from sea to freshwater habitats during the juvenile stage. We used quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the gene expression profile of Na(+)-K(+)-ATPase, Hsp70 (heat shock protein 70) and Hsp90 in megalopa exposed to salinities of 0, 2, 5, 10, and 15 parts per thousand. Both low and high salinities markedly stimulated expression of Na(+)-K(+)-ATPase, Hsp70 and Hsp90 genes of Chinese mitten crab megalopa; salinity had different effects on Na(+)-K(+)-ATPase, Hsp70 and Hsp90 levels depending on the duration of salinity stress, implying that Na(+)-K(+)-ATPase, Hsp70 and Hsp90 may play an important role in salinity tolerance in this crab species. PMID:22576924

  3. Inhibition of Plasmodium falciparum Hsp90 Contributes to the Antimalarial Activities of Aminoalcohol-carbazoles.

    PubMed

    Wang, Tai; Mäser, Pascal; Picard, Didier

    2016-07-14

    Malaria caused by the protozoan parasite Plasmodium falciparum (Pf) remains a major public health problem throughout the developing world. One molecular target that should receive more attention is the molecular chaperone Hsp90. It is essential and highly conserved in all eukaryotes, including in protozoan parasites. We have identified an amino-alcohol carbazole (N-CBZ) as a PfHsp90-selective inhibitor by virtually docking a large set of antimalarial compounds, previously found in a phenotypic screen, into a PfHsp90-specific pocket. By correlating the ability of 30 additional N-CBZ derivatives to bind directly to PfHsp90 with their Pf-inhibitory activity, we found that these types of compounds are more likely to inhibit Pf growth if they bind PfHsp90. For plausible targets such as PfHsp90, our workflow may help identifying the molecular target for compounds found by screening large chemical libraries for a desired biological effect and, conversely, ensuring biological effectiveness for compounds affecting a particular target. PMID:27312008

  4. Gedunin, a novel hsp90 inhibitor: semisynthesis of derivatives and preliminary structure-activity relationships.

    PubMed

    Brandt, Gary E L; Schmidt, Matthew D; Prisinzano, Thomas E; Blagg, Brian S J

    2008-10-23

    Gedunin (1), a tetranortriterpenoid isolated from the Indian neem tree ( Azadirachta indica), was recently shown to manifest anticancer activity via inhibition of the 90 kDa heat shock protein (Hsp90) folding machinery and to induce the degradation of Hsp90-dependent client proteins similar to other Hsp90 inhibitors. The mechanism of action by which gedunin induces client protein degradation remains undetermined, however, prior studies have demonstrated that it does not bind competitively versus ATP. In an effort to further probe the mechanism of action, 19 semisynthetic derivatives of gedunin were prepared and their antiproliferative activity against MCF-7 and SkBr3 breast cancer cells determined. Although no compound was found to exhibit antiproliferative activity more effective than the natural product, functionalities critical for antiproliferative activity have been identified. PMID:18816111

  5. Hsp90-binding immunophilin FKBP51 forms complexes with hTERT enhancing telomerase activity.

    PubMed

    Lagadari, Mariana; Zgajnar, Nadia R; Gallo, Luciana I; Galigniana, Mario D

    2016-08-01

    FK506-binding proteins are members of the immunophilin family of proteins. Those immunophilins associated to the 90-kDa-heat-shock protein, Hsp90, have been proposed as potential modulators of signalling cascade factors chaperoned by Hsp90. FKBP51 and FKBP52 are the best characterized Hsp90-bound immunophilins first described associated to steroid-receptors. The reverse transcriptase subunit of telomerase, hTERT, is also an Hsp90 client-protein and is highly expressed in cancer cells, where it is required to compensate the loss of telomeric DNA after each successive cell division. Because FKBP51 is also a highly expressed protein in cancer tissues, we analyzed its potential association with hTERT·Hsp90 complexes and its possible biological role. In this study it is demonstrated that both immunophilins, FKBP51 and FKBP52, co-immunoprecipitate with hTERT. The Hsp90 inhibitor radicicol disrupts the heterocomplex and favors the partial cytoplasmic relocalization of hTERT in similar manner as the overexpression of the TPR-domain peptide of the immunophilin. While confocal microscopy images show that FKBP51 is primarily localized in mitochondria and hTERT is totally nuclear, upon the onset of oxidative stress, FKBP51 (but not FKBP52) becomes mostly nuclear colocalizing with hTERT, and longer exposure times to peroxide favors hTERT export to mitochondria. Importantly, telomerase activity of hTERT is significantly enhanced by FKBP51. These observations support the emerging role assigned to FKBP51 as antiapoptotic factor in cancer development and progression, and describe for the first time the potential role of this immunophilin favoring the clonal expansion by enhancing telomerase activity. PMID:27233944

  6. The non-Geldanamycin Hsp90 inhibitors enhanced the antifungal activity of fluconazole

    PubMed Central

    Li, Liping; An, Maomao; Shen, Hui; Huang, Xin; Yao, Xueya; Liu, Jian; Zhu, Fang; Zhang, Shiqun; Chen, Simin; He, Lijuan; Zhang, Jundong; Zou, Zui; Jiang, Yuanying

    2015-01-01

    The molecular chaperone heat shock protein 90 (Hsp90) is highly conserved in eukaryotes and facilitates the correct folding, productive assembly and maturation of a diverse cellular proteins. In fungi, especially the most prevalent human fungal pathogen Candida albicans, Hsp90 influences development and modulates drug resistance. Here, we mainly explore the effect of non-Geldanamycin Hsp90 inhibitor HSP990 on the activity of fluconazole (FLC) against Candida albicans and investigate the underlying mechanism. We demonstrate that HSP990 has potent synergistic antifungal activity with FLC against FLC-resistant C. albicans through the checkerboard microdilution assay,agar diffusion tests and time-kill curves, and shows low cytotoxicity to human umbilical vein endothelial cells. Further study shows that the activity of FLC against C. albicans biofilm formation in vitro is significantly enhanced when used in combination with HSP990. In a murine model of disseminated candidiasis, the therapeutic efficacy of FLC is also enhanced by the pharmacological inhibition of C. albicans Hsp90 function with HSP990. Thus, the combined use of small molecule compound and existing antifungal drugs may provide a potential therapeutic strategy for fungal infectious disease. PMID:26885259

  7. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

    PubMed

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A; Prakash, Thazha P; Nichols, Joshua G; Crooke, Stanley T

    2016-05-01

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs. PMID:26945041

  8. Hsp90: Friends, clients and natural foes.

    PubMed

    Verma, Sharad; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-08-01

    Hsp90, a homodimeric ATPase, is responsible for the correct folding of a number of newly synthesized polypeptides in addition to the correct folding of denatured/misfolded client proteins. It requires several co-chaperones and other partner proteins for chaperone activity. Due to the involvement of Hsp90-dependent client proteins in a variety of oncogenic signaling pathways, Hsp90 inhibition has emerged as one of the leading strategies for anticancer chemotherapeutics. Most of Hsp90 inhibitors blocks the N terminal ATP binding pocket and prevents the conformational changes which are essential for the loading of co-chaperones and client proteins. Several other inhibitors have also been reported which disrupt chaperone cycle in ways other than binding to N terminal ATP binding pocket. The Hsp90 inhibition is associated with heat shock response, mediated by HSF-1, to overcome the loss of Hsp90 and sustain cell survival. This review is an attempt to give an over view of all the important players of chaperone cycle. PMID:27295069

  9. Bioactive Metabolites from Chaetomium aureum: Structure Elucidation and Inhibition of the Hsp90 Machine Chaperoning Activity

    PubMed Central

    Kabbaj, Fatima Zahra; Lu, Su; Faouzi, My El Abbés; Meddah, Bouchra; Proksch, Peter; Cherrah, Yahya; Altenbach, Hans-Josef; Aly, Amal H.; Chadli, Ahmed; Debbab, Abdessamad

    2014-01-01

    Chemical investigation of the EtOAc extract of the fungus Chaetomium aureum, an endophyte of the Moroccan medicinal plant Thymelaea lythroides, afforded one new resorcinol derivative named chaetorcinol, together with five known metabolites. The structures of the isolated compounds were determined on the basis of one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry as well as by comparison with the literature. All compounds were tested for their activity towards the Hsp90 chaperoning machine in vitro using the progesterone receptor (PR) and rabbit reticulocyte lysate (RRL). Among the isolated compounds, only sclerotiorin efficiently inhibited the Hsp90 machine chaperoning activity. However, sclerotiorin showed no cytotoxic effect on breast cancer Hs578T, MDA-MB-231 and prostate cancer LNCaP cell lines. Interestingly, deacetylation of sclerotiorin increased its cytotoxicity toward the tested cell lines over a period of 48h. PMID:25482429

  10. Stromal cell–derived factor 2 is critical for Hsp90-dependent eNOS activation

    PubMed Central

    Siragusa, Mauro; Fröhlich, Florian; Park, Eon Joo; Schleicher, Michael; Walther, Tobias C.; Sessa, William C.

    2016-01-01

    Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell–derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser1177, a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser1177 in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells. PMID:26286023

  11. Histone Deacetylase 6-Controlled Hsp90 Acetylation Significantly Alters Mineralocorticoid Receptor Subcellular Dynamics But Not its Transcriptional Activity.

    PubMed

    Jiménez-Canino, Rubén; Lorenzo-Díaz, Fabián; Jaisser, Frederic; Farman, Nicolette; Giraldez, Teresa; Alvarez de la Rosa, Diego

    2016-06-01

    The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily that transduces the biological effects of corticosteroids. Its best-characterized role is to enhance transepithelial sodium reabsorption in response to increased aldosterone levels. In addition, MR participates in other aldosterone- or glucocorticoid-controlled processes such as cardiovascular homeostasis, adipocyte differentiation or neurogenesis, and regulation of neuronal activity in the hippocampus. Like other steroid receptors, MR forms cytosolic heterocomplexes with heat shock protein (Hsp) 90), Hsp70, and other proteins such as immunophilins. Interaction with Hsp90 is thought to maintain MR in a ligand-binding competent conformation and to regulate ligand-dependent and -independent nucleocytoplasmatic shuttling. It has previously been shown that acetylation of residue K295 in Hsp90 regulates its interaction with the androgen receptor and glucocorticoid receptor (GR). In this work we hypothesized that Hsp90 acetylation provides a regulatory step to modulate MR cellular dynamics and activity. We used Hsp90 acetylation mimic mutant K295Q or nonacetylatable mutant K295R to examine whether MR nucleocytoplasmatic shuttling and gene transactivation are affected. Furthermore, we manipulated endogenous Hsp90 acetylation levels by controlling expression or activity of histone deacetylase 6 (HDAC6), the enzyme responsible for deacetylation of Hsp90-K295. Our data demonstrates that HDAC6-mediated Hsp90 acetylation regulates MR cellular dynamics but it does not alter its function. This stands in contrast with the down-regulation of GR by HDAC6, suggesting that Hsp90 acetylation may play a role in balancing relative MR and GR activity when both factors are co-expressed in the same cell. PMID:27100623

  12. Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3)*

    PubMed Central

    Dutta, Dipanjan; Chattopadhyay, Shiladitya; Bagchi, Parikshit; Halder, Umesh Chandra; Nandi, Satabdi; Mukherjee, Anupam; Kobayashi, Nobumichi; Taniguchi, Koki; Chawla-Sarkar, Mamta

    2011-01-01

    Heat shock protein 90 (Hsp90) has been reported to positively regulate rotavirus replication by modulating virus induced PI3K/Akt and NFκB activation. Here, we report the active association of Hsp90 in the folding and stabilization of rotavirus nonstructural protein 3 (NSP3). In pCD-NSP3-transfected cells, treatment with Hsp90 inhibitor (17-N,N-dimethylethylenediamine-geldanamycin (17DMAG)) resulted in the proteasomal degradation of NSP3. Sequence analysis and deletion mutations revealed that the region spanning amino acids 225–258 within the C-terminal eIF4G-binding domain of NSP3 is a putative Hsp90 binding region. Co-immunoprecipitation and mammalian two-hybrid experiments revealed direct interaction of the C-terminal 12-kDa domain of Hsp90 (C90) with residues 225–258 of NSP3. NSP3-Hsp90 interaction is important for the formation of functionally active mature NSP3, because full-length NSP3 in the presence of the Hsp90 inhibitor or NSP3 lacking the amino acid 225–258 region did not show NSP3 dimers following in vitro coupled transcription-translation followed by chase. Disruption of residues 225–258 within NSP3 also resulted in poor RNA binding and eIF4G binding activity. In addition, inhibition of Hsp90 by 17DMAG resulted in reduced nuclear translocation of poly(A)-binding protein and translation of viral proteins. These results highlight the crucial role of Hsp90 chaperone in the regulation of assembly and functionality of a viral protein during the virus replication and propagation in host cells. PMID:21489987

  13. Mechanisms of Hsp90 regulation

    PubMed Central

    Prodromou, Chrisostomos

    2016-01-01

    Heat shock protein 90 (Hsp90) is a molecular chaperone that is involved in the activation of disparate client proteins. This implicates Hsp90 in diverse biological processes that require a variety of co-ordinated regulatory mechanisms to control its activity. Perhaps the most important regulator is heat shock factor 1 (HSF1), which is primarily responsible for upregulating Hsp90 by binding heat shock elements (HSEs) within Hsp90 promoters. HSF1 is itself subject to a variety of regulatory processes and can directly respond to stress. HSF1 also interacts with a variety of transcriptional factors that help integrate biological signals, which in turn regulate Hsp90 appropriately. Because of the diverse clientele of Hsp90 a whole variety of co-chaperones also regulate its activity and some are directly responsible for delivery of client protein. Consequently, co-chaperones themselves, like Hsp90, are also subject to regulatory mechanisms such as post translational modification. This review, looks at the many different levels by which Hsp90 activity is ultimately regulated. PMID:27515256

  14. Anticancer Inhibitors of Hsp90 Function: Beyond the Usual Suspects.

    PubMed

    Garg, Gaurav; Khandelwal, Anuj; Blagg, Brian S J

    2016-01-01

    The 90-kDa heat-shock protein (Hsp90) is a molecular chaperone responsible for the stability and function of a wide variety of client proteins that are critical for cell growth and survival. Many of these client proteins are frequently mutated and/or overexpressed in cancer cells and are therefore being actively pursued as individual therapeutic targets. Consequently, Hsp90 inhibition offers a promising strategy for simultaneous degradation of several anticancer targets. Currently, most Hsp90 inhibitors under clinical evaluation act by blocking the binding of ATP to the Hsp90 N-terminal domain and thereby, induce the degradation of many Hsp90-dependent oncoproteins. Although, they have shown some promising initial results, clinical challenges such as induction of the heat-shock response, retinopathy, and gastrointestinal tract toxicity are emerging from human trials, which constantly raise concerns about the future development of these inhibitors. Novobiocin derivatives, which do not bind the chaperone's N-terminal ATPase pocket, have emerged over the past decade as an alternative strategy to inhibit Hsp90, but to date, no derivative has been investigated in the clinical setting. In recent years, a number of natural or synthetic compounds have been identified that modulate Hsp90 function via various mechanisms. These compounds not only offer new chemotypes for the development of future Hsp90 inhibitors but can also serve as chemical probes to unravel the biology of Hsp90. This chapter presents a synopsis of inhibitors that directly, allosterically, or even indirectly alters Hsp90 function, and highlights their proposed mechanisms of action. PMID:26916001

  15. Asymmetric dimethylarginine inhibits HSP90 activity in Pulmonary Arterial Endothelial Cells: Role of Mitochondrial Dysfunction

    PubMed Central

    Sud, Neetu; Wells, Sandra M.; Wiseman, Dean A.; Wilham, Jason; Black, Stephen M.

    2013-01-01

    Increased ADMA levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus, the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that that ADMA increased NOS uncoupling both in recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Further, we found that this eNOS uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of HSP90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states. PMID:18385287

  16. Pharmacological inhibition of HSP90 and ras activity as a new strategy in the treatment of HNSCC.

    PubMed

    Misso, Gabriella; Giuberti, Gaia; Lombardi, Angela; Grimaldi, Anna; Ricciardiello, Filippo; Giordano, Antonio; Tagliaferri, Pierosandro; Abbruzzese, Alberto; Caraglia, Michele

    2013-01-01

    Advanced head and neck squamous cell cancer (HNSCC) is currently treated with taxane-based chemotherapy. We have previously shown that docetaxel (DTX) induces a ras-dependent survival signal that can be antagonized by farnesyl transferase inhibitors (FTI) such as tipifarnib (TIP). Here we show that the synergistic TIP/DTX combination determines synergistic apoptotic conditions but, at the same time, it modulates the expression of the components of the multichaperone complex that is, in turn, involved in the regulation of the stability of members of the ras-mediated pathway. Therefore, we have stably transfected HNSCC KB and Hep-2 cells with a plasmid encoding for HSP90. The expression of the protein was increased in both transfected cell lines but its activation status was increased in Hep-2 clones and decreased in KB clones. On the basis of these results, we have treated both parental and HSP90-transfected cells with a HSP90 inhibitor geldanamycin (GA). We have found that the antiproliferative activity of GA is dependent upon the activation status of HSP90 and that it is strongly synergistic when added in combination with TIP but not with DTX in cells overexpressing HSP90 and even more in cells with increased HSP90 activity. These data were paralleled by the decreased expression and activity of the components belonging to the ras→mediated signal transduction pathway. The present results suggest that multichaperone complex activation could be a resistance mechanism to the anti-proliferative and apoptotic effects induced by TIP and that the combination of FTIs such as TIP with GA could be a suitable therapeutic strategy in the treatment of HSP90-overexpressing HNSCC. PMID:22566192

  17. Cdc37-Hsp90 Complexes Are Responsive to Nucleotide-induced Conformational Changes and Binding of Further Cofactors*

    PubMed Central

    Gaiser, Andreas M.; Kretzschmar, Anja; Richter, Klaus

    2010-01-01

    Hsp90 is an ATP-dependent molecular chaperone, which facilitates the activation and stabilization of hundreds of client proteins in cooperation with a defined set of cofactors. Many client proteins are protein kinases, which are activated and stabilized by Hsp90 in cooperation with the kinase-specific co-chaperone Cdc37. Other Hsp90 co-chaperones, like the ATPase activator Aha1, also are implicated in kinase activation, and it is not yet clear how Cdc37 is integrated into Hsp90 co-chaperone complexes. Here, we studied the interaction between Cdc37, Hsp90, and other Hsp90 co-chaperones from the nematode Caenorhabditis elegans. Nematode Cdc37 binds with high affinity to Hsp90 and strongly inhibits the ATPase activity. In contrast to the human Hsp90 system, we observed binding of Cdc37 to open and closed Hsp90 conformations, potentially reflecting two different binding modes. Using a novel ultracentrifugation setup, which allows accurate analysis of multifactorial protein complexes, we show that cooperative and competitive interactions exist between other co-chaperones and Cdc37-Hsp90 complexes in the C. elegans system. We observed strong competitive interactions between Cdc37 and the co-chaperones p23 and Sti1, whereas the binding of the phosphatase Pph5 and the ATPase activator Aha1 to Cdc37-Hsp90 complexes is possible. The ternary Aha1-Cdc37-Hsp90 complex is disrupted by the nucleotide-induced closing reaction at the N terminus of Hsp90. This implies a carefully regulated exchange process of cofactors during the chaperoning of kinase clients by Hsp90. PMID:20880838

  18. HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death

    PubMed Central

    Jacobsen, A V; Lowes, K N; Tanzer, M C; Lucet, I S; Hildebrand, J M; Petrie, E J; van Delft, M F; Liu, Z; Conos, S A; Zhang, J-G; Huang, D C S; Silke, J; Lessene, G; Murphy, J M

    2016-01-01

    Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3. PMID:26775703

  19. HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death.

    PubMed

    Jacobsen, A V; Lowes, K N; Tanzer, M C; Lucet, I S; Hildebrand, J M; Petrie, E J; van Delft, M F; Liu, Z; Conos, S A; Zhang, J-G; Huang, D C S; Silke, J; Lessene, G; Murphy, J M

    2016-01-01

    Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3. PMID:26775703

  20. Design, synthesis, and anticancer activity of C8-substituted-4'-thionucleosides as potential HSP90 inhibitors.

    PubMed

    Qu, Shuhao; Mulamoottil, Varughese A; Nayak, Akshata; Ryu, Seungyeon; Hou, Xiyan; Song, Jayoung; Yu, Jinha; Sahu, Pramod K; Zhao, Long Xuan; Choi, Sun; Lee, Sang Kook; Jeong, Lak Shin

    2016-08-15

    A series of C8-substituted-4'-thioadenosine analogs 3a-3g, 15, and 17 and their truncated derivatives 4a-4j, 23-25, and 27 have been successfully synthesized from d-ribose and d-mannose, respectively, employing Pummerer type or Vorbrüggen condensation reactions and the functionalization at the C8-position of nucleobase via Stille coupling or nucleophilic aromatic substitution reactions as key steps. All the synthesized compounds were assayed for their HSP90 inhibitory activity, but they were found to be inactive up to 100μM. However, the 8-iodo derivatives 15, 17, and 27 exhibited potent anticancer activity, indicating that different mechanism of action might be involved in their biological activity. PMID:27283788

  1. The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90

    PubMed Central

    Haslbeck, Veronika; Eckl, Julia M.; Drazic, Adrian; Rutz, Daniel A.; Lorenz, Oliver R.; Zimmermann, Kerstin; Kriehuber, Thomas; Lindemann, Claudia; Madl, Tobias; Richter, Klaus

    2015-01-01

    Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex. PMID:26593036

  2. Cruentaren A Binds F1F0 ATP Synthase To Modulate the Hsp90 Protein Folding Machinery

    PubMed Central

    2015-01-01

    The molecular chaperone Hsp90 requires the assistance of immunophilins, co-chaperones, and partner proteins for the conformational maturation of client proteins. Hsp90 inhibition represents a promising anticancer strategy due to the dependence of numerous oncogenic signaling pathways upon Hsp90 function. Historically, small molecules have been designed to inhibit ATPase activity at the Hsp90 N-terminus; however, these molecules also induce the pro-survival heat shock response (HSR). Therefore, inhibitors that exhibit alternative mechanisms of action that do not elicit the HSR are actively sought. Small molecules that disrupt Hsp90-co-chaperone interactions can destabilize the Hsp90 complex without induction of the HSR, which leads to inhibition of cell proliferation. In this article, selective inhibition of F1F0 ATP synthase by cruentaren A was shown to disrupt the Hsp90-F1F0 ATP synthase interaction and result in client protein degradation without induction of the HSR. PMID:24450340

  3. The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation

    PubMed Central

    Lu, Xin-an; Wang, Xiaofeng; Zhuo, Wei; Jia, Lin; Jiang, Yushan; Fu, Yan; Luo, Yongzhang

    2013-01-01

    It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr115/Thr425/Thr603, of Hsp90α is specifically phosphorylated by PKCγ, and, more interestingly, this threonine residue set serves as a ‘phosphorylation switch’ for Hsp90α binding or release of PKCγ. Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells. PMID:24117238

  4. Distinct functions of the 90 kDa heat-shock protein (hsp90) in oestrogen and mineralocorticosteroid receptor activity: effects of hsp90 deletion mutants.

    PubMed

    Binart, N; Lombès, M; Baulieu, E E

    1995-11-01

    Recent studies have confirmed that the 90 kDa heat-shock protein (hsp90) interacts both in vitro and in vivo with steroid receptors, encouraging further detailed physicochemical and functional analysis of its chaperone role. Thus, to explore the relationship between hsp90 and receptors, the baculovirus system was used to overexpress the chick hsp90 alpha (chsp90) along with the chick oestradiol receptor (cER) or the human mineralocorticosteroid receptor (hMR). These receptors were able to form 9 S complexes with chsp90, demonstrating the association of the co-expressed recombinant proteins. Three mutants of chsp90 (delta A, delta B and delta Z) have been created by deletion of the A (residues 221-290) and B (530-581) regions, rich in charged amino acids, and the Z (392-419) region, a putative leucine zipper. After co-expression, anti-receptor antibodies immunoprecipitated the cER or hMR complexed with the wild-type chsp90, the delta B or the delta Z mutant, but not with the delta A chsp90, indicating that deletion of the A region of chsp90 leads to a lack of interaction with these receptors. The hormone binding capacity of the cER was unaffected after its co-expression with each of the three mutants. In contrast, the hMR co-expressed with the delta B mutant failed to bind aldosterone, a finding confirmed in vivo by the absence of hormone-induced hMR nuclear translocation. Thus the B region is required for high-affinity ligand binding by the hMR. Our results suggest that the A region (but not the B or Z regions) is involved in binding of chsp90 to the cER and hMR, while the B region is essential for hormone binding by the hMR, consistent with a chaperone function for hsp90. PMID:7487934

  5. Methylmercury Alters the Activities of Hsp90 Client Proteins, Prostaglandin E Synthase/p23 (PGES/23) and nNOS

    PubMed Central

    Caito, Samuel; Zeng, Heng; Aschner, Judy L.; Aschner, Michael

    2014-01-01

    Methylmercury (MeHg) is a persistent pollutant with known neurotoxic effects. We have previously shown that astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS) by altering glutamate signaling, generating oxidative stress, depleting glutathione (GSH) and initiating lipid peroxidation. Interestingly, all of these pathways can be regulated by the constitutively expressed, 90-kDa heat shock protein, Hsp90. As Hsp90 function is regulated by oxidative stress, we hypothesized that MeHg disrupts Hsp90-client protein functions. Astrocytes were treated with MeHg and expression of Hsp90, as well as the abundance of complexes of Hsp90-neuronal nitric oxide synthase (nNOS) and Hsp90-prostaglandin E synthase/p23 (PGES/p23) were assessed. MeHg exposure decreased Hsp90 protein expression following 12 h of treatment while shorter exposures had no effect on Hsp90 protein expression. Interestingly, following 1 or 6 h of MeHg exposure, Hsp90 binding to PGES/p23 or nNOS was significantly increased, resulting in increased prostaglandin E2 (PGE2) synthesis from MeHg-treated astrocytes. These effects were attenuated by the Hsp90 antagonist, geldanmycin. NOS activity was increased following MeHg treatment while cGMP formation was decreased. This was accompanied by an increase in •O2− and H2O2 levels, suggesting that MeHg uncouples NO formation from NO-dependent signaling and increases oxidative stress. Altogether, our data demonstrates that Hsp90 interactions with client proteins are increased following MeHg exposure, but over time Hsp90 levels decline, contributing to oxidative stress and MeHg-dependent excitotoxicity. PMID:24852575

  6. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity

    PubMed Central

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A.; Prakash, Thazha P.; Nichols, Joshua G.; Crooke, Stanley T.

    2016-01-01

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2′-modifications in 2–5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2′-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2′-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2′-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2′-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2′ modifications, e.g. (S)-cEt or LNA, in the 5′-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5′-LNA or 5′-cEt wings, but not with 5′-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs. PMID:26945041

  7. Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

    SciTech Connect

    Qin Zhiqiang; DeFee, Michael; Isaacs, Jennifer S.; Parsons, Chris

    2010-07-20

    The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90 and Hsp90-specific antibodies, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses.

  8. Deguelin Analogue SH-1242 Inhibits Hsp90 Activity and Exerts Potent Anticancer Efficacy with Limited Neurotoxicity.

    PubMed

    Lee, Su-Chan; Min, Hye-Young; Choi, Hoon; Bae, Song Yi; Park, Kwan Hee; Hyun, Seung Yeob; Lee, Ho Jin; Moon, Jayoung; Park, Shin-Hyung; Kim, Jun Yong; An, Hongchan; Park, So-Jung; Seo, Ji Hae; Lee, Seungbeom; Kim, Young-Myeong; Park, Hyun-Ju; Lee, Sang Kook; Lee, Jeewoo; Lee, Jeeyeon; Kim, Kyu-Won; Suh, Young-Ger; Lee, Ho-Young

    2016-02-01

    The Hsp90 facilitates proper folding of signaling proteins associated with cancer progression, gaining attention as a target for therapeutic intervention. The natural rotenoid deguelin was identified as an Hsp90 inhibitor, but concerns about neurotoxicity have limited prospects for clinical development. In this study, we report progress on deguelin analogues that address this limitation, focusing on the novel analogue SH-1242 as a candidate to broadly target human lung cancer cells, including those that are chemoresistant or harboring KRAS mutations. In a KRAS-driven mouse model of lung cancer, SH-1242 administration reduced tumor multiplicity, volume, and load. Similarly, in human cell line-based or patient-derived tumor xenograft models, SH-1242 induced apoptosis and reduced tumor vasculature in the absence of detectable toxicity. In contrast to deguelin, SH-1242 toxicity was greatly reduced in normal cells and when administered to rats did not produce obvious histopathologic features in the brain. Mechanistic studies revealed that SH-1242 bound to the C-terminal ATP-binding pocket of Hsp90, disrupting the ability to interact with its co-chaperones and clients and triggering a degradation of client proteins without affecting Hsp70 expression. Taken together, our findings illustrate the superior properties of SH-1242 as an Hsp90 inhibitor and as an effective antitumor and minimally toxic agent, providing a foundation for advancing further preclinical and clinical studies. PMID:26645561

  9. Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90

    PubMed Central

    Blackburn, Elizabeth A.; Wear, Martin A.; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L.; Walkinshaw, Malcolm D.

    2015-01-01

    Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal–EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the–MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when–MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. PMID:26330616

  10. Hsp90 and PKM2 Drive the Expression of Aromatase in Li-Fraumeni Syndrome Breast Adipose Stromal Cells.

    PubMed

    Subbaramaiah, Kotha; Brown, Kristy A; Zahid, Heba; Balmus, Gabriel; Weiss, Robert S; Herbert, Brittney-Shea; Dannenberg, Andrew J

    2016-07-29

    Li-Fraumeni syndrome (LFS) patients harbor germ line mutations in the TP53 gene and are at increased risk of hormone receptor-positive breast cancers. Recently, elevated levels of aromatase, the rate-limiting enzyme for estrogen biosynthesis, were found in the breast tissue of LFS patients. Although p53 down-regulates aromatase expression, the underlying mechanisms are incompletely understood. In the present study, we found that LFS stromal cells expressed higher levels of Hsp90 ATPase activity and aromatase compared with wild-type stromal cells. Inhibition of Hsp90 ATPase suppressed aromatase expression. Silencing Aha1 (activator of Hsp90 ATPase 1), a co-chaperone of Hsp90 required for its ATPase activity, led to both inhibition of Hsp90 ATPase activity and reduced aromatase expression. In comparison with wild-type stromal cells, increased levels of the Hsp90 client proteins, HIF-1α, and PKM2 were found in LFS stromal cells. A complex comprised of HIF-1α and PKM2 was recruited to the aromatase promoter II in LFS stromal cells. Silencing either HIF-1α or PKM2 suppressed aromatase expression in LFS stromal cells. CP-31398, a p53 rescue compound, suppressed levels of Aha1, Hsp90 ATPase activity, levels of PKM2 and HIF-1α, and aromatase expression in LFS stromal cells. Consistent with these in vitro findings, levels of Hsp90 ATPase activity, Aha1, HIF-1α, PKM2, and aromatase were increased in the mammary glands of p53 null versus wild-type mice. PKM2 and HIF-1α were shown to co-localize in the nucleus of stromal cells of LFS breast tissue. Taken together, our results show that the Aha1-Hsp90-PKM2/HIF-1α axis mediates the induction of aromatase in LFS. PMID:27467582

  11. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

    PubMed Central

    Woodford, Mark R.; Dunn, Diana M.; Blanden, Adam R.; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F.; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A.; Loh, Stewart N.; Bourboulia, Dimitra; Schmidt, Laura S.; Marston Linehan, W.; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  12. The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding.

    PubMed

    Woodford, Mark R; Dunn, Diana M; Blanden, Adam R; Capriotti, Dante; Loiselle, David; Prodromou, Chrisostomos; Panaretou, Barry; Hughes, Philip F; Smith, Aaron; Ackerman, Wendi; Haystead, Timothy A; Loh, Stewart N; Bourboulia, Dimitra; Schmidt, Laura S; Marston Linehan, W; Bratslavsky, Gennady; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors. PMID:27353360

  13. Synthesis and Evaluation of Novologues as C-Terminal Hsp90 Inhibitors with Cytoprotective Activity against Sensory Neuron Glucotoxicity

    PubMed Central

    Kusuma, Bhaskar Reddy; Zhang, Liang; Sundstrom, Teather; Peterson, Laura B.; Dobrowsky, Rick T.; Blagg, Brian S. J.

    2012-01-01

    Compound 2 (KU-32) is a first-generation novologue (a novobiocin-based, C-terminal, heat shock protein 90 (Hsp90) inhibitor), that decreases glucose-induced death of primary sensory neurons and reverses numerous clinical indices of diabetic peripheral neuropathy in mice. The current study sought to exploit the C-terminal binding site of Hsp90 to determine whether the optimization of hydrogen bonding and hydrophobic interactions of second generation novologues could enhance neuroprotective activity. Using a series of substituted phenylboronic acids to replace the coumarin lactone of 2, we identified electronegative atoms placed at the meta-position of the B-ring exhibit improved cytoprotective activity, which is believed to result from favorable interactions with Lys539 in the Hsp90 C-terminal binding pocket. Consistent with these results, a meta-3-fluorophenyl substituted novologue (13b) exhibited a 14-fold lower ED50 compared to 2 for protection against glucose-induced toxicity of primary sensory neurons. PMID:22702513

  14. BIIB021, a synthetic Hsp90 inhibitor, induces mutant ataxin-1 degradation through the activation of heat shock factor 1.

    PubMed

    Ding, Ying; Adachi, Hiroaki; Katsuno, Masahisa; Sahashi, Kentaro; Kondo, Naohide; Iida, Madoka; Tohnai, Genki; Nakatsuji, Hideaki; Sobue, Gen

    2016-07-01

    Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract in ataxin-1 (ATXN1). The pathological hallmarks of SCA1 are the loss of cerebellar Purkinje cells and neurons in the brainstem and the presence of nuclear aggregates containing the polyQ-expanded ATXN1 protein. Heat shock protein 90 (Hsp90) inhibitors have been shown to reduce polyQ-induced toxicity. This study was designed to examine the therapeutic effects of BIIB021, a purine-scaffold Hsp90 inhibitor, on the protein homeostasis of polyQ-expanded mutant ATXN1 in a cell culture model of SCA1. Our results demonstrated that BIIB021 activated heat shock factor 1 (HSF1) and suppressed the abnormal accumulation of ATXN1 and its toxicity. The pharmacological degradation of mutant ATXN1 via activated HSF1 was dependent on both the proteasome and autophagy systems. These findings indicate that HSF1 is a key molecule in the regulation of the protein homeostasis of the polyQ-expanded mutant ATXN1 and that Hsp90 has potential as a novel therapeutic target in patients with SCA1. PMID:27058144

  15. Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

    PubMed Central

    Qin, Zhiqiang; DeFee, Michael; Isaacs, Jennifer; Parsons, Chris

    2010-01-01

    The Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses. PMID:20451233

  16. Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

    PubMed Central

    Blacklock, Kristin; Verkhivker, Gennady M.

    2013-01-01

    Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity

  17. Multiple Conformations of E. Coli Hsp90 in Solution: Insights Into the Conformational Dynamics of Hsp90

    SciTech Connect

    Krukenberg, K.A.; Forster, F.; Rice, L.M.; Sali, A.; Agard, D.A.

    2009-05-20

    Hsp90, an essential eukaryotic chaperone, depends upon its intrinsic ATPase activity for function. Crystal structures of the bacterial Hsp90 homolog, HtpG, and the yeast Hsp90 reveal large domain rearrangements between the nucleotide-free and the nucleotide-bound forms. We used small-angle X-ray scattering and recently developed molecular modeling methods to characterize the solution structure of HtpG and demonstrate how it differs from known Hsp90 conformations. In addition to this HtpG conformation, we demonstrate that under physiologically relevant conditions, multiple conformations coexist in equilibrium. In solution, nucleotide-free HtpG adopts a more extended conformation than observed in the crystal, and upon the addition of AMPPNP, HtpG is in equilibrium between this open state and a closed state that is in good agreement with the yeast AMPPNP crystal structure. These studies provide a unique view of Hsp90 conformational dynamics and provide a model for the role of nucleotide in effecting conformational change.

  18. p23/Sba1p Protects against Hsp90 Inhibitors Independently of Its Intrinsic Chaperone Activity▿ ‡

    PubMed Central

    Forafonov, Fedor; Toogun, Oyetunji A.; Grad, Iwona; Suslova, Elena; Freeman, Brian C.; Picard, Didier

    2008-01-01

    The molecular chaperone Hsp90 assists a subset of cellular proteins and is essential in eukaryotes. A cohort of cochaperones contributes to and regulates the multicomponent Hsp90 machine. Unlike the biochemical activities of the cochaperone p23, its in vivo functions and the structure-function relationship remain poorly understood, even in the genetically tractable model organism Saccharomyces cerevisiae. The SBA1 gene that encodes the p23 ortholog in this species is not an essential gene. We found that in the absence of p23/Sba1p, yeast and mammalian cells are hypersensitive to Hsp90 inhibitors. This protective function of Sba1p depends on its abilities to bind Hsp90 and to block the Hsp90 ATPase and inhibitor binding. In contrast, the protective function of Sba1p does not require the Hsp90-independent molecular chaperone activity of Sba1p. The structure-function analysis suggests that Sba1p undergoes considerable structural rearrangements upon binding Hsp90 and that the large size of the p23/Sba1p-Hsp90 interaction surface facilitates maintenance of high affinity despite sequence divergence during evolution. The large interface may also contribute to preserving a protective function in an environment in which Hsp90 inhibitory compounds can be produced by various microorganisms. PMID:18362168

  19. The Hsp90 molecular chaperone complex regulates maltose induction and stability of the Saccharomyces MAL gene transcription activator Mal63p.

    PubMed

    Bali, Mehtap; Zhang, Bin; Morano, Kevin A; Michels, Corinne A

    2003-11-28

    Induction of the Saccharomyces MAL structural genes encoding maltose permease and maltase requires the MAL activator, a DNA-binding transcription activator. Genetic analysis of MAL activator mutations suggested that protein folding and stability play an important role in MAL activator regulation and led us to explore the role of the Hsp90 molecular chaperone complex in the regulation of the MAL activator. Strains carrying mutations in genes encoding components of the Hsp90 chaperone complex, hsc82 Delta hsp82-T101I and hsc82 Delta cpr7 Delta, are defective for maltase induction and exhibit significantly reduced growth rates on media containing a limiting concentration of maltose (0.05%). This growth defect is suppressed by providing maltose in excess. Using epitope-tagged alleles of the MAL63 MAL activator, we showed that Mal63p levels are drastically reduced following depletion of cellular Hsp90. Overexpression ( approximately 3-fold) of Mal63p in the hsc82 Delta hsp82-T101I and hsc82 Delta cpr7 Delta strains suppresses their Mal- growth phenotype, suggesting that Mal63p levels are limiting for maltose utilization in strains with abrogated Hsp90 activity. Consistent with this, the half-life of Mal63p is significantly shorter in the hsc82 Delta cpr7 Delta strain (reduced about 6-fold) and modestly affected in the Hsp90-ts strain (reduced about 2-fold). Most importantly, triple hemagglutinin-tagged Mal63p protein is found in association with Hsp90 as demonstrated by co-immunoprecipitation. Taken together, these results identify the inducible MAL activator as a client protein of the Hsp90 molecular chaperone complex and point to a critical role for chaperone function in alternate carbon source utilization in Saccharomyces cerevisiae. PMID:14500708

  20. Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β

    PubMed Central

    Peng, Yi-Jheng; Huang, Jing-Jia; Wu, Hao-Han; Hsieh, Hsin-Ying; Wu, Chia-Ying; Chen, Shu-Ching; Chen, Tsung-Yu; Tang, Chih-Yung

    2016-01-01

    Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation. PMID:27580824

  1. Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

    PubMed

    Peng, Yi-Jheng; Huang, Jing-Jia; Wu, Hao-Han; Hsieh, Hsin-Ying; Wu, Chia-Ying; Chen, Shu-Ching; Chen, Tsung-Yu; Tang, Chih-Yung

    2016-01-01

    Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation. PMID:27580824

  2. Identification of the Plant Compound Geraniin as a Novel Hsp90 Inhibitor

    PubMed Central

    Vassallo, Antonio; Vaccaro, Maria Carmela; De Tommasi, Nunziatina; Dal Piaz, Fabrizio; Leone, Antonella

    2013-01-01

    Besides its function in normal cellular growth, the molecular chaperone heat shock protein 90 (Hsp90) binds to a large number of client proteins required for promoting cancer cell growth and/or survival. In an effort to discover new small molecules able to inhibit the Hsp90 ATPase and chaperoning activities, we screened, by a surface plasmon resonance assay, a small library including different plant polyphenols. The ellagitannin geraniin, was identified as the most promising molecule, showing a binding affinity to Hsp90α similar to that of 17-(allylamino)-17-demethoxygeldanamycin (17AGG). Geraniin was able to inhibit in vitro the Hsp90α ATPase activity in a dose−dependent manner, with an inhibitory efficiency comparable to that measured for 17-AAG. In addition, this compound compromised the chaperone activity of Hsp90α, monitored by the citrate synthase thermal induced aggregation assay. Geraniin decreased the viability of HeLa and Jurkat cell lines and caused an arrest in G2/M phase. We also proved that following exposure to different concentrations of geraniin, the level of expression of the client proteins c-Raf, pAkt, and EGFR was strongly down−regulated in both the cell lines. These results, along with the finding that geraniin did not exert any appreciable cytotoxicity on normal cells, encourage further studies on this compound as a promising chemical scaffold for the design of new Hsp90 inhibitors. PMID:24066128

  3. Exploiting conformational dynamics in drug discovery: design of C-terminal inhibitors of Hsp90 with improved activities

    PubMed Central

    Moroni, Elisabetta; Zhao, Huiping; Blagg, Brian S.J.; Colombo, Giorgio

    2014-01-01

    The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated anti-proliferative activity at ~150 nanomolar concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site. PMID:24397468

  4. Intracellular dynamics of the Hsp90 co-chaperone p23 is dictated by Hsp90

    SciTech Connect

    Picard, Didier . E-mail: picard@cellbio.unige.ch

    2006-01-15

    p23 is a component of the Hsp90 molecular chaperone machine. It binds and stabilizes the ATP-bound dimeric form of Hsp90. Since Hsp90 binds protein substrates in the ATP conformation, p23 has been proposed to stabilize Hsp90-substrate complexes. In addition, p23 can also function as a molecular chaperone by itself and even possesses an unrelated enzymatic activity. Whether it fulfills the latter functions in cells while bound to Hsp90 remains unknown and is difficult to extrapolate from cell-free biochemical experiments. Using the 'fluorescence recovery after photobleaching' (FRAP) technology, I have examined the dynamics of human p23, expressed as a fusion protein with the green fluorescent protein (GFP), in living human HeLa cells. GFP-p23 is distributed throughout the cell, and its mobility is identical in the cytoplasm and in the nucleus. When the Hsp90 interaction is disrupted either with the Hsp90 inhibitor geldanamycin or by introduction of point mutations into p23, the mobility of p23 is greatly accelerated. Under these conditions, its intracellular movement may be diffusion-controlled. In contrast, when wild-type p23 is able to bind Hsp90, a more complex FRAP behavior is observed, suggesting that it is quantitatively bound in Hsp90 complexes undergoing a multitude of other interactions.

  5. Hsp90 as a Potential Therapeutic Target in Retinal Disease.

    PubMed

    Aguilà, Mònica; Cheetham, Michael E

    2016-01-01

    The molecular chaperone heat shock protein 90 (Hsp90) is a pivotal cellular regulator involved in the folding, activation and assembly of a wide range of proteins. Hsp90 has multiple roles in the retina and the use of different Hsp90 inhibitors has been shown to prevent retinal degeneration in models of retinitis pigmentosa and age-related macular degeneration. Hsp90 is also a potential target in uveal melanoma. Mechanistically, Hsp90 inhibition can evoke a dual response in the retina; stimulating a stress response with molecular chaperone expression. Thereby leading to an improvement in visual function and photoreceptor survival; however, prolonged inhibition can also stimulate the degradation of Hsp90 client proteins potentially deleteriously affect vision. Here, we review the multiple roles of Hsp90 in the retina and the therapeutic potential of Hsp90 as a target. PMID:26427407

  6. Heat shock protein 90 (HSP90) inhibitors activate the heat shock factor 1 (HSF1) stress response pathway and improve glucose regulation in diabetic mice.

    PubMed

    Lee, Jee-Hyung; Gao, Jiaping; Kosinski, Penelope A; Elliman, Stephen J; Hughes, Thomas E; Gromada, Jesper; Kemp, Daniel M

    2013-01-18

    The cytoprotective stress response factor HSF1 regulates the transcription of the chaperone HSP70, which exhibits anti-inflammatory effects and improves insulin sensitivity. We tested the therapeutic potential of this pathway in rodent models of diabetes using pharmacological tools. Activation of the HSF1 pathway was achieved using potent inhibitors of the upstream regulatory protein, HSP90. Treatment with AUY922, a selective HSP90 inhibitor led to robust inhibition of JNK1 phosphorylation, cytoprotection and improved insulin signaling in cells, consistent with effects observed with HSP70 treatment. Chronic dosing with HSP90 inhibitors reversed hyperglycemia in the diabetic db/db mouse model, and improved insulin sensitivity in the diet-induced obese mouse model of insulin resistance, further supporting the concept that the HSF1 pathway is a potentially viable anti-diabetes target. PMID:23261432

  7. 4-Hydroxybenzoic acid derivatives as HDAC6-specific inhibitors modulating microtubular structure and HSP90α chaperone activity against prostate cancer.

    PubMed

    Seidel, Carole; Schnekenburger, Michael; Mazumder, Aloran; Teiten, Marie-Hélène; Kirsch, Gilbert; Dicato, Mario; Diederich, Marc

    2016-01-01

    Histone deacetylase (HDAC)6 is a unique isoenzyme targeting specific substrates including α-tubulin and heat shock protein (HSP)90. HDAC6 is involved in protein trafficking and degradation, cell shape and migration. Deregulation of HDAC6 activity is associated with a variety of diseases including cancer leading to a growing interest for developing HDAC6 inhibitors. Here, we identified two new structurally related 4-hydroxybenzoic acids as selective HDAC6 inhibitors reducing proliferation, colony and spheroid formation as well as viability of prostate cancer cells. Both compounds strongly enhanced α-tubulin acetylation leading to remodeling of microtubular organization. Furthermore, 4-hydroxybenzoic acids decreased HSP90α regulation of the human androgen receptor in prostate cancer cells by increasing HSP90α acetylation levels. Collectively, our data support the potential of 4-hydroxybenzoic acid derivatives as HDAC6-specific inhibitors with anti-cancer properties. PMID:26549368

  8. Identification of Limonol Derivatives as Heat Shock Protein 90 (Hsp90) Inhibitors through a Multidisciplinary Approach.

    PubMed

    Chini, Maria G; Malafronte, Nicola; Vaccaro, Maria C; Gualtieri, Maria J; Vassallo, Antonio; Vasaturo, Michele; Castellano, Sabrina; Milite, Ciro; Leone, Antonietta; Bifulco, Giuseppe; De Tommasi, Nunziatina; Dal Piaz, Fabrizio

    2016-09-01

    The identification of inhibitors of Hsp90 is currently a primary goal in the development of more effective drugs for the treatment of various types of multidrug resistant malignancies. In an attempt to identify new small molecules modulating the activity of Hsp90, we screened a small library of tetranortriterpenes. A high-affinity interaction with Hsp90 inducible form was uncovered for eight of these compounds, five of which are described here for the first time. By monitoring the ATPase activity and the citrate synthase thermal induced aggregation, compound 1 (cedrelosin A), 3 (7α-limonylacetate), and 5 (cedrelosin B), containing a limonol moiety, were found to be the most effective in compromising the Hsp90α chaperone activity. Consistent with these findings, the three compounds caused a depletion of c-Raf and pAkt Hsp90 client proteins in HeLa and MCF/7 cell lines. Induced fit docking protocol and molecular dynamics were used to rationalize the structural basis of the biological activity of the limonol derivatives. Taken together, these results point to limonol-derivatives as promising scaffolds for the design of novel Hsp90α inhibitors. PMID:27492719

  9. Prediction of dual agents as an activator of mutant p53 and inhibitor of Hsp90 by docking, molecular dynamic simulation and virtual screening.

    PubMed

    Abbasi, Maryam; Sadeghi-Aliabadi, Hojjat; Hassanzadeh, Farshid; Amanlou, Massoud

    2015-09-01

    Heat shock protein90s (Hsp90s) play a crucial role in the development of cancer, and their inhibitors are a main target for tumor suppression. P53 also is a tumor suppressor, but in cancer cells, mutations in the p53 gene lead to the inactivation and accumulation of protein. For instance, the ninth p53 cancer mutation, Y220C, destabilizes the p53 core domain. Small molecules have been assumed to bind to Y220C DNA-binding domain and reactivate cellular mutant p53 functions. In this study, one of the mutant p53 activators is suggested as an Hsp90 inhibitor according to a pyrazole scaffold. To confirm a new ligand as a dual agent, molecular docking and molecular dynamic simulations were performed on both proteins (p53 and Hsp90). Molecular dynamic simulations were also conducted to evaluate the obtained results on the other two pyrazole structures, one known as Hsp90 inhibitor and the other as the reported mutant p53 activator. The findings indicate that the new ligand was stable in the active site of both proteins. Finally, a virtual screening was performed on ZINC database, and a set of new dual agents was proposed according to the new ligand scaffold. PMID:26277488

  10. Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition

    PubMed Central

    2010-01-01

    Background Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer. Methods Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression. Results The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer. Conclusion In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor. PMID

  11. Mutational analysis of hsp90 binding to the progesterone receptor.

    PubMed

    Sullivan, W P; Toft, D O

    1993-09-25

    The 90-kDa heat shock protein, hsp90, is known to associate with steroid receptors that are in the inactive state. While the biochemical function of hsp90 is unclear, this association is believed to be significant because dissociation of hsp90 occurs when receptors are activated by hormone. Complexes between hsp90 and the progesterone receptor can be formed in vitro in rabbit reticulocyte lysate. This has been shown to be an ATP-dependent process, and dissociation of the complex occurs when progesterone is added to the system. We now show that hsp90 synthesized by in vitro translation in reticulocyte lysate can form complexes with progesterone receptor that are sensitive to hormone. This system was used to analyze several mutant forms of hsp90. A series of NH2-terminal deletions showed that amino acids 1-380 can be removed from hsp90 without substantial loss of receptor binding activity. However, several deletions in the COOH-terminal half of hsp90 resulted in a partial or complete loss of this activity. Two regions, amino acids 381-441 and 601-677, appear to be particularly important for receptor binding. These studies describe a convenient and reliable method for the initial screening of hsp90 mutants, and they provide important clues to the identification of domains on hsp90 that interact with other proteins. PMID:8376394

  12. PRKD2: A two-pronged kinase crucial for the tumor-supporting activity of HSP90

    PubMed Central

    Azoitei, Ninel; Fröhling, Stefan; Scholl, Claudia; Seufferlein, Thomas

    2015-01-01

    PRKD2 plays an important role in tumor cell survival, proliferation, migration, and angiogenesis. We recently reported that cell death and impaired blood vessel formation evoked by inhibition of the HSP90 chaperone in human cancer cells of various tissue origins is mediated by destabilization of PRKD2. PMID:27308444

  13. Spatiotemporal Regulation of Hsp90–Ligand Complex Leads to Immune Activation

    PubMed Central

    Tamura, Yasuaki; Yoneda, Akihiro; Takei, Norio; Sawada, Kaori

    2016-01-01

    Although heat shock proteins (HSPs) primarily play a pivotal role in the maintenance of cellular homeostasis while reducing extracellular as well as intracellular stresses, their role in immunologically relevant scenarios, including activation of innate immunity as danger signals, antitumor immunity, and autoimmune diseases, is now gaining much attention. The most prominent feature of HSPs is that they function both in their own and as an HSP–ligand complex. We here show as a unique feature of extracellular HSPs that they target chaperoned molecules into a particular endosomal compartment of dendritic cells, thereby inducing innate and adaptive immune responses via spatiotemporal regulation. PMID:27252703

  14. Extracellular heat shock protein HSP90{beta} secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-{beta}1

    SciTech Connect

    Suzuki, Shigeki; Kulkarni, Ashok B.

    2010-07-30

    Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-{beta} signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understanding of the latent TGF-{beta} activation process. In this study, we have identified heat shock protein 90 {beta} (HSP90{beta}) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90{beta} into extracellular space which inhibits the activation of latent TGF-{beta}1, and that there is a subsequent decrease in cell proliferation. TGF-{beta}1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90{beta}. Thus, extracellular HSP90{beta} is a negative regulator for the activation of latent TGF-{beta}1 modulating TGF-{beta} signaling in the extracellular domain. -- Research highlights: {yields} Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex. {yields} This complex consists of latency-associated peptide (LAP) and the mature ligand. {yields} The release of the mature ligand from LAP is the first step in TGF-{beta} activation. {yields} We identified for the first time a novel mechanism for this activation process. {yields} Heat shock protein 90 {beta} is discovered as a negative regulator for this process.

  15. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis.

    PubMed

    Wang, Xiaodong; Venable, John; LaPointe, Paul; Hutt, Darren M; Koulov, Atanas V; Coppinger, Judith; Gurkan, Cemal; Kellner, Wendy; Matteson, Jeanne; Plutner, Helen; Riordan, John R; Kelly, Jeffery W; Yates, John R; Balch, William E

    2006-11-17

    The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, DeltaF508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of DeltaF508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the "chaperome") is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease. PMID:17110338

  16. A systematic protocol for the characterization of Hsp90 modulators.

    PubMed

    Matts, Robert L; Brandt, Gary E L; Lu, Yuanming; Dixit, Anshuman; Mollapour, Mehdi; Wang, Suiquan; Donnelly, Alison C; Neckers, Leonard; Verkhivker, Gennady; Blagg, Brian S J

    2011-01-01

    Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA. PMID:21129982

  17. N-Aryl-benzimidazolones as novel small molecule HSP90 inhibitors

    SciTech Connect

    Bruncko, Milan; Tahir, Stephen K.; Song, Xiaohong; Chen, Jun; Ding, Hong; Huth, Jeffrey R.; Jin, Sha; Judge, Russell A.; Madar, David J.; Park, Chang H.; Park, Cheol-Min; Petros, Andrew M.; Tse, Christin; Rosenberg, Saul H.; Elmore, Steven W.

    2012-03-16

    We describe the development of a novel series of N-aryl-benzimidazolone HSP90 inhibitors (9) targeting the N-terminal ATP-ase site. SAR development was influenced by structure-based design based around X-ray structures of ligand bound HSP90 complexes. Lead compounds exhibited high binding affinities, ATP-ase inhibition and cellular client protein degradation.

  18. Hsp90 inhibition protects against inherited retinal degeneration

    PubMed Central

    Aguilà, Mònica; Bevilacqua, Dalila; McCulley, Caroline; Schwarz, Nele; Athanasiou, Dimitra; Kanuga, Naheed; Novoselov, Sergey S.; Lange, Clemens A.K.; Ali, Robin R.; Bainbridge, James W.; Gias, Carlos; Coffey, Peter J.; Garriga, Pere; Cheetham, Michael E.

    2014-01-01

    The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1−/− cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function. PMID:24301679

  19. Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells.

    PubMed

    Thangjam, Gagan S; Birmpas, Charalampos; Barabutis, Nektarios; Gregory, Betsy W; Clemens, Mary Ann; Newton, Joseph R; Fulton, David; Catravas, John D

    2016-05-15

    The ability of anti-heat shock protein 90 (Hsp90) drugs to attenuate NF-κB-mediated transcription is the major basis for their anti-inflammatory properties. While the molecular mechanisms underlying this effect are not clear, they appear to be distinct in human endothelial cells. We now show for the first time that type 2 sirtuin (Sirt-2) histone deacetylase binds human NF-κB target gene promoter and prevents the recruitment of NF-κB proteins and subsequent assembly of RNA polymerase II complex in human lung microvascular endothelial cells. Hsp90 inhibitors stabilize the Sirt-2/promoter interaction and impose a "transcriptional block," which is reversed by either inhibition or downregulation of Sirt-2 protein expression. Furthermore, this process is independent of NF-κB (p65) Lysine 310 deacetylation, suggesting that it is distinct from known Sirt-2-dependent mechanisms. We demonstrate that Sirt-2 is recruited to NF-κB target gene promoter via interaction with core histones. Upon inflammatory challenge, chromatin remodeling and core histone H3 displacement from the promoter region removes Sirt-2 and allows NF-κB/coactivator recruitment essential for RNA Pol II-dependent mRNA induction. This novel mechanism may have important implications in pulmonary inflammation. PMID:27036868

  20. Synthesis and SAR study of 4-hydroxy-3-(2-hydroxynapthalene-1-yl)phenyl)-arylsulfonamides: Heat shock protein 90 (Hsp90) inhibitors with submicromolar activity in an in vitro assay

    PubMed Central

    Ganesh, Thota; Thepchatri, Pahk; Li, Lian; Du, Yuhong; Fu, Haian; Snyder, James P.; Sun, Aiming

    2008-01-01

    Heat shock protein 90 is emerging as an important target in cancer chemotherapy. In a program directed towards identifying novel chemical probes for Hsp90, we found 4-hydroxy-3-(2-hydroxynapthalene-1-yl)phenyl)benzene sulfonamide as an Hsp90 inhibitor with very weak activity. In this report we present a new and general method for the synthesis of variety of analogs around this scaffold and discuss their structure activity relationships. PMID:18762423

  1. Molecular analysis of Plasmodium falciparum co-chaperone Aha1 supports its interaction with and regulation of Hsp90 in the malaria parasite.

    PubMed

    Chua, Chun Song; Low, Huiyu; Lehming, Norbert; Sim, T S

    2012-01-01

    The recent recognition of Plasmodium falciparum Hsp90 (PfHsp90) as a promising anti-malaria drug target has sparked interest in identifying factors that regulate its function and drug-interaction. Co-chaperones are well-known regulators of Hsp90's chaperone function, and certain members have been implicated in conferring protection against lethal cellular effects of Hsp90-specific inhibitors. In this context, studies on PfHsp90's co-chaperones are imperative to gain insight into the regulation of the chaperone in the malaria parasite. In this study, a putative co-chaperone P. falciparum Aha1 (PfAha1) was identified and investigated for its interaction and regulation of PfHsp90. A previous genome-wide yeast two-hybrid study failed to identify PfAha1's association with PfHsp90, which prompted us to use a directed assay to investigate their interaction. PfAha1 was shown to interact with PfHsp90 via the in vivo split-ubiquitin assay and the association was confirmed in vitro by GST pull-down experiments. The GST pull-down assay further revealed PfAha1's interaction with PfHsp90 to be dependent on MgCl(2) and ATP, and was competed by co-chaperone Pfp23 that binds PfHsp90 under the same condition. In addition, the PfHsp90-PfAha1 complex was found to be sensitive to disruption by high salt, indicating a polar interaction between them. Using bio-computational modelling coupled with site-directed mutagenesis, the polar residue N108 in PfAha1 was found to be strategically located and essential for PfHsp90 interaction. The functional significance of PfAha1's interaction was clearly that of exerting a stimulatory effect on the ATPase activity of PfHsp90, likely to be essential for promoting the activation of PfHsp90's client proteins. PMID:22100910

  2. New, non-quinone fluorogeldanamycin derivatives strongly inhibit Hsp90.

    PubMed

    Hermane, Jekaterina; Bułyszko, Ilona; Eichner, Simone; Sasse, Florenz; Collisi, Wera; Poso, Antti; Schax, Emilia; Walter, Johanna-Gabriela; Scheper, Thomas; Kock, Klaus; Herrmann, Christian; Aliuos, Pooyan; Reuter, Günter; Zeilinger, Carsten; Kirschning, Andreas

    2015-01-19

    Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA-blocked mutant with all possible monofluoro 3-aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies. PMID:25572106

  3. Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities.

    PubMed

    Batista, Fernanda A H; Almeida, Glessler S; Seraphim, Thiago V; Silva, Kelly P; Murta, Silvane M F; Barbosa, Leandro R S; Borges, Júlio C

    2015-01-01

    The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90. PMID:25369258

  4. Quantitation of the interaction of the immunosuppressant deoxyspergualin and analogs with Hsc70 and Hsp90.

    PubMed

    Nadeau, K; Nadler, S G; Saulnier, M; Tepper, M A; Walsh, C T

    1994-03-01

    Deoxyspergualin (DSG), a spermidinyl, alpha-hydroxyglycyl, 7-guanidinoheptanoyl peptidomimetic, shows immunosuppressive activity. In confirmation of a recent report that immobilized methoxyDSG selectively retains the heat shock protein Hsc70, we report here quantitative binding of DSG and analogs to both Hsc70 and the 90-kDa heat shock protein Hsp90. We have utilized affinity capillary electrophoresis to obtain Kd values for DSG and analogs, and stimulation of the ATPase activity of Hsc70 to obtain Km values for DSG, that are comparable and corroborative. Kd values are 4 microM for DSG binding to Hsc70 and 5 microM for DSG binding to Hsp90. Two active analogs, methoxy- and glycylDSG, bind with similar affinities. Glyoxylylspermidine and des(aminopropyl)DSG, two inactive metabolites, have much reduced affinity for Hsc70 and Hsp90. These data validate binding of these novel immunosuppressant agents to these molecular chaperones, at concentrations in the range of pharmacologically active doses, and indicate that further characterization of Hsc70 and/or Hsp90 as potential targets for DSG is warranted. PMID:8117717

  5. Structure–Activity Relationship in a Purine-Scaffold Compound Series with Selectivity for the Endoplasmic Reticulum Hsp90 Paralog Grp94

    PubMed Central

    Patel, Hardik J.; Patel, Pallav D.; Ochiana, Stefan O.; Yan, Pengrong; Sun, Weilin; Patel, Maulik R.; Shah, Smit K.; Tramentozzi, Elisa; Brooks, James; Bolaender, Alexander; Shrestha, Liza; Stephani, Ralph; Finotti, Paola; Leifer, Cynthia; Li, Zihai; Gewirth, Daniel T.; Taldone, Tony; Chiosis, Gabriela

    2015-01-01

    Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 μM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/β and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor. PMID:25901531

  6. Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

    PubMed Central

    Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

    2016-01-01

    The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

  7. Tumor-Intrinsic and Tumor-Extrinsic Factors Impacting Hsp90-Targeted Therapy

    PubMed Central

    Alarcon, S. V.; Mollapour, M.; Lee, M.-J.; Tsutsumi, S.; Lee, S.; Kim, Y. S.; Prince, T.; Apolo, A.; Giaccone, G.; Xu, W.; Neckers, L. M.; Trepel, J. B.

    2012-01-01

    In 1994 the first heat shock protein 90 (Hsp90) inhibitor was identified and Hsp90 was reported to be a target for anticancer therapeutics. In the past 18 years there have been 17 distinct Hsp90 inhibitors entered into clinical trial, and the small molecule Hsp90 inhibitors have been highly valuable as probes of the role of Hsp90 and its client proteins in cancer. Although no Hsp90 inhibitor has achieved regulatory approval, recently there has been significant progress in Hsp90 inhibitor clinical development, and in the past year RECIST responses have been documented in HER2-positive breast cancer and EML4-ALK-positive non-small cell lung cancer. All of the clinical Hsp90 inhibitors studied to date are specific in their target, i.e. they bind exclusively to Hsp90 and two related heat shock proteins. However, Hsp90 inhibitors are markedly pleiotropic, causing degradation of over 200 client proteins and impacting critical multiprotein complexes. Furthermore, it has only recently been appreciated that Hsp90 inhibitors can, paradoxically, cause transient activation of the protein kinase clients they are chaperoning, resulting in initiation of signal transduction and significant physiological events in both tumor and tumor microenvironment. An additional area of recent progress in Hsp90 research is in studies of the posttranslational modifications of Hsp90 itself and Hsp90 co-chaperone proteins. Together, a picture is emerging in which the impact of Hsp90 inhibitors is shaped by the tumor intracellular and extracellular milieu, and in which Hsp90 inhibitors impact tumor and host on a microenvironmental and systems level. Here we review the tumor intrinsic and extrinsic factors that impact the efficacy of small molecules engaging the Hsp90 chaperone machine. PMID:22804236

  8. Thermodynamic Analysis of the Geldanamycin-Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach.

    PubMed

    Xu, Yingrong; Wallace, M Ariel Geer; Fitzgerald, Michael C

    2016-10-01

    Geldanamycin is a natural product with well-established and potent anti-cancer activities. Heat shock protein 90 (Hsp90) is the known target of geldanamycin, which directly binds to Hsp90's N-terminal ATP binding domain and inhibits Hsp90's ATPase activity. The affinity of geldanamycin for Hsp90 has been measured in multiple studies. However, there have been large discrepancies between the reported dissociation constants (i.e., Kd values), which have ranged from low nanomolar to micromolar. Here the stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to measure the binding affinity of geldanamycin to unpurified Hsp90 in an MCF-7 cell lysate. The Kd values determined here were dependent on how long geldanamycin was equilibrated with the lysate prior to SPROX analysis. The Kd values determined using equilibration times of 0.5 and 24 h were 1 and 0.03 μM, respectively. These Kd values, which are similar to those previously reported in a geldanamycin-Hsp90 binding study that involved the use of a fluorescently labeled geldanamycin analogue, establish that the slow-tight binding behavior previously observed for the fluorescently labeled geldanamycin analogue is not an artifact of the fluorescent label, but rather an inherent property of the geldanamycin-Hsp90 binding interaction. The slow-tight binding property of this complex may be related to time-dependent conformational changes in Hsp90 and/or to time-dependent chemical changes in geldanamycin, both of which have been previously proposed to explain the slow-tight binding behavior of the geldanamycin-Hsp90 complex. Graphical Abstract ᅟ. PMID:27530778

  9. Critical regulation of TGFbeta signaling by Hsp90.

    PubMed

    Wrighton, Katharine H; Lin, Xia; Feng, Xin-Hua

    2008-07-01

    Transforming growth factor beta (TGFbeta) controls a diverse set of cellular processes by activating TGFbeta type I (TbetaRI) and type II (TbetaRII) serine-threonine receptor kinases. Canonical TGFbeta signaling is mediated by Smad2 and Smad3, which are phosphorylated in their SXS motif by activated TbetaRI. The 90-kDa heat-shock protein (Hsp90) is a molecular chaperone facilitating the folding and stabilization of many protein kinases and intracellular signaling molecules. Here, we present evidence identifying a critical role for Hsp90 in TGFbeta signaling. Inhibition of Hsp90 function by using small-molecule inhibitors such as 17-allylamino-17-demethoxygeldanamycin (17AAG), and also at the genetic level, blocks TGFbeta-induced signaling and transcriptional responses. Furthermore, we identify TbetaRI and TbetaRII as Hsp90-interacting proteins in vitro and in vivo and demonstrate that inhibition of Hsp90 function increases TbetaR ubiquitination and degradation dependent on the Smurf2 ubiquitin E3 ligase. Our data reveal an essential level of TGFbeta signaling regulation mediated by Hsp90 by its ability to chaperone TbetaRs and also implicate the use of Hsp90 inhibitors in blocking undesired activation of TGFbeta signaling in diseases. PMID:18591668

  10. Alternative approaches to Hsp90 modulation for the treatment of cancer

    PubMed Central

    Hall, Jessica A; Forsberg, Leah K; Blagg, Brian SJ

    2015-01-01

    Hsp90 is responsible for the conformational maturation of newly synthesized polypeptides (client proteins) and the re-maturation of denatured proteins via the Hsp90 chaperone cycle. Inhibition of the Hsp90 N-terminus has emerged as a clinically relevant strategy for anticancer chemotherapeutics due to the involvement of clients in a variety of oncogenic pathways. Several immunophilins, co-chaperones and partner proteins are also necessary for Hsp90 chaperoning activity. Alternative strategies to inhibit Hsp90 function include disruption of the C-terminal dimerization domain and the Hsp90 heteroprotein complex. C-terminal inhibitors and Hsp90 co-chaperone disruptors prevent cancer cell proliferation similar to N-terminal inhibitors and destabilize client proteins without induction of heat shock proteins. Herein, current Hsp90 inhibitors, the chaperone cycle, and regulation of this cycle will be discussed. PMID:25367392

  11. Tethered Hsp90 Inhibitors Carrying Optical or Radioiodinated Probes Reveal Selective Internalization of Ectopic Hsp90 in Malignant Breast Tumor Cells

    PubMed Central

    Barrott, Jared J.; Hughes, Philip F.; Osada, Takuya; Yang, Xiao-Yi; Hartman, Zachary C.; Loiselle, David R.; Spector, Neil L.; Neckers, Len; Rajaram, Narasimhan; Hu, Fangyao; Ramanujam, Nimmi; Vaidyanathan, Ganesan; Zalutsky, Michael R.; Lyerly, H. Kim; Haystead, Timothy A.

    2013-01-01

    Summary Hsp90 inhibitors have demonstrated unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained but could be influenced by ectopically expressed Hsp90 in tumors. We have synthesized novel Hsp90 inhibitors that can carry optical or radioiodinated probes via a PEG tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein is occurring at the plasma membrane. In mice, we show exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors targeting ectopic Hsp90 can be used to detect breast cancer malignancies through non-invasive imaging. PMID:24035283

  12. A model for monitoring of Hsp90-buffered genetic variations

    NASA Astrophysics Data System (ADS)

    Kozeko, Liudmyla

    Genetic material of terrestrial organisms can be considerably injured by cosmic rays and UV-radiation in the space environment. Organisms onboard are also exposed to the entire complex of negative physical factors which can generate genetic variations and affect morphogenesis. However, species phenotypes must be robust to genetic variation, requiring "buffering" systems to ensure normal development. The molecular chaperone Hsp90 can serve as such "a buffer". It is important in the maturation and conformational regulation of a diverse set of signal transducers. The requirement of many principal regulatory proteins for Hsp90 renders entire metabolic pathways sensitive to impairment of its function. So inhibition of Hsp90 function can open cryptic genetic variations and produce morphological changes. In this paper, we present a model for monitoring of cryptic Hsp90-buffered genetic variations arising during exposure to space and spaceflight factors. This model has been developed with Arabidopsis thaliana seeds gathered in natural habitats with high anthropogenic pressure and wild type (Col-0) seeds subjected to negative influences (UV, heavy metals) experimentally. The phenotypic traits of early seedlings grown under reduction of Hsp90 activity were characterized to estimate Hsp90-buffered genetic variations. Geldanamycin was used as an inhibitor of Hsp90 function.

  13. Interactions of Hsp90 with histones and related peptides.

    PubMed

    Schnaider, T; Oikarinen, J; Ishiwatari-Hayasaka, H; Yahara, I; Csermely, P

    1999-01-01

    The 90 kDa heat shock protein (Hsp90) induces the condensation of the chromatin structure [Csermely, P., Kajtár, J., Hollósi, M., Oikarinen, J., and Somogyi, J. (1994) Biochem. Biophys. Res. Commun. 202, 1657-1663]. In our present studies we used surface plasmon resonance measurements to demonstrate that Hsp90 binds histones H1, H2A, H2B, H3 and H4 with high affinity having dissociation constants in the submicromolar range. Strong binding of the C-terminal peptide of histone H1 containing the SPKK-motif and a pentaeicosa-peptide including the Hsp90 bipartite nuclear localization signal sequence was also observed. However, a lysine/arginine-rich peptide of casein, and the lysine-rich platelet factor 4 did not display a significant interaction with Hsp90. Histones and positively charged peptides modulated the Hsp90-associated kinase activity. Interactions between Hsp90, histones, and high mobility group (HMG) protein-derived peptides raise the possibility of the involvement of Hsp90 in chromatin reorganization during steroid action, mitosis, or after cellular stress. PMID:10597896

  14. Artificial accelerators of the molecular chaperone Hsp90 facilitate rate-limiting conformational transitions.

    PubMed

    Zierer, Bettina K; Weiwad, Matthias; Rübbelke, Martin; Freiburger, Lee; Fischer, Gunter; Lorenz, Oliver R; Sattler, Michael; Richter, Klaus; Buchner, Johannes

    2014-11-01

    The molecular chaperone Hsp90 undergoes an ATP-driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well-established small-molecule inhibitors of Hsp90 compete with ATP-binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET-based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co-chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate-limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors. PMID:25244159

  15. Comparison of the activity of three different HSP70 inhibitors on apoptosis, cell cycle arrest, autophagy inhibition, and HSP90 inhibition

    PubMed Central

    Budina-Kolomets, Anna; Balaburski, Gregor M; Bondar, Anastasia; Beeharry, Neil; Yen, Tim; Murphy, Maureen E

    2014-01-01

    The chaperone HSP70 promotes the survival of cells exposed to many different types of stresses, and is also potently anti-apoptotic. The major stress-induced form of this protein, HSP70–1, is overexpressed in a number of human cancers, yet is negligibly expressed in normal cells. Silencing of the gene encoding HSP70–1 (HSPA1A) is cytotoxic to transformed but not normal cells. Therefore, HSP70 is considered to be a promising cancer drug target, and there has been active interest in the identification and characterization of HSP70 inhibitors for cancer therapy. Because HSP70 behaves in a relatively non-specific manner in the control of protein folding, to date there are no reliably-identified “clients” of this protein, nor is there consensus as to what the phenotypic effects of HSP70 inhibitors are on a cancer cell. Here for the first time we compare three recently-identified HSP70 inhibitors, PES-Cl, MKT-077, and Ver-155008, for their ability to impact some of the known and reported functions of this chaperone; specifically, the ability to inhibit autophagy, to influence the level of HSP90 client proteins, to induce cell cycle arrest, and to inhibit the enzymatic activity of the anaphase-promoting complex/cyclosome (APC/C). We report that all three of these compounds can inhibit autophagy and cause reduced levels of HSP90 client proteins; however, only PES-Cl can inhibit the APC/C and induce G2/M arrest. Possible reasons for these differences, and the implications for the further development of these prototype compounds as anti-cancer agents, are discussed. PMID:24100579

  16. Comparison of the activity of three different HSP70 inhibitors on apoptosis, cell cycle arrest, autophagy inhibition, and HSP90 inhibition.

    PubMed

    Budina-Kolomets, Anna; Balaburski, Gregor M; Bondar, Anastasia; Beeharry, Neil; Yen, Tim; Murphy, Maureen E

    2014-02-01

    The chaperone HSP70 promotes the survival of cells exposed to many different types of stresses, and is also potently anti-apoptotic. The major stress-induced form of this protein, HSP70-1, is overexpressed in a number of human cancers, yet is negligibly expressed in normal cells. Silencing of the gene encoding HSP70-1 (HSPA1A) is cytotoxic to transformed but not normal cells. Therefore, HSP70 is considered to be a promising cancer drug target, and there has been active interest in the identification and characterization of HSP70 inhibitors for cancer therapy. Because HSP70 behaves in a relatively non-specific manner in the control of protein folding, to date there are no reliably-identified "clients" of this protein, nor is there consensus as to what the phenotypic effects of HSP70 inhibitors are on a cancer cell. Here for the first time we compare three recently-identified HSP70 inhibitors, PES-Cl, MKT-077, and Ver-155008, for their ability to impact some of the known and reported functions of this chaperone; specifically, the ability to inhibit autophagy, to influence the level of HSP90 client proteins, to induce cell cycle arrest, and to inhibit the enzymatic activity of the anaphase-promoting complex/cyclosome (APC/C). We report that all three of these compounds can inhibit autophagy and cause reduced levels of HSP90 client proteins; however, only PES-Cl can inhibit the APC/C and induce G 2/M arrest. Possible reasons for these differences, and the implications for the further development of these prototype compounds as anti-cancer agents, are discussed. PMID:24100579

  17. Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.

    PubMed

    Olesen, Sanne H; Ingles, Donna J; Zhu, Jin-Yi; Martin, Mathew P; Betzi, Stephane; Georg, Gunda I; Tash, Joseph S; Schönbrunn, Ernst

    2015-01-01

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. PMID:25608045

  18. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    PubMed Central

    Olesen, Sanne H.; Ingles, Donna J.; Zhu, Jin-Yi; Martin, Mathew P.; Betzi, Stephane; Georg, Gunda I.; Tash, Joseph S.; Schönbrunn, Ernst

    2015-01-01

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in-vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. PMID:25608045

  19. Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis.

    PubMed

    Batista, Fernanda A H; Seraphim, Thiago V; Santos, Clelton A; Gonzaga, Marisvanda R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2016-06-15

    Parasites belonging to the genus Leishmania are subjected to extensive environmental changes during their life cycle; molecular chaperones/co-chaperones act as protagonists in this scenario to maintain cellular homeostasis. Hop/Sti1 is a co-chaperone that connects the Hsp90 and Hsp70 systems, modulating their ATPase activities and affecting the fate of client proteins because it facilitates their transfer from the Hsp70 to the Hsp90 chaperone. Hop/Sti1 is one of the most prevalent co-chaperones, highlighting its importance despite the relatively low sequence identity among orthologue proteins. This multi-domain protein comprises three tetratricopeptides domains (TPR1, TPR2A and TPR2B) and two Asp/Pro-rich domains. Given the importance of Hop/Sti1 for the chaperone system and for Leishmania protozoa viability, the Leishmania braziliensis Hop (LbHop) and a truncated mutant (LbHop(TPR2AB)) were characterized. Structurally, both proteins are α-helix-rich and highly elongated monomeric proteins. Functionally, they inhibited the ATPase activity of Leishmania braziliensis Hsp90 (LbHsp90) to a similar extent, and the thermodynamic parameters of their interactions with LbHsp90 were similar, indicating that TPR2A-TPR2B forms the functional center for the LbHop interaction with LbHsp90. These results highlight the structural and functional similarity of Hop/Sti1 proteins, despite their low sequence conservation compared to the Hsp70 and Hsp90 systems, which are phylogenetic highly conserved. PMID:27103305

  20. Molecular docking study, synthesis and biological evaluation of Mannich bases as Hsp90 inhibitors.

    PubMed

    Gupta, Sayan Dutta; Bommaka, Manish Kumar; Mazaira, Gisela I; Galigniana, Mario D; Subrahmanyam, Chavali Venkata Satya; Gowrishankar, Naryanasamy Lachmana; Raghavendra, Nulgumnalli Manjunathaiah

    2015-09-01

    The ubiquitously expressed heat shock protein 90 is an encouraging target for the development of novel anticancer agents. In a program directed towards uncovering novel chemical scaffolds against Hsp90, we performed molecular docking studies using Tripos-Sybyl drug designing software by including the required conserved water molecules. The results of the docking studies predicted Mannich bases derived from 2,4-dihydroxy acetophenone/5-chloro 2,4-dihydroxy acetophenone as potential Hsp90 inhibitors. Subsequently, a few of them were synthesized (1-6) and characterized by IR, (1)H NMR, (13)C NMR and mass spectral analysis. The synthesized Mannich compounds were evaluated for their potential to suppress Hsp90 ATPase activity by the colorimetric Malachite green assay. Subsequently, the molecules were screened for their antiproilferative effect against PC3 pancreatic carcinoma cells by adopting the 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The activity profile of the identified derivatives correlated well with their docking results. PMID:26116388

  1. Hsp-90 and the biology of nematodes

    PubMed Central

    Him, Nik AIIN; Gillan, Victoria; Emes, Richard D; Maitland, Kirsty; Devaney, Eileen

    2009-01-01

    Background Hsp-90 from the free-living nematode Caenorhabditis elegans is unique in that it fails to bind to the specific Hsp-90 inhibitor, geldanamycin (GA). Here we surveyed 24 different free-living or parasitic nematodes with the aim of determining whether C. elegans Hsp-90 was the exception or the norm amongst the nematodes. We combined these data with codon evolution models in an attempt to identify whether hsp-90 from GA-binding and non-binding species has evolved under different evolutionary constraints. Results We show that GA-binding is associated with life history: free-living nematodes and those parasitic species with free-living larval stages failed to bind GA. In contrast, obligate parasites and those worms in which the free-living stage in the environment is enclosed within a resistant egg, possess a GA-binding Hsp-90. We analysed Hsp-90 sequences from fifteen nematode species to determine whether nematode hsp-90s have undergone adaptive evolution that influences GA-binding. Our data provide evidence of rapid diversifying selection in the evolution of the hsp-90 gene along three separate lineages, and identified a number of residues showing significant evidence of adaptive evolution. However, we were unable to prove that the selection observed is correlated with the ability to bind geldanamycin or not. Conclusion Hsp-90 is a multi-functional protein and the rapid evolution of the hsp-90 gene presumably correlates with other key cellular functions. Factors other than primary amino acid sequence may influence the ability of Hsp-90 to bind to geldanamycin. PMID:19849843

  2. Hsp90, an unlikely ally in the war on cancer

    PubMed Central

    Barrott, Jared J.; Haystead, Timothy A. J.

    2013-01-01

    On the surface heat shock protein 90 (Hsp90) is an unlikely drug target for the treatment of any disease, let alone cancer. Hsp90 is highly conserved and ubiquitously expressed in all cells. There are two major isoforms α and β encoded by distinct genes and together they may constitute 1%–3% of the cellular protein. Deletion of the protein is embryonic lethal and there are no recognized polymorphisms suggesting an association or causal relationship with any human disease. With respect to cancer, the proteins absence from two recent high profile articles, ‘Hallmarks of cancer: the next generation’ [Hanahan & Weinberg (2011) Cell 144, 646–674] and ‘Comprehensive molecular portraits of human breast tumours’ [Koboldt et al. (2012) Nature] underlines the perception that it is an unlikely bona fide target to treat this disease. Yet, to date, there are 17 distinct Hsp90 inhibitors in clinical trials for multiple indications in cancer. The protein has been championed for over 20 years by the National Cancer Institute (Bethesda, MD, USA) as a cancer target since the discovery of the antitumor activity of the natural product geldanamycin. This review aims to look at the conundrum of why Hsp90 can even be considered a druggable target for the treatment of cancer. We propose that in contrast to the majority of chemotherapeutics our growing armamentarium of investigational Hsp90 drugs represents an elegant choice that offers real hope in the long-term treatment of certain cancers. PMID:23356585

  3. Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants

    PubMed Central

    Lee, Sunmin; Tsutsumi, Shinji; Yim, Kendrick; Rivas, Candy; Alarcon, Sylvia; Schwartz, Harvey; Khamit-Kush, Kofi; Scroggins, Bradley T.; Beebe, Kristin; Trepel, Jane B.; Neckers, Len

    2015-01-01

    The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states. PMID:26517842

  4. Hsp90 is involved in apoptosis of Candida albicans by regulating the calcineurin-caspase apoptotic pathway.

    PubMed

    Dai, BaoDi; Wang, Yan; Li, DeDong; Xu, Yi; Liang, RongMei; Zhao, LanXue; Cao, YongBing; Jia, JianHui; Jiang, YuanYing

    2012-01-01

    Candida albicans is the most common human fungal pathogen. Recent evidence has revealed the occurrence of apoptosis in C. albicans that is inducible by environmental stresses such as hydrogen peroxide, acetic acid, and amphotericin B. Apoptosis is regulated by the calcineurin-caspase pathway in C. albicans, and calcineurin is under the control of Hsp90 in echinocandin resistance. However, the role of Hsp90 in apoptosis of C. albicans remains unclear. In this study, we investigated the role of Hsp90 in apoptosis of C. albicans by using an Hsp90-compromised strain tetO-HSP90/hsp90 and found that upon apoptotic stimuli, including hydrogen peroxide, acetic acid or amphotericin B treatment, less apoptosis occurred, less ROS was produced, and more cells survived in the Hsp90-compromised strain compared with the Hsp90/Hsp90 wild-type strain. In addition, Hsp90-compromised cells were defective in up-regulating caspase-encoding gene CaMCA1 expression and activating caspase activity upon the apoptotic stimuli. Investigations on the relationship between Hsp90 and calcineurin revealed that activation of calcineurin could up-regulate apoptosis but could not further down-regulate apoptosis in Hsp90-compromised cells, indicating that calcineurin was downstream of Hsp90. Hsp90 inhibitor geldanamycin (GdA) could further decrease the apoptosis in calcineurin-pathway-defect strains, indicating that compromising Hsp90 function had a stronger effect than compromising calcineurin function on apoptosis. Collectively, this study demonstrated that compromised Hsp90 reduced apoptosis in C. albicans, partially through downregulating the calcineurin-caspase pathway. PMID:23028789

  5. Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.

    PubMed

    Verba, Kliment A; Wang, Ray Yu-Ruei; Arakawa, Akihiko; Liu, Yanxin; Shirouzu, Mikako; Yokoyama, Shigeyuki; Agard, David A

    2016-06-24

    The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions. PMID:27339980

  6. Cryptic variation in morphological evolution: HSP90 as a capacitor for loss of eyes in cavefish.

    PubMed

    Rohner, Nicolas; Jarosz, Dan F; Kowalko, Johanna E; Yoshizawa, Masato; Jeffery, William R; Borowsky, Richard L; Lindquist, Susan; Tabin, Clifford J

    2013-12-13

    In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting. PMID:24337296

  7. The co-chaperone Cdc37 regulates the rabies virus phosphoprotein stability by targeting to Hsp90AA1 machinery

    PubMed Central

    Xu, Yunbin; Liu, Fei; Liu, Juan; Wang, Dandan; Yan, Yan; Ji, Senlin; Zan, Jie; Zhou, Jiyong

    2016-01-01

    Cdc37, as a kinase-specific co-chaperone of the chaperone Hsp90AA1 (Hsp90), actively aids with the maturation, stabilization and activation of the cellular or viral kinase/kinase-like targets. Phosphoprotein (P) of rabies virus (RABV) is a multifunctional, non-kinase protein involved in interferon antagonism, viral transcription and replication. Here, we demonstrated that the RABV non-kinase P is chaperoned by Cdc37 and Hsp90 during infection. We found that Cdc37 and Hsp90 affect the RABV life cycle directly. Activity inhibition and knockdown of Cdc37 and Hsp90 increased the instability of the viral P protein. Overexpression of Cdc37 and Hsp90 maintained P’s stability but did not increase the yield of infectious RABV virions. We further demonstrated that the non-enzymatic polymerase cofactor P protein of all the genotypes of lyssaviruses is a target of the Cdc37/Hsp90 complex. Cdc37, phosphorylated or unphosphorylated on Ser13, aids the P protein to load onto the Hsp90 machinery, with or without Cdc37 binding to Hsp90. However, the interaction between Cdc37 and Hsp90 appears to have additional allosteric regulation of the conformational switch of Hsp90. Our study highlighted a novel mechanism in which Cdc37/Hsp90 chaperones a non-kinase target, which has significant implications for designing therapeutic targets against Rabies. PMID:27251758

  8. The co-chaperone Cdc37 regulates the rabies virus phosphoprotein stability by targeting to Hsp90AA1 machinery.

    PubMed

    Xu, Yunbin; Liu, Fei; Liu, Juan; Wang, Dandan; Yan, Yan; Ji, Senlin; Zan, Jie; Zhou, Jiyong

    2016-01-01

    Cdc37, as a kinase-specific co-chaperone of the chaperone Hsp90AA1 (Hsp90), actively aids with the maturation, stabilization and activation of the cellular or viral kinase/kinase-like targets. Phosphoprotein (P) of rabies virus (RABV) is a multifunctional, non-kinase protein involved in interferon antagonism, viral transcription and replication. Here, we demonstrated that the RABV non-kinase P is chaperoned by Cdc37 and Hsp90 during infection. We found that Cdc37 and Hsp90 affect the RABV life cycle directly. Activity inhibition and knockdown of Cdc37 and Hsp90 increased the instability of the viral P protein. Overexpression of Cdc37 and Hsp90 maintained P's stability but did not increase the yield of infectious RABV virions. We further demonstrated that the non-enzymatic polymerase cofactor P protein of all the genotypes of lyssaviruses is a target of the Cdc37/Hsp90 complex. Cdc37, phosphorylated or unphosphorylated on Ser13, aids the P protein to load onto the Hsp90 machinery, with or without Cdc37 binding to Hsp90. However, the interaction between Cdc37 and Hsp90 appears to have additional allosteric regulation of the conformational switch of Hsp90. Our study highlighted a novel mechanism in which Cdc37/Hsp90 chaperones a non-kinase target, which has significant implications for designing therapeutic targets against Rabies. PMID:27251758

  9. 2.4 Å resolution crystal structure of human TRAP1NM, the Hsp90 paralog in the mitochondrial matrix.

    PubMed

    Sung, Nuri; Lee, Jungsoon; Kim, Ji Hyun; Chang, Changsoo; Tsai, Francis T F; Lee, Sukyeong

    2016-08-01

    TRAP1 is an organelle-specific Hsp90 paralog that is essential for neoplastic growth. As a member of the Hsp90 family, TRAP1 is presumed to be a general chaperone facilitating the late-stage folding of Hsp90 client proteins in the mitochondrial matrix. Interestingly, TRAP1 cannot replace cytosolic Hsp90 in protein folding, and none of the known Hsp90 co-chaperones are found in mitochondria. Thus, the three-dimensional structure of TRAP1 must feature regulatory elements that are essential to the ATPase activity and chaperone function of TRAP1. Here, the crystal structure of a human TRAP1NM dimer is presented, featuring an intact N-domain and M-domain structure, bound to adenosine 5'-β,γ-imidotriphosphate (ADPNP). The crystal structure together with epitope-mapping results shows that the TRAP1 M-domain loop 1 contacts the neighboring subunit and forms a previously unobserved third dimer interface that mediates the specific interaction with mitochondrial Hsp70. PMID:27487821

  10. Hsp90 Inhibitors and Drug Resistance in Cancer: The Potential Benefits of Combination Therapies of Hsp90 Inhibitors and Other Anti-Cancer Drugs

    PubMed Central

    Lu, Xiangyi; Xiao, Li; Wang, Luan; Ruden, Douglas M.

    2012-01-01

    Hsp90 is a chaperone protein that interacts with client proteins that are known to be in the cell cycle, signaling and chromatin-remodeling pathways. Hsp90 inhibitors act additively or synergistically with many other drugs in the treatment of both solid tumors and leukemias in murine tumor models and humans. Hsp90 inhibitors potentiate the actions of anti-cancer drugs that target Hsp90 client proteins, including trastuzumab (Herceptin™) which targets Her2/Erb2B, as Hsp90 inhibition elicits the drug effects in cancer cell lines that are otherwise resistant to the drug. A Phase II study of the Hsp90 inhibitor 17-AAG and trastuzumab showed that this combination therapy has anticancer activity in patients with HER2-positive metastatic breast cancer progressing on trastuzumab. In this review, we discuss the results of Hsp90 inhibitors in combination with trastuzumab and other cancer drugs. We also discuss recent results from yeast focused on the genetics of drug resistance when Hsp90 is inhibited and the implications that this might have in understanding the effects of genetic variation in treating cancer in humans. PMID:22120678

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

    PubMed Central

    Trcka, Filip; Durech, Michal; Man, Petr; Hernychova, Lenka; Muller, Petr; Vojtesek, Borivoj

    2014-01-01

    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. PMID:24567332

  12. Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

    PubMed Central

    Zhao, X M; Chen, Z; Zhao, J B; Zhang, P P; Pu, Y F; Jiang, S H; Hou, J J; Cui, Y M; Jia, X L; Zhang, S Q

    2016-01-01

    The pseudokinase mixed lineage kinase domain-like protein (MLKL) is a key component of tumor necrosis factor (TNF)-induced necroptosis and plays a crucial role in necroptosis execution. However, the mechanisms that control MLKL activity are not completely understood. Here, we identify the molecular chaperone Hsp90 as a novel MLKL-interacting protein. We show that Hsp90 associates with MLKL and is required for MLKL stability. Moreover, we find that Hsp90 also regulates the stability of the upstream RIP3 kinase. Interference with Hsp90 function with the 17AAG inhibitor destabilizes MLKL and RIP3, resulting in their degradation by the proteasome pathway. Furthermore, we find that Hsp90 is required for TNF-stimulated necrosome assembly. Disruption of Hsp90 function prevents necrosome formation and strongly reduces MLKL phosphorylation and inhibits TNF-induced necroptosis. Consistent with a positive role of Hsp90 in necroptosis, coexpression of Hsp90 increases MLKL oligomerization and plasma membrane translocation and enhances MLKL-mediated necroptosis. Our findings demonstrate that an efficient necrotic response requires a functional Hsp90. PMID:26866270

  13. Experimentally guided structural modeling and dynamics analysis of Hsp90-p53 interactions: allosteric regulation of the Hsp90 chaperone by a client protein.

    PubMed

    Blacklock, Kristin; Verkhivker, Gennady M

    2013-11-25

    A fundamental role of the Hsp90 chaperone system in mediating maturation of protein clients is essential for the integrity of signaling pathways involved in cell cycle control and organism development. Molecular characterization of Hsp90 interactions with client proteins is fundamental to understanding the activity of many tumor-inducing signaling proteins and presents an active area of structural and biochemical studies. In this work, we have probed mechanistic aspects of allosteric regulation of Hsp90 by client proteins via a detailed computational study of Hsp90 interactions with the tumor suppressor protein p53. Experimentally guided protein docking and molecular dynamics structural refinement have reconstructed the recognition-competent states of the Hsp90-p53 complexes that are consistent with the NMR studies. Protein structure network analysis has identified critical interacting networks and specific residues responsible for structural integrity and stability of the Hsp90-p53 complexes. Coarse-grained modeling was used to characterize the global dynamics of the regulatory complexes and map p53-induced changes in the conformational equilibrium of Hsp90. The variations in the functional dynamics profiles of the Hsp90-p53 complexes are consistent with the NMR studies and could explain differences in the functional role of the alternative binding sites. Despite the overall similarity of the collective movements and the same global interaction footprint, p53 binding at the C-terminal interaction site of Hsp90 may have a more significant impact on the chaperone dynamics, which is consistent with the stronger allosteric effect of these interactions revealed by the experimental studies. The results suggest that p53-induced modulation of the global dynamics and structurally stable interaction networks can target the regulatory hinge regions and facilitate stabilization of the closed Hsp90 dimer that underlies the fundamental stimulatory effect of the p53 client. PMID

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

    PubMed Central

    Bohen, Sean P.

    1998-01-01

    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. PMID:9584173

  15. Plant Hsp90 Proteins Interact with B-Cells and Stimulate Their Proliferation

    PubMed Central

    Corigliano, Mariana G.; Maglioco, Andrea; Laguía Becher, Melina; Goldman, Alejandra; Martín, Valentina; Angel, Sergio O.; Clemente, Marina

    2011-01-01

    Background The molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro. Methodology Plant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction. Conclusions Our results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of

  16. Tumor-secreted Hsp90 Subverts Polycomb Function to Drive Prostate Tumor Growth and Invasion*

    PubMed Central

    Nolan, Krystal D.; Franco, Omar E.; Hance, Michael W.; Hayward, Simon W.; Isaacs, Jennifer S.

    2015-01-01

    Prostate cancer remains the second highest contributor to male cancer-related lethality. The transition of a subset of tumors from indolent to invasive disease is associated with a poor clinical outcome. Activation of the epithelial to mesenchymal transition (EMT) genetic program is a major risk factor for cancer progression. We recently reported that secreted extracellular Hsp90 (eHsp90) initiates EMT in prostate cancer cells, coincident with its enhanced expression in mesenchymal models. Our current work substantially extended these findings in defining a pathway linking eHsp90 signaling to EZH2 function, a methyltransferase of the Polycomb repressor complex. EZH2 is also implicated in EMT activation, and its up-regulation represents one of the most frequent epigenetic alterations during prostate cancer progression. We have now highlighted a novel epigenetic function for eHsp90 via its modulation of EZH2 expression and activity. Mechanistically, eHsp90 initiated sustained activation of MEK/ERK, a signal critical for facilitating EZH2 transcriptional up-regulation and recruitment to the E-cadherin promoter. We further demonstrated that an eHsp90-EZH2 pathway orchestrates an expanded repertoire of EMT-related events including Snail and Twist expression, tumor cell motility, and anoikis resistance. To evaluate the role of eHsp90 in vivo, eHsp90 secretion was stably enforced in a prostate cancer cell line resembling indolent disease. Remarkably, eHsp90 was sufficient to induce tumor growth, suppress E-cadherin, and initiate localized invasion, events that are exquisitely dependent upon EZH2 function. In summary, our findings illuminate a hitherto unknown epigenetic function for eHsp90 and support a model wherein tumor eHsp90 functions as a rheostat for EZH2 expression and activity to orchestrate mesenchymal properties and coincident aggressive behavior. PMID:25670862

  17. Mapping the Hsp90 Genetic Network Reveals Ergosterol Biosynthesis and Phosphatidylinositol-4-Kinase Signaling as Core Circuitry Governing Cellular Stress

    PubMed Central

    O’Meara, Teresa R.; Valaei, Seyedeh Fereshteh; Diezmann, Stephanie; Cowen, Leah E.

    2016-01-01

    Candida albicans is a leading human fungal pathogen that causes life-threatening systemic infections. A key regulator of C. albicans stress response, drug resistance, morphogenesis, and virulence is the molecular chaperone Hsp90. Targeting Hsp90 provides a powerful strategy to treat fungal infections, however, the therapeutic utility of current inhibitors is compromised by toxicity due to inhibition of host Hsp90. To identify components of the Hsp90-dependent circuitry governing virulence and drug resistance that are sufficiently divergent for selective targeting in the pathogen, we pioneered chemical genomic profiling of the Hsp90 genetic network in C. albicans. Here, we screen mutant collections covering ~10% of the genome for hypersensitivity to Hsp90 inhibition in multiple environmental conditions. We identify 158 HSP90 chemical genetic interactors, most of which are important for growth only in specific environments. We discovered that the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 are HSP90 genetic interactors under multiple conditions, suggesting a function upstream of Hsp90. By systematic analysis of the ergosterol biosynthetic cascade, we demonstrate that defects in ergosterol biosynthesis induce cellular stress that overwhelms Hsp90’s functional capacity. By analysis of the phosphatidylinositol pathway, we demonstrate that there is a genetic interaction between the PI4K Stt4 and Hsp90. We also establish that Stt4 is required for normal actin polarization through regulation of Wal1, and suggest a model in which defects in actin remodeling induces stress that creates a cellular demand for Hsp90 that exceeds its functional capacity. Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors. We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis. This work uncovers novel circuitry regulating Hsp90

  18. Mapping the Hsp90 Genetic Network Reveals Ergosterol Biosynthesis and Phosphatidylinositol-4-Kinase Signaling as Core Circuitry Governing Cellular Stress.

    PubMed

    O'Meara, Teresa R; Veri, Amanda O; Polvi, Elizabeth J; Li, Xinliu; Valaei, Seyedeh Fereshteh; Diezmann, Stephanie; Cowen, Leah E

    2016-06-01

    Candida albicans is a leading human fungal pathogen that causes life-threatening systemic infections. A key regulator of C. albicans stress response, drug resistance, morphogenesis, and virulence is the molecular chaperone Hsp90. Targeting Hsp90 provides a powerful strategy to treat fungal infections, however, the therapeutic utility of current inhibitors is compromised by toxicity due to inhibition of host Hsp90. To identify components of the Hsp90-dependent circuitry governing virulence and drug resistance that are sufficiently divergent for selective targeting in the pathogen, we pioneered chemical genomic profiling of the Hsp90 genetic network in C. albicans. Here, we screen mutant collections covering ~10% of the genome for hypersensitivity to Hsp90 inhibition in multiple environmental conditions. We identify 158 HSP90 chemical genetic interactors, most of which are important for growth only in specific environments. We discovered that the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 are HSP90 genetic interactors under multiple conditions, suggesting a function upstream of Hsp90. By systematic analysis of the ergosterol biosynthetic cascade, we demonstrate that defects in ergosterol biosynthesis induce cellular stress that overwhelms Hsp90's functional capacity. By analysis of the phosphatidylinositol pathway, we demonstrate that there is a genetic interaction between the PI4K Stt4 and Hsp90. We also establish that Stt4 is required for normal actin polarization through regulation of Wal1, and suggest a model in which defects in actin remodeling induces stress that creates a cellular demand for Hsp90 that exceeds its functional capacity. Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors. We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis. This work uncovers novel circuitry regulating Hsp90

  19. Co- and Post-translocation Roles for HSP90 in Cholera Intoxication*

    PubMed Central

    Burress, Helen; Taylor, Michael; Banerjee, Tuhina; Tatulian, Suren A.; Teter, Ken

    2014-01-01

    Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the rest of the toxin. CTA1 then unfolds and passes through an ER translocon pore to reach its cytosolic target. Due to its intrinsic instability, cytosolic CTA1 must be refolded to achieve an active conformation. The cytosolic chaperone Hsp90 is involved with the ER to cytosol export of CTA1, but the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Moreover, potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 have not been explored. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and refolding of CTA1 by Hsp90 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allows disordered CTA1 to attain an active state, which is further enhanced by ADP-ribosylation factor 6, a host cofactor for CTA1. Our data indicate CTA1 translocation involves a process that couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. The molecular basis for toxin translocation elucidated in this study may also apply to several ADP-ribosylating toxins that move from the endosomes to the cytosol in an Hsp90-dependent process. PMID:25320090

  20. Profiling Hsp90 differential expression and the molecular effects of the Hsp90 inhibitor IPI-504 in high-grade glioma models.

    PubMed

    Di, Kaijun; Keir, Stephen T; Alexandru-Abrams, Daniela; Gong, Xing; Nguyen, Howard; Friedman, Henry S; Bota, Daniela A

    2014-12-01

    Retaspimycin hydrochloride (IPI-504), an Hsp90 (heat shock protein 90) inhibitor, has shown activity in multiple preclinical cancer models, such as lung, breast and ovarian cancers. However, its biological effects in gliomas and normal brain derived cellular populations remain unknown. In this study, we profiled the expression pattern of Hsp90α/β mRNA in stable glioma cell lines, multiple glioma-derived primary cultures and human neural stem/progenitor cells. The effects of IPI-504 on cell proliferation, apoptosis, motility and expression of Hsp90 client proteins were evaluated in glioma cell lines. In vivo activity of IPI-504 was investigated in subcutaneous glioma xenografts. Our results showed Hsp90α and Hsp90β expression levels to be patient-specific, higher in high-grade glioma-derived primary cells than in low-grade glioma-derived primary cells, and strongly correlated with CD133 expression and differentiation status of cells. Hsp90 inhibition by IPI-504 induced apoptosis, blocked migration and invasion, and significantly decreased epidermal growth factor receptor levels, mitogen-activated protein kinase and/or Akt activities, and secretion of vascular endothelial growth factor in glioma cell lines. In vivo study showed that IPI-504 could mildly attenuate tumor growth in immunocompromised mice. These findings suggest that targeting Hsp90 by IPI-504 has the potential to become an active therapeutic strategy in gliomas in a selective group of patients, but further research into combination therapies is still needed. PMID:25115740

  1. Hsp90 Inhibitors for the Treatment of Chronic Myeloid Leukemia

    PubMed Central

    Khajapeer, Kalubai Vari; Baskaran, Rajasekaran

    2015-01-01

    Chronic myeloid leukemia (CML) is a hematological malignancy that arises due to reciprocal translocation of 3′ sequences from c-Abelson (ABL) protooncogene of chromosome 9 with 5′ sequence of truncated break point cluster region (BCR) on chromosome 22. BCR-ABL is a functional oncoprotein p210 that exhibits constitutively activated tyrosine kinase causing genomic alteration of hematopoietic stem cells. BCR-ABL specific tyrosine kinase inhibitors (TKIs) successfully block CML progression. However, drug resistance owing to BCR-ABL mutations and overexpression is still an issue. Heat-shock proteins (Hsps) function as molecular chaperones facilitating proper folding of nascent polypeptides. Their increased expression under stressful conditions protects cells by stabilizing unfolded or misfolded peptides. Hsp90 is the major mammalian protein and is required by BCR-ABL for stabilization and maturation. Hsp90 inhibitors destabilize the binding of BCR-ABL protein thus leading to the formation of heteroprotein complex that is eventually degraded by the ubiquitin-proteasome pathway. Results of many novel Hsp90 inhibitors that have entered into various clinical trials are encouraging. The present review targets the current development in the CML treatment by availing Hsp90 specific inhibitors. PMID:26770832

  2. Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As{sup +3}- and MMA{sup +3}-induced apoptosis through inhibition of telomerase activity via JNK activation

    SciTech Connect

    Shen, S.-C.; Yang, L.-Y.; Lin, H.-Y.; Wu, C.-Y.; Su, T.-H.; Chen, Y.-C.

    2008-06-01

    The effects of six arsenic compounds including As{sup +3}, MMA{sup +3}, DMA{sup +3}, As{sup +5}, MMA{sup +5}, and DMA{sup +5} on the viability of NIH3T3 cells were examined. As{sup +3} and MMA{sup +3}, but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As{sup +3} and MMA{sup +3} were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As{sup +3} and MMA{sup +3} treatments. An increase in the intracellular peroxide level was examined in As{sup +3}- and MMA{sup +3}-treated NIH3T3 cells, and As{sup +3}- and MMA{sup +3}-induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As{sup +3}- and MMA{sup +3}-induced cytotoxicity. Suppression of JNKs significantly inhibited As{sup +3}- and MMA{sup +3}-induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As{sup +3}- and MMA{sup +3}-induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As{sup +3} or MMA{sup +3}. These data provide the first evidence to indicate that apoptosis induced by As{sup +3} and MMA{sup +3} is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved.

  3. Hsp90: A New Player in DNA Repair?

    PubMed

    Pennisi, Rosa; Ascenzi, Paolo; di Masi, Alessandra

    2015-01-01

    Heat shock protein 90 (Hsp90) is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis), transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR) pathways that include: (i) cell cycle arrest; (ii) transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii) triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted. PMID:26501335

  4. The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

    SciTech Connect

    Song, Xiaomin; Luo, Yongzhang

    2010-07-16

    Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.

  5. Anti-Hsp90 therapy in autoimmune and inflammatory diseases: a review of preclinical studies.

    PubMed

    Tukaj, Stefan; Węgrzyn, Grzegorz

    2016-03-01

    Heat shock protein 90 (Hsp90), a 90-kDa molecular chaperone, is responsible for biological activities of key signaling molecules (clients) such as protein kinases, ubiquitin ligases, steroid receptors, cell cycle regulators, and transcription factors regulating various cellular processes, including growth, survival, differentiation, and apoptosis. Because Hsp90 is also involved in stabilization of oncogenic 'client' proteins, its specific chaperone activity blockers are currently being tested as anticancer agents in advanced clinical trials. Recent in vitro and in vivo studies have shown that Hsp90 is also involved in activation of innate and adaptive cells of the immune system. For these reasons, pharmacological inhibition of Hsp90 has been evaluated in murine models of autoimmune and inflammatory diseases. This mini-review summarizes current knowledge of the effects of Hsp90 inhibitors on autoimmune and inflammatory diseases' features and is based solely on preclinical studies. PMID:26786410

  6. Identification of a new scaffold for hsp90 C-terminal inhibition.

    PubMed

    Zhao, Huiping; Moroni, Elisabetta; Colombo, Giorgio; Blagg, Brian S J

    2014-01-01

    Inhibition of Hsp90 C-terminal function is an advantageous therapeutic paradigm for the treatment of cancer. Currently, the majority of Hsp90 C-terminal inhibitors are derived from novobiocin, a natural product traditionally used as an antibiotic. Assisted by molecular docking studies, a scaffold containing a biphenyl moiety in lieu of the coumarin ring system found in novobiocin was identified for development of new Hsp90 C-terminal inhibitors. Initial structure-activity studies led to derivatives that manifest good antiproliferative activity against two breast cancer cell lines through Hsp90 inhibition. This platform serves as a scaffold upon which new Hsp90 C-terminal inhibitors can be readily assembled for further investigation. PMID:24900777

  7. Targeting Hsp90 in urothelial carcinoma

    PubMed Central

    Skotnicki, Kamil; Landas, Steve; Bratslavsky, Gennady; Bourboulia, Dimitra

    2015-01-01

    Urothelial carcinoma, or transitional cell carcinoma, is the most common urologic malignancy that carries significant morbidity, mortality, recurrence risk and associated health care costs. Despite use of current chemotherapies and immunotherapies, long-term remission in patients with muscle-invasive or metastatic disease remains low, and disease recurrence is common. The molecular chaperone Heat Shock Protein-90 (Hsp90) may offer an ideal treatment target, as it is a critical signaling hub in urothelial carcinoma pathogenesis and potentiates chemoradiation. Preclinical testing with Hsp90 inhibitors has demonstrated reduced proliferation, enhanced apoptosis and synergism with chemotherapies and radiation. Despite promising preclinical data, clinical trials utilizing Hsp90 inhibitors for other malignancies had modest efficacy. Therefore, we propose that Hsp90 inhibition would best serve as an adjuvant treatment in advanced muscle-invasive or metastatic bladder cancers to potentiate other therapies. An overview of bladder cancer biology, current treatments, molecular targeted therapies, and the role for Hsp90 inhibitors in the treatment of urothelial carcinoma is the focus of this review. PMID:25909217

  8. Intra- And Inter-Monomer Interactions are Required to Synergistically Facilitate ATP Hydrolysis in HSP90

    SciTech Connect

    Cunningham, C.N.; Krukenberg, K.A.; Agard, D.A.

    2009-05-12

    Nucleotide-dependent conformational changes of the constitutively dimeric molecular chaperone Hsp90 are integral to its molecular mechanism. Recent full-length crystal structures (Protein Data Bank codes 2IOQ, 2CG9, AND 2IOP) of Hsp90 homologs reveal large scale quaternary domain rearrangements upon the addition of nucleotides. Although previous work has shown the importance of C-terminal domain dimerization for efficient ATP hydrolysis, which should imply cooperativity, other studies suggest that the two ATPases function independently. Using the crystal structures as a guide, we examined the role of intra- and intermonomer interactions in stabilizing the ATPase activity of a single active site within an intact dimer. This was accomplished by creating heterodimers that allow us to differentially mutate each monomer, probing the context in which particular residues are important for ATP hydrolysis. Although the ATPase activity of each monomer can function independently, we found that the activity of one monomer could be inhibited by the mutation of hydrophobic residues on the trans N-terminal domain (opposite monomer). Furthermore, these trans interactions are synergistically mediated by a loop on the cis middle domain. This loop contains hydrophobic residues as well as a critical arginine that provides a direct linkage to the {gamma}-phosphate of bound ATP. Small angle x-ray scattering demonstrates that deleterious mutations block domain closure in the presence of AMPPNP (5{prime}-adenylyl-{beta},{gamma}-imidodiphosphate), providing a direct linkage between structural changes and functional consequences. Together, these data indicate that both the cis monomer and the trans monomer and the intradomain and interdomain interactions cooperatively stabilize the active conformation of each active site and help explain the importance of dimer formation.

  9. GRP94: an HSP90-like protein specialized for protein folding and quality control in the Endoplasmic Reticulum

    PubMed Central

    Marzec, Michal; Eletto, Davide; Argon, Yair

    2011-01-01

    Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitates by the conditions in the endoplasmic reticulum. GRP94’s mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remain obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. PMID:22079671

  10. Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox.

    PubMed

    Okada, Miki; Itoh, Hideaki; Hatakeyama, Takashi; Tokumitsu, Hiroshi; Kobayashi, Ryoji

    2003-09-01

    Hsp90 (heat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but in vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5-3.0 microM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both in vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90. PMID:12803546

  11. Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox.

    PubMed Central

    Okada, Miki; Itoh, Hideaki; Hatakeyama, Takashi; Tokumitsu, Hiroshi; Kobayashi, Ryoji

    2003-01-01

    Hsp90 (heat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but in vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5-3.0 microM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both in vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90. PMID:12803546

  12. Resistance to HSP90 inhibition involving loss of MCL1 addiction.

    PubMed

    Busacca, S; Law, E W P; Powley, I R; Proia, D A; Sequeira, M; Le Quesne, J; Klabatsa, A; Edwards, J M; Matchett, K B; Luo, J L; Pringle, J H; El-Tanani, M; MacFarlane, M; Fennell, D A

    2016-03-24

    Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality. PMID:26096930

  13. Resistance to HSP90 inhibition involving loss of MCL1 addiction

    PubMed Central

    Busacca, S; Law, E W P; Powley, I R; Proia, D A; Sequeira, M; Le Quesne, J; Klabatsa, A; Edwards, J M; Matchett, K B; Luo, J L; Pringle, J H; El-Tanani, M; MacFarlane, M; Fennell, D A

    2016-01-01

    Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality. PMID:26096930

  14. Structures of GRP94-Nucleotide Complexes Reveal Mechanistic Differences Between the Hsp90 Chaperones

    SciTech Connect

    Dollins, D.E.; Warren, J.J.; Immormino, R.M.; Gewirth, D.T.

    2009-06-02

    GRP94, an essential endoplasmic reticulum chaperone, is required for the conformational maturation of proteins destined for cell-surface display or export. The extent to which GRP94 and its cytosolic paralog, Hsp90, share a common mechanism remains controversial. GRP94 has not been shown conclusively to hydrolyze ATP or bind cochaperones, and both activities, by contrast, result in conformational changes and N-terminal dimerization in Hsp90 that are critical for its function. Here, we report the 2.4 {angstrom} crystal structure of mammalian GRP94 in complex with AMPPNP and ADP. The chaperone is conformationally insensitive to the identity of the bound nucleotide, adopting a 'twisted V' conformation that precludes N-terminal domain dimerization. We also present conclusive evidence that GRP94 possesses ATPase activity. Our observations provide a structural explanation for GRP94's observed rate of ATP hydrolysis and suggest a model for the role of ATP binding and hydrolysis in the GRP94 chaperone cycle.

  15. MicroRNA-27b plays a role in pulmonary arterial hypertension by modulating peroxisome proliferator-activated receptor γ dependent Hsp90-eNOS signaling and nitric oxide production

    SciTech Connect

    Bi, Rui; Bao, Chunrong; Jiang, Lianyong; Liu, Hao; Yang, Yang; Mei, Ju; Ding, Fangbao

    2015-05-01

    Pulmonary artery endothelial dysfunction is associated with pulmonary arterial hypertension (PAH). Based on recent studies showing that microRNA (miR)-27b is aberrantly expressed in PAH, we hypothesized that miR-27b may contribute to pulmonary endothelial dysfunction and vascular remodeling in PAH. The effect of miR-27b on pulmonary endothelial dysfunction and the underlying mechanism were investigated in human pulmonary artery endothelial cells (HPAECs) in vitro and in a monocrotaline (MCT)-induced model of PAH in vivo. miR-27b expression was upregulated in MCT-induced PAH and inversely correlated with the levels of peroxisome proliferator-activated receptor (PPAR)-γ, and miR-27b inhibition attenuated MCT-induced endothelial dysfunction and remodeling and prevented PAH associated right ventricular hypertrophy and systolic pressure in rats. PPARγ was confirmed as a direct target of miR-27b in HPAECs and shown to mediate the effect of miR-27b on the disruption of endothelial nitric oxide synthase (eNOS) coupling to Hsp90 and the suppression of NO production associated with the PAH phenotype. We showed that miR-27b plays a role endothelial function and NO release and elucidated a potential mechanism by which miR-27b regulates Hsp90-eNOS and NO signaling by modulating PPARγ expression, providing potential therapeutic targets for the treatment of PAH. - Highlights: • miR-27b plays a role in endothelial function and NO release. • miR-27b inhibition ameliorates MCT-induced endothelial dysfunction and PAH. • miR-27b targets PPARγ in HPAECs. • miR-27b regulates PPARγ dependent Hsp90-eNOS and NO signaling.

  16. Expression of heat shock protein (Hsp90) paralogues is regulated by amino acids in skeletal muscle of Atlantic salmon.

    PubMed

    Garcia de la Serrana, Daniel; Johnston, Ian A

    2013-01-01

    Heat shock proteins 90 (Hsp90) have an essential role in sarcomere formation and differentiation in skeletal muscle and also act as molecular chaperones during protein folding impacting a wide range of physiological processes. We characterised and provided a phylogenetically consistent nomenclature for the complete repertoire of six Hsp90 paralogues present in duplicated salmonid fish genomes (Hsp90α1a, Hsp90α1b, Hsp90α2a, Hsp90α2b, Hsp90ß1a and Hsp90ß1b). The expression of paralogues in fast skeletal muscle was investigated using in vivo fasting-feeding experiments and primary myogenic cultures. Fasted juvenile Atlantic salmon (Salmo salar) showed a transient 2 to 8-fold increase in the expression of all 4 Hsp90α paralogues within 24h of satiation feeding. Hsp90α1a and hsp90α1b also showed a pronounced secondary increase in expression after 10 days, concomitant with muscle differentiation and the expression of myogenin and sarcomeric proteins (mlc2, myhc). Hsp90ß1b was constitutively expressed whereas Hsp90ß1a expression was downregulated 10-fold between fasted and fed individuals. Hsp90α1a and Hsp90α1b were upregulated 10 to 15-fold concomitant with myotube formation and muscle differentiation in vitro whereas other Hsp90 paralogues showed no change in expression. In cells starved of amino acid (AA) and serum for 72h the addition of AA, but not insulin-like growth factor 1, increased phosphorylation of mTor and expression of all 4 hsp90α paralogues and associated co-chaperones including hsp30, tbcb, pdia4, pdia6, stga and fk504bp1, indicating a general activation of the protein folding response. In contrast, Hsp90ß1a expression in vitro was unresponsive to AA treatment indicating that some other as yet uncharacterised signal(s) regulate its expression in response to altered nutritional state. PMID:24040223

  17. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein.

    PubMed

    McCready, Jessica; Wong, Daniel S; Burlison, Joseph A; Ying, Weiwen; Jay, Daniel G

    2014-01-01

    Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion. PMID:24785146

  18. C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis

    PubMed Central

    Harriss, June; Das, Chhaya; Zhu, Li; Edwards, Melissa; Shaaban, Salam; Tucker, Haley

    2015-01-01

    The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3′s oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors. PMID:25738358

  19. A Hyperactive Signalosome in Acute Myeloid Leukemia Drives Addiction to a Tumor-Specific Hsp90 Species.

    PubMed

    Zong, Hongliang; Gozman, Alexander; Caldas-Lopes, Eloisi; Taldone, Tony; Sturgill, Eric; Brennan, Sarah; Ochiana, Stefan O; Gomes-DaGama, Erica M; Sen, Siddhartha; Rodina, Anna; Koren, John; Becker, Michael W; Rudin, Charles M; Melnick, Ari; Levine, Ross L; Roboz, Gail J; Nimer, Stephen D; Chiosis, Gabriela; Guzman, Monica L

    2015-12-15

    Acute myeloid leukemia (AML) is a heterogeneous and fatal disease with an urgent need for improved therapeutic regimens given that most patients die from relapsed disease. Irrespective of mutation status, the development of aggressive leukemias is enabled by increasing dependence on signaling networks. We demonstrate that a hyperactive signalosome drives addiction of AML cells to a tumor-specific Hsp90 species (teHsp90). Through genetic, environmental, and pharmacologic perturbations, we demonstrate a direct and quantitative link between hyperactivated signaling pathways and apoptotic sensitivity of AML to teHsp90 inhibition. Specifically, we find that hyperactive JAK-STAT and PI3K-AKT signaling networks are maintained by teHsp90 and, in fact, gradual activation of these networks drives tumors increasingly dependent on teHsp90. Thus, although clinically aggressive AML survives via signalosome activation, this addiction creates a vulnerability that can be exploited with Hsp90-directed therapy. PMID:26628369

  20. Drosophila Spag is the homolog of RNA polymerase II-associated protein 3 (RPAP3) and recruits the heat shock proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries.

    PubMed

    Benbahouche, Nour El Houda; Iliopoulos, Ioannis; Török, István; Marhold, Joachim; Henri, Julien; Kajava, Andrey V; Farkaš, Robert; Kempf, Tore; Schnölzer, Martina; Meyer, Philippe; Kiss, István; Bertrand, Edouard; Mechler, Bernard M; Pradet-Balade, Bérengère

    2014-02-28

    The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA(+)-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes. PMID:24394412

  1. Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries* ♦

    PubMed Central

    Benbahouche, Nour El Houda; Iliopoulos, Ioannis; Török, István; Marhold, Joachim; Henri, Julien; Kajava, Andrey V.; Farkaš, Robert; Kempf, Tore; Schnölzer, Martina; Meyer, Philippe; Kiss, István; Bertrand, Edouard; Mechler, Bernard M.; Pradet-Balade, Bérengère

    2014-01-01

    The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA+-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes. PMID:24394412

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

    PubMed Central

    Nguyen, M T; Csermely, P; Sőti, C

    2013-01-01

    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. PMID:24096869

  3. Targeting HSF1 disrupts HSP90 chaperone function in chronic lymphocytic leukemia

    PubMed Central

    Yacoub, Abdulraheem; Kambhampati, Suman; Shi, Huidong; Dandawate, Prasad; Padhye, Subhash; Saluja, Ashok K.; McGuirk, Joseph; Rao, Rekha

    2015-01-01

    CLL is a disease characterized by chromosomal deletions, acquired copy number changes and aneuploidy. Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. Interestingly, several independent studies have demonstrated that HSF1 expression and activity also affects the chaperoning of HSP90 kinase clients, although the mechanism underlying this observation is unclear. Here, we determined how HSF1 regulates HSP90 function using CLL as a model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. We demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). Treatment with triptolide or HSF1 knockdown disrupts the cytosolic complex between HSF1, p97, HSP90 and the HSP90 deacetylase- Histone deacetylase 6 (HDAC6). Consequently, HSF1 inhibition results in HSP90 acetylation and abrogation of its chaperone function. Finally, tail vein injection of Mec-1 cells into Rag2−/−IL2Rγc−/− mice followed by treatment with minnelide (a pro-drug of triptolide), reduced leukemia, increased survival and attenuated HSP90-dependent survival signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 and test the activity of minnelide against human CLL. PMID:26397138

  4. Targeting HSF1 disrupts HSP90 chaperone function in chronic lymphocytic leukemia.

    PubMed

    Ganguly, Siddhartha; Home, Trisha; Yacoub, Abdulraheem; Kambhampati, Suman; Shi, Huidong; Dandawate, Prasad; Padhye, Subhash; Saluja, Ashok K; McGuirk, Joseph; Rao, Rekha

    2015-10-13

    CLL is a disease characterized by chromosomal deletions, acquired copy number changes and aneuploidy. Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. Interestingly, several independent studies have demonstrated that HSF1 expression and activity also affects the chaperoning of HSP90 kinase clients, although the mechanism underlying this observation is unclear. Here, we determined how HSF1 regulates HSP90 function using CLL as a model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. We demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). Treatment with triptolide or HSF1 knockdown disrupts the cytosolic complex between HSF1, p97, HSP90 and the HSP90 deacetylase- Histone deacetylase 6 (HDAC6). Consequently, HSF1 inhibition results in HSP90 acetylation and abrogation of its chaperone function. Finally, tail vein injection of Mec-1 cells into Rag2-/-IL2Rγc-/- mice followed by treatment with minnelide (a pro-drug of triptolide), reduced leukemia, increased survival and attenuated HSP90-dependent survival signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 and test the activity of minnelide against human CLL. PMID:26397138

  5. The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells.

    PubMed

    Gomez-Casal, Roberto; Bhattacharya, Chitralekha; Epperly, Michael W; Basse, Per H; Wang, Hong; Wang, Xinhui; Proia, David A; Greenberger, Joel S; Socinski, Mark A; Levina, Vera

    2015-01-01

    The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors. PMID:26010604

  6. The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells

    PubMed Central

    Gomez-Casal, Roberto; Bhattacharya, Chitralekha; Epperly, Michael W.; Basse, Per H.; Wang, Hong; Wang, Xinhui; Proia, David A.; Greenberger, Joel S.; Socinski, Mark A.; Levina, Vera

    2015-01-01

    The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors. PMID:26010604

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

    PubMed

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    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. PMID:22066472

  8. Protozoan HSP90-heterocomplex: molecular interaction network and biological significance.

    PubMed

    Figueras, Maria J; Echeverria, Pablo C; Angel, Sergio O

    2014-05-01

    The HSP90 chaperone is a highly conserved protein from bacteria to higher eukaryotes. In eukaryotes, this chaperone participates in different large complexes, such as the HSP90 heterocomplex, which has important biological roles in cell homeostasis and differentiation. The HSP90-heterocomplex is also named the HSP90/HSP70 cycle because different co-chaperones (HIP, HSP40, HOP, p23, AHA1, immunophilins, PP5) participate in this complex by assembling sequentially, from the early to the mature complex. In this review, we analyze the conservation and relevance of HSP90 and the HSP90-heterocomplex in several protozoan parasites, with emphasis in Plasmodium spp., Toxoplasma spp., Leishmania spp. and Trypanosoma spp. In the last years, there has been an outburst of studies based on yeast two-hybrid methodology, co-immunoprecipitation-mass spectrometry and bioinformatics, which have generated a most comprehensive protein-protein interaction (PPI) network of HSP90 and its co-chaperones. This review analyzes the existing PPI networks of HSP90 and its co-chaperones of some protozoan parasites and discusses the usefulness of these powerful tools to analyze the biological role of the HSP90-heterocomplex in these parasites. The generation of a T. gondii HSP90 heterocomplex PPI network based on experimental data and a recent Plasmodium HSP90 heterocomplex PPI network are also included and discussed. As an example, the putative implication of nuclear transport and chromatin (histones and Sir2) as HSP90-heterocomplex interactors is here discussed. PMID:24694366

  9. Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

    PubMed Central

    Boczek, Edgar E.; Reefschläger, Lasse G.; Dehling, Marco; Struller, Tobias J.; Häusler, Elisabeth; Seidl, Andreas; Kaila, Ville R. I.; Buchner, Johannes

    2015-01-01

    Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated. PMID:26056257

  10. Approaches for Defining the Hsp90-dependent Proteome

    PubMed Central

    Hartson, Steven D.; Matts, Robert L.

    2011-01-01

    Hsp90 is the target of ongoing drug discovery studies seeking new compounds to treat cancer, neurodegenerative diseases, and protein folding disorders. To better understand Hsp90’s roles in cellular pathologies and in normal cells, numerous studies have utilized proteomics assays and related high-throughput tools to characterize its physical and functional protein partnerships. This review surveys these studies, and summarizes the strengths and limitations of the individual attacks. We also include downloadable spreadsheets compiling all of the Hsp90-interacting proteins identified in more than 23 studies. These tools include cross-references among gene aliases, human homologues of yeast Hsp90-interacting proteins, hyperlinks to database entries, summaries of canonical pathways that are enriched in the Hsp90 interactome, and additional bioinformatic annotations. In addition to summarizing Hsp90 proteomics studies performed to date and the insights they have provided, we identify gaps in our current understanding of Hsp90-mediated proteostasis. PMID:21906632

  11. Heat shock protein 90 (Hsp90): A novel antifungal target against Aspergillus fumigatus.

    PubMed

    Lamoth, Frédéric; Juvvadi, Praveen R; Steinbach, William J

    2016-01-01

    Invasive aspergillosis is a life-threatening and difficult to treat infection in immunosuppressed patients. The efficacy of current anti-Aspergillus therapies, targeting the cell wall or membrane, is limited by toxicity (polyenes), fungistatic activity and some level of basal resistance (echinocandins), or the emergence of acquired resistance (triazoles). The heat shock protein 90 (Hsp90) is a conserved molecular chaperone involved in the rapid development of antifungal resistance in the yeast Candida albicans. Few studies have addressed its role in filamentous fungi such as Aspergillus fumigatus, in which mechanisms of resistance may differ substantially. Hsp90 is at the center of a complex network involving calcineurin, lysine deacetylases (KDAC) and other client proteins, which orchestrate compensatory repair mechanisms of the cell wall in response to the stress induced by antifungals. In A. fumigatus, Hsp90 is a trigger for resistance to high concentrations of caspofungin, known as the paradoxical effect. Disrupting Hsp90 circuitry by different means (Hsp90 inhibitors, KDAC inhibitors and anti-calcineurin drugs) potentiates the antifungal activity of caspofungin, thus representing a promising novel antifungal approach. This review will discuss the specific features of A. fumigatus Hsp90 and the potential for antifungal strategies of invasive aspergillosis targeting this essential chaperone. PMID:25243616

  12. Combined HSP90 and kinase inhibitor therapy: Insights from The Cancer Genome Atlas.

    PubMed

    Schwartz, Harvey; Scroggins, Brad; Zuehlke, Abbey; Kijima, Toshiki; Beebe, Kristin; Mishra, Alok; Neckers, Len; Prince, Thomas

    2015-09-01

    The merging of knowledge from genomics, cellular signal transduction and molecular evolution is producing new paradigms of cancer analysis. Protein kinases have long been understood to initiate and promote malignant cell growth and targeting kinases to fight cancer has been a major strategy within the pharmaceutical industry for over two decades. Despite the initial success of kinase inhibitors (KIs), the ability of cancer to evolve resistance and reprogram oncogenic signaling networks has reduced the efficacy of kinase targeting. The molecular chaperone HSP90 physically supports global kinase function while also acting as an evolutionary capacitor. The Cancer Genome Atlas (TCGA) has compiled a trove of data indicating that a large percentage of tumors overexpress or possess mutant kinases that depend on the HSP90 molecular chaperone complex. Moreover, the overexpression or mutation of parallel activators of kinase activity (PAKA) increases the number of components that promote malignancy and indirectly associate with HSP90. Therefore, targeting HSP90 is predicted to complement kinase inhibitors by inhibiting oncogenic reprogramming and cancer evolution. Based on this hypothesis, consideration should be given by both the research and clinical communities towards combining kinase inhibitors and HSP90 inhibitors (H90Ins) in combating cancer. The purpose of this perspective is to reflect on the current understanding of HSP90 and kinase biology as well as promote the exploration of potential synergistic molecular therapy combinations through the utilization of The Cancer Genome Atlas. PMID:26070366

  13. HSP90 functions in the circadian clock through stabilization of the client F-box protein ZEITLUPE

    PubMed Central

    Kim, Tae-sung; Kim, Woe Yeon; Fujiwara, Sumire; Kim, Jeongsik; Cha, Joon-Yung; Park, Jin Ho; Lee, Sang Yeol; Somers, David E.

    2011-01-01

    The autoregulatory loops of the circadian clock consist of feedback regulation of transcription/translation circuits but also require finely coordinated cytoplasmic and nuclear proteostasis. Although protein degradation is important to establish steady-state levels, maturation into their active conformation also factors into protein homeostasis. HSP90 facilitates the maturation of a wide range of client proteins, and studies in metazoan clocks implicate HSP90 as an integrator of input or output. Here we show that the Arabidopsis circadian clock-associated F-box protein ZEITLUPE (ZTL) is a unique client for cytoplasmic HSP90. The HSP90-specific inhibitor geldanamycin and RNAi-mediated depletion of cytoplasmic HSP90 reduces levels of ZTL and lengthens circadian period, consistent with ztl loss-of-function alleles. Transient transfection of artificial microRNA targeting cytoplasmic HSP90 genes similarly lengthens period. Proteolytic targets of SCFZTL, TOC1 and PRR5, are stabilized in geldanamycin-treated seedlings, whereas the levels of closely related clock proteins, PRR3 and PRR7, are unchanged. An in vitro holdase assay, typically used to demonstrate chaperone activity, shows that ZTL can be effectively bound, and aggregation prevented, by HSP90. GIGANTEA, a unique stabilizer of ZTL, may act in the same pathway as HSP90, possibly linking these two proteins to a similar mechanism. Our findings establish maturation of ZTL by HSP90 as essential for proper function of the Arabidopsis circadian clock. Unlike metazoan systems, HSP90 functions here within the core oscillator. Additionally, F-box proteins as clients may place HSP90 in a unique and more central role in proteostasis. PMID:21949396

  14. Crowding Activates Heat Shock Protein 90.

    PubMed

    Halpin, Jackson C; Huang, Bin; Sun, Ming; Street, Timothy O

    2016-03-18

    Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulum-specific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro. PMID:26797120

  15. The N-terminal adenosine triphosphate binding domain of Hsp90 is necessary and sufficient for interaction with estrogen receptor.

    PubMed

    Bouhouche-Chatelier, L; Chadli, A; Catelli, M G

    2001-10-01

    To understand how the molecular chaperone Hsp90 participates in conformational maturation of the estrogen receptor (ER), we analyzed the interaction of immobilized purified avian Hsp90 with mammalian cytosolic ER. Hsp90 was either immunoadsorbed to BF4 antibody-Sepharose or GST-Hsp90 fusion protein (GST.90) was adsorbed to glutathione-Sepharose. GST.90 was able to retain specifically ER, similarly to immunoadsorbed Hsp90. When cells were treated with estradiol and the hormone treatment was maintained during cell homogenization, binding, and washing steps, GST.90 still interacted efficiently with ER, suggesting that ER may form complexes with Hsp90 even after its activation by hormone and salt extraction from nuclei. The GST.90-ER interaction was consistently reduced in the presence of increasing concentrations of potassium chloride or when cytosolic ER-Hsp90 complexes were previously stabilized by molybdate, indicating that GST.90-ER complexes behave like cytosolic Hsp90-ER complexes. A purified thioredoxin-ER fusion protein was also able to form complexes with GST.90, suggesting that the presence of other chaperones is not required. ER was retained only by GST.90 deletion mutants bearing an intact Hsp90 N-terminal region (1-224), the interaction being more efficient when the charged region A was present in the mutant (1-334). The N-terminal fragment 1-334, devoid of the dimeric GST moiety, was also able to interact with ER, pointing to the monomeric N-terminal adenosine triphosphate binding region of Hsp90 (1-224) as the region necessary and sufficient for interaction. These results contribute to understand the Hsp90-dependent process responsible for conformational competence of ER. PMID:11795466

  16. Extracellular Hsp90 (eHsp90) as the Actual Target in Clinical Trials: Intentionally or Unintentionally

    PubMed Central

    Li, Wei; Tsen, Fred; Sahu, Divya; Bhatia, Ayesha; Chen, Mei; Multhoff, Gabriele; Woodley, David T.

    2014-01-01

    Despite extensive investigative studies and clinical trials over the past two decades, we still do not understand why cancer cells are more sensitive to the cellular toxicity of Hsp90 inhibitors than normal cells. We still do not understand why only some cancer cells are sensitive to the Hsp90 inhibitors. Based on studies of the past few years, we argue that the selected sensitivity of cancer cells to Hsp90 inhibitors, such as 17-N-allylamino-17-demethoxygeldanamycin, is due to inhibition of the extracellular Hsp90 (eHsp90) rather than intracellular Hsp90 by these inhibitors. Because not all tumor cells utilize eHsp90 for motility, invasion and metastasis, only the group of “eHsp90-dependent” cancer cells is sensitive to Hsp90 inhibitors. If these notions prove to be true, pharmaceutical agents that selectively target eHsp90 should be more effective on tumor cells and less toxic on normal cells than current inhibitors that nondiscriminatively target both extracellular and intracellular Hsp90. PMID:23445811

  17. Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

    PubMed Central

    Donzé, Olivier; Picard, Didier

    1999-01-01

    The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2. PMID:10567567

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

    PubMed

    Zhang, Fengxue; Snead, Connie M; Catravas, John D

    2012-06-15

    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. PMID:22496241

  19. p50cdc37 Acting in Concert with Hsp90 Is Required for Raf-1 Function†

    PubMed Central

    Grammatikakis, Nicholas; Lin, Jun-Hsiang; Grammatikakis, Aliki; Tsichlis, Philip N.; Cochran, Brent H.

    1999-01-01

    Genetic screens in Drosophila have identified p50cdc37 to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50cdc37 in this pathway has been defined. In this study, we examined the role of p50cdc37 and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50cdc37 with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50cdc37 greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50cdc37 is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50cdc37 mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50cdc37-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50cdc37 or by GA. Thus, formation of a ternary Raf-1–p50cdc37–Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50cdc37-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular. PMID:10022854

  20. Effects of treatment with an Hsp90 inhibitor in tumors based on 15 phase II clinical trials

    PubMed Central

    Wang, He; Lu, Mingjie; Yao, Mengqian; Zhu, Wei

    2016-01-01

    Heat shock protein (Hsp)90 serves as a chaperone protein that promotes the proper folding of proteins involved in a variety of signal transduction processes involved in cell growth. Hsp90 inhibitors, which inhibit the activity of critical client proteins, have emerged as the accessory therapeutic agents for multiple human cancer types. To better understand the effects of Hsp90 inhibitors in cancer treatment, the present study reviewed 15 published phase II clinical trials to investigate whether Hsp90 inhibitors will benefit patients with cancer. Information of complete response, partial response, stable disease, objective response and objective response rate was collected to evaluate clinical outcomes. Overall, Hsp90 inhibitors are effective against a variety of oncogene-addicted cancers, including those that have developed resistance to specific receptors.

  1. NMNAT2:HSP90 Complex Mediates Proteostasis in Proteinopathies

    PubMed Central

    Ali, Yousuf O.; Allen, Hunter M.; Yu, Lei; Li-Kroeger, David; McCabe, Cristin; Xu, Jishu; Bjorklund, Nicole; Taglialatela, Giulio; Bennett, David A.; De Jager, Philip L.; Shulman, Joshua M.; Bellen, Hugo J.; Lu, Hui-Chen

    2016-01-01

    Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is neuroprotective in numerous preclinical models of neurodegeneration. Here, we show that brain nmnat2 mRNA levels correlate positively with global cognitive function and negatively with AD pathology. In AD brains, NMNAT2 mRNA and protein levels are reduced. NMNAT2 shifts its solubility and colocalizes with aggregated Tau in AD brains, similar to chaperones, which aid in the clearance or refolding of misfolded proteins. Investigating the mechanism of this observation, we discover a novel chaperone function of NMNAT2, independent from its enzymatic activity. NMNAT2 complexes with heat shock protein 90 (HSP90) to refold aggregated protein substrates. NMNAT2’s refoldase activity requires a unique C-terminal ATP site, activated in the presence of HSP90. Furthermore, deleting NMNAT2 function increases the vulnerability of cortical neurons to proteotoxic stress and excitotoxicity. Interestingly, NMNAT2 acts as a chaperone to reduce proteotoxic stress, while its enzymatic activity protects neurons from excitotoxicity. Taken together, our data indicate that NMNAT2 exerts its chaperone or enzymatic function in a context-dependent manner to maintain neuronal health. PMID:27254664

  2. Targeting the Hsp90-associated viral oncoproteome in gammaherpesvirus-associated malignancies.

    PubMed

    Nayar, Utthara; Lu, Pin; Goldstein, Rebecca L; Vider, Jelena; Ballon, Gianna; Rodina, Anna; Taldone, Tony; Erdjument-Bromage, Hediye; Chomet, Max; Blasberg, Ronald; Melnick, Ari; Cerchietti, Leandro; Chiosis, Gabriela; Wang, Y Lynn; Cesarman, Ethel

    2013-10-17

    PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies. PMID:23943653

  3. A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter*

    PubMed Central

    El-Kasaby, Ali; Koban, Florian; Sitte, Harald H.; Freissmuth, Michael; Sucic, Sonja

    2014-01-01

    Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay. PMID:25202009

  4. A cytosolic relay of heat shock proteins HSP70-1A and HSP90β monitors the folding trajectory of the serotonin transporter.

    PubMed

    El-Kasaby, Ali; Koban, Florian; Sitte, Harald H; Freissmuth, Michael; Sucic, Sonja

    2014-10-17

    Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay. PMID:25202009

  5. Design and synthesis of pyrimidinyl acyl thioureas as novel Hsp90 inhibitors in invasive ductal breast cancer and its bone metastasis.

    PubMed

    Koca, İrfan; Özgür, Aykut; Er, Muhammet; Gümüş, Mehmet; Açikalin Coşkun, Kübra; Tutar, Yusuf

    2016-10-21

    Invasive ductal carcinoma is the most common breast malignancies tumors and has tendency to bone metastases. Many oncogenic client proteins involved in formation of metastatic pathways. Stabilization, regulation, and maintenance of these oncogenic client proteins are provided with Heat Shock Protein 90 (Hsp90). Hsp90 perform these processes through its ATP binding and subsequent hydrolysis energy. Therefore, designing Hsp90 inhibitors is a novel cancer treatment method. However, many Hsp90 inhibitors have solubility problems and showed adverse effects in clinical trials. Thus, we designed and synthesized novel pyrimidinyl acyl thiourea derivatives to selectively inhibit Hsp90 alpha in human invasive ductal breast (MCF-7) and human bone osteosarcoma (Saos-2) cell lines. In vitro experiments showed that the compounds inhibited cell proliferation, ATP hydrolysis, and exhibited cytotoxic effect on these cancer cell lines. Further, gene expression was analyzed by microarray studies on MCF-7 cell lines. Several genes that play vital roles in breast cancer pathogenesis displayed altered gene expression in the presence of a selected pyrimidinyl acyl thiourea compound. Molecular docking studies were also performed to determine interaction between Hsp90 ATPase domain and pyrimidinyl acyl thiourea derivatives. The results indicated that the compounds are able to interact with Hsp90 ATP binding pocket and inhibit ATPase function. The designed compounds powerfully inhibit Hsp90 by an average of 1 μM inhibition constant. And further, the compounds perturb Hsp90 N terminal domain proper orientation and ATP may not provide required conformational change for Hsp90 function as evidenced by in silico experiments. Therefore, the designed compounds effectively inhibited both invasive ductal breast carcinoma and bone metastasis. Pyrimidinyl acyl thiourea derivatives may provide a drug template for effective treatment of invasive ductal breast carcinoma and its bone metastasis as

  6. The 90 kDa heat-shock protein (hsp90) modulates the binding of the oestrogen receptor to its cognate DNA.

    PubMed

    Sabbah, M; Radanyi, C; Redeuilh, G; Baulieu, E E

    1996-02-15

    The role of heat-shock protein 90 (hsp90) in the regulation of the oestrogen receptor (ER) function is less well understood than for other steroid-hormone receptors because hsp90 is not involved in the stabilization or induction of a high-affinity ligand-binding state of ER nor in the inhibition of receptor dimerization. Electrophoretic mobility-shift assays, using purified ER and hsp90, were employed to investigate directly the effect of hsp90 on the ability of ER to bind to the oestrogen-response element (ERE) from the vitellogenin A2 gene. Contrary to models in which hsp90 binds to and passively inactivates steroid-hormone receptors, our studies show that the binding of ER to ERE is inversely dependent on the relative concentration of hsp90. Exposure of purified ER-hsp90 complexes to ERE led to the dissociation of hsp90 and concomitant specific binding of ER to ERE. We demonstrate that the amount of ER-ERE complex decreased with increasing concentrations of hsp90. Furthermore hsp90 dissociated preformed high-affinity ER-ERE complexes. Kinetic dissociation experiments indicate the hsp90 acts in a dynamic and specific process rather than by simple trapping of ER owing to its inherent off-rate. The receptor released from the ERE-bound state by hsp90 was recovered associated with hsp90 and was able to rebind to ERE. These results indicate that hsp90 does not suppress ER function merely by steric hindrance. On the basis of these results and others, we propose that, in vivo, hsp90 may play a dual role in ER function: (i) at a physiological temperature, hsp90 stabilizes an active form of the receptor in accordance with its general molecular chaperone role; (ii) at elevated temperatures or under other environmental stress, the increased cellular concentration of hsp90 negatively interferes with ER-dependent transcription, in accordance with the inhibition of gene transcription attributed to hsp90 after heat shock. PMID:8660284

  7. Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33

    PubMed Central

    Azoitei, Ninel; Hoffmann, Christopher M.; Ellegast, Jana M.; Ball, Claudia R.; Obermayer, Kerstin; Gößele, Ulrike; Koch, Britta; Faber, Katrin; Genze, Felicitas; Schrader, Mark; Kestler, Hans A.; Döhner, Hartmut; Chiosis, Gabriela; Glimm, Hanno

    2012-01-01

    Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential. PMID:22451720

  8. Downregulation of the evolutionary capacitor Hsp90 is mediated by social cues

    PubMed Central

    Eggert, Hendrik

    2015-01-01

    The relationship between robustness and evolvability is a long-standing question in evolution. Heat shock protein 90 (HSP90), a molecular chaperone, has been identified as a potential capacitor for evolution, since it allows for the accumulation and release of cryptic genetic variation, and also for the regulation of novel genetic variation through transposon activity. However, to date, it is unknown whether Hsp90 expression is regulated upon demand (i.e. when the release of cryptic genetic variation is most needed). Here, we show that Hsp90 has reduced transcription under conditions where the mobilization of genetic variation could be advantageous. We designed a situation that indicates a stressful environment but avoids the direct effects of stress, by placing untreated (focal) red flour beetles, Tribolium castaneum, into groups together with wounded conspecifics, and found a consistent reduction in expression of two Hsp90 genes (Hsp83 and Hsp90) in focal beetles. We moreover observed a social transfer of immunity in this non-eusocial insect: there was increased activity of the phenoloxidase enzyme and downregulation of the immune regulator, imd. Our study poses the exciting question of whether evolvability might be regulated through the use of information derived from the social environment. PMID:26582024

  9. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    PubMed

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. PMID:26722004

  10. Quantitative Proteomics Reveals That Hsp90 Inhibition Preferentially Targets Kinases and the DNA Damage Response*

    PubMed Central

    Sharma, Kirti; Vabulas, R. Martin; Macek, Boris; Pinkert, Stefan; Cox, Jürgen; Mann, Matthias; Hartl, F. Ulrich

    2012-01-01

    Despite the increasing importance of heat shock protein 90 (Hsp90) inhibitors as chemotherapeutic agents in diseases such as cancer, their global effects on the proteome remain largely unknown. Here we use high resolution, quantitative mass spectrometry to map protein expression changes associated with the application of the Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG). In depth data obtained from five replicate SILAC experiments enabled accurate quantification of about 6,000 proteins in HeLa cells. As expected, we observed activation of a heat shock response with induced expression of molecular chaperones, which refold misfolded proteins, and proteases, which degrade irreparably damaged polypeptides. Despite the broad range of known Hsp90 substrates, bioinformatics analysis revealed that particular protein classes were preferentially affected. These prominently included proteins involved in the DNA damage response, as well as protein kinases and especially tyrosine kinases. We followed up on this observation with a quantitative phosphoproteomic analysis of about 4,000 sites, which revealed that Hsp90 inhibition leads to much more down- than up-regulation of the phosphoproteome (34% down versus 6% up). This study defines the cellular response to Hsp90 inhibition at the proteome level and sheds light on the mechanisms by which it can be used to target cancer cells. PMID:22167270

  11. The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function

    PubMed Central

    Jahn, Markus; Rehn, Alexandra; Pelz, Benjamin; Hellenkamp, Björn; Richter, Klaus; Rief, Matthias; Buchner, Johannes; Hugel, Thorsten

    2014-01-01

    The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90’s chaperone cycle. A unique linker, called charged linker (CL), connects the N- and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 kBT (2.7 kJ/mol) through binding of the CL to the N-domain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90’s chaperone cycle governing client activation, cell viability, and stress tolerance. PMID:25468961

  12. A rat retinal damage model predicts for potential clinical visual disturbances induced by Hsp90 inhibitors

    SciTech Connect

    Zhou, Dan; Liu, Yuan; Ye, Josephine; Ying, Weiwen; Ogawa, Luisa Shin; Inoue, Takayo; Tatsuta, Noriaki; Wada, Yumiko; Koya, Keizo; Huang, Qin; Bates, Richard C.; Sonderfan, Andrew J.

    2013-12-01

    In human trials certain heat shock protein 90 (Hsp90) inhibitors, including 17-DMAG and NVP-AUY922, have caused visual disorders indicative of retinal dysfunction; others such as 17-AAG and ganetespib have not. To understand these safety profile differences we evaluated histopathological changes and exposure profiles of four Hsp90 inhibitors, with or without clinical reports of adverse ocular effects, using a rat retinal model. Retinal morphology, Hsp70 expression (a surrogate marker of Hsp90 inhibition), apoptotic induction and pharmacokinetic drug exposure analysis were examined in rats treated with the ansamycins 17-DMAG and 17-AAG, or with the second-generation compounds NVP-AUY922 and ganetespib. Both 17-DMAG and NVP-AUY922 induced strong yet restricted retinal Hsp70 up-regulation and promoted marked photoreceptor cell death 24 h after the final dose. In contrast, neither 17-AAG nor ganetespib elicited photoreceptor injury. When the relationship between drug distribution and photoreceptor degeneration was examined, 17-DMAG and NVP-AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates, such that 51% of 17-DMAG and 65% of NVP-AUY922 present at 30 min post-injection were retained in the retina 6 h post-dose. For 17-AAG and ganetespib, retinal elimination was rapid (90% and 70% of drugs eliminated from the retina at 6 h, respectively) which correlated with lower R/P ratios. These findings indicate that prolonged inhibition of Hsp90 activity in the eye results in photoreceptor cell death. Moreover, the results suggest that the retina/plasma exposure ratio and retinal elimination rate profiles of Hsp90 inhibitors, irrespective of their chemical class, may predict for ocular toxicity potential. - Highlights: • In human trials some Hsp90 inhibitors cause visual disorders, others do not. • Prolonged inhibition of Hsp90 in the rat eye results in photoreceptor cell death. • Retina/plasma ratio and retinal

  13. Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression*

    PubMed Central

    Lin, Jeffrey J.; Hemenway, Charles S.

    2010-01-01

    AF9/MLLT3 contributes to the regulation of the gene encoding the epithelial sodium channel α, ENaCα, in renal tubular cells. Specifically, increases in AF9 protein lead to a reduction in ENaCα expression and changes in AF9 activity appear to be an important component of aldosterone signaling in the kidney. Whereas AF9 is found in the nucleus where it interacts with the histone H3 lysine 79 methyltransferase, Dot1, AF9 is also present in the cytoplasm. Data presented in this report indicate that the heat shock protein Hsp90 directly and specifically interacts with AF9 as part of an Hsp90-Hsp70-p60/Hop chaperone complex. Experimental manipulation of Hsp90 function by the inhibitor novobiocin, but not 17-AAG, results in redistribution of AF9 from a primarily nuclear to cytoplasmic location. Knockdown of Hsp90 with siRNA mimics the effect elicited by novobiocin. As expected, a shift in AF9 from the nucleus to the cytoplasm in response to Hsp90 interference leads to increased ENaCα expression. This is accompanied by a decrease in AF9 occupancy at the ENaCα promoter. Our data suggest that the interaction of Hsp90, Hsp70, and p60/Hop with AF9 is necessary for the proper subnuclear localization and activity of AF9. AF9 is among a growing number of nuclear proteins recognized to rely on the Hsp90 complex for nuclear targeting. PMID:20159978

  14. In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

    PubMed Central

    Kang, K I; Devin, J; Cadepond, F; Jibard, N; Guiochon-Mantel, A; Baulieu, E E; Catelli, M G

    1994-01-01

    In target tissue extracts, heat shock protein hsp90 has been found associated to all unliganded steroid receptors. Modulation of important functions of these receptors, including prevention of DNA binding and optimization of transcriptional activity, has been attributed to hsp90. However no unequivocal in vivo demonstration of interaction between receptors and hsp90 has been presented. We targeted chicken hsp90, a mainly cytoplasmic protein, with the nucleoplasmin nuclear localization signal (90NLS). After transfection into COS-7 cells, 90NLS was found in the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cytoplasmic. Coexpression of 90NLS and of the cytoplasmic human glucocorticosteroid receptor mutant led to complete nuclear localization of the receptor, indicating its piggyback transport by 90NLS and thus physical and functional interaction between the two proteins in the absence of hormone. The same nuclear localization was obtained after cotransfection of 90NLS and a cytoplasmic rabbit progesterone receptor mutant. Finally, coexpression of wild-type rabbit progesterone receptor (nuclear) and wildtype hsp90 (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp90 into the nucleus. These experiments lay the groundwork on which to study hsp90 as a chaperone, regulating activities of steroid receptors and possibly participating in their nuclear-cytoplasmic shuttling. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8278390

  15. Hsp90 regulates the dynamics of its cochaperone Sti1 and the transfer of Hsp70 between modules

    PubMed Central

    Röhl, Alina; Wengler, Daniela; Madl, Tobias; Lagleder, Stephan; Tippel, Franziska; Herrmann, Monika; Hendrix, Jelle; Richter, Klaus; Hack, Gordon; Schmid, Andreas B.; Kessler, Horst; Lamb, Don C.; Buchner, Johannes

    2015-01-01

    The cochaperone Sti1/Hop physically links Hsp70 and Hsp90. The protein exhibits one binding site for Hsp90 (TPR2A) and two binding sites for Hsp70 (TPR1 and TPR2B). How these sites are used remained enigmatic. Here we show that Sti1 is a dynamic, elongated protein that consists of a flexible N-terminal module, a long linker and a rigid C-terminal module. Binding of Hsp90 and Hsp70 regulates the Sti1 conformation with Hsp90 binding determining with which site Hsp70 interacts. Without Hsp90, Sti1 is more compact and TPR2B is the high-affinity interaction site for Hsp70. In the presence of Hsp90, Hsp70 shifts its preference. The linker connecting the two modules is crucial for the interaction with Hsp70 and for client activation in vivo. Our results suggest that the interaction of Hsp70 with Sti1 is tightly regulated by Hsp90 to assure transfer of Hsp70 between the modules, as a prerequisite for the efficient client handover. PMID:25851214

  16. The association of Hsp90 expression induced by aspirin with anti-stress damage in chicken myocardial cells

    PubMed Central

    Zhang, Xiao-hui; Zhu, Huai-sen; Qian, Zhuang; Tang, Shu; Wu, Di; Kemper, Nicole; Hartung, Joerg

    2016-01-01

    The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes. PMID:27051338

  17. Combination of JAK2 and HSP90 inhibitors: an effective therapeutic option in drug-resistant chronic myelogenous leukemia

    PubMed Central

    Perazzona, Bastianella; Sun, Xiaoping; Lin, Yu-Hsi; Arlinghaus, Ralph B.

    2016-01-01

    Recent studies suggest that JAK2 serves as a novel therapeutic target in Bcr-Abl+ chronic myelogenous leukemia (CML). We have reported the existence of an HSP90- associated high molecular weight network complex (HMWNC) that is composed of HSP90 client proteins BCR-ABL, JAK2, and STAT3 in wild type Bcr-Abl+ leukemic cells. Here we showed that the HSP90-HMWNC is present in leukemia cells from CML patients in blast stage, and in Imatinib (IM)-resistant 32Dp210 (T315I) leukemia cells. We found that the HSP90-HMWNC could be disassembled by depleting JAK2 with either Jak2-specific shRNA or treatment with JAK2 inhibitors (TG101209 or Ruxolitinib) and HSP90 inhibitor (AUY922). Combinational treatment with JAK2 and HSP90 inhibitors diminished the activation of BCR-ABL, JAK2 and its downstream targets. As a result, the IM-resistant 32Dp210 T315I cells underwent apoptosis. When administered in mice bearing 32Dp210 T315I leukemia, combinational therapy using Ruxolitinib and AUY922 prolonged the survival significantly. Thus, a combination of JAK2 and HSP90 inhibitors could be a powerful strategy for the treatment of CML, especially in IM-resistant patients. PMID:27551334

  18. Hsp90 regulates the dynamics of its cochaperone Sti1 and the transfer of Hsp70 between modules.

    PubMed

    Röhl, Alina; Wengler, Daniela; Madl, Tobias; Lagleder, Stephan; Tippel, Franziska; Herrmann, Monika; Hendrix, Jelle; Richter, Klaus; Hack, Gordon; Schmid, Andreas B; Kessler, Horst; Lamb, Don C; Buchner, Johannes

    2015-01-01

    The cochaperone Sti1/Hop physically links Hsp70 and Hsp90. The protein exhibits one binding site for Hsp90 (TPR2A) and two binding sites for Hsp70 (TPR1 and TPR2B). How these sites are used remained enigmatic. Here we show that Sti1 is a dynamic, elongated protein that consists of a flexible N-terminal module, a long linker and a rigid C-terminal module. Binding of Hsp90 and Hsp70 regulates the Sti1 conformation with Hsp90 binding determining with which site Hsp70 interacts. Without Hsp90, Sti1 is more compact and TPR2B is the high-affinity interaction site for Hsp70. In the presence of Hsp90, Hsp70 shifts its preference. The linker connecting the two modules is crucial for the interaction with Hsp70 and for client activation in vivo. Our results suggest that the interaction of Hsp70 with Sti1 is tightly regulated by Hsp90 to assure transfer of Hsp70 between the modules, as a prerequisite for the efficient client handover. PMID:25851214

  19. The association of Hsp90 expression induced by aspirin with anti-stress damage in chicken myocardial cells.

    PubMed

    Zhang, Xiao-Hui; Zhu, Huai-Sen; Qian, Zhuang; Tang, Shu; Wu, Di; Kemper, Nicole; Hartung, Joerg; Bao, En-Dong

    2016-03-01

    The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes. PMID:27051338

  20. HSP90α plays an important role in piRNA biogenesis and retrotransposon repression in mouse

    PubMed Central

    Ichiyanagi, Tomoko; Ichiyanagi, Kenji; Ogawa, Ayako; Kuramochi-Miyagawa, Satomi; Nakano, Toru; Chuma, Shinichiro; Sasaki, Hiroyuki; Udono, Heiichiro

    2014-01-01

    HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins. We demonstrated that HSP90α, one of two paralogs duplicated in vertebrates, plays an important role in the biogenesis of fetal PIWI-interacting RNAs (piRNA), which act against the transposon activities, in mouse male germ cells. The knockout mutation of Hsp90α resulted in a large reduction in the expression of primary and secondary piRNAs and mislocalization of MIWI2, a PIWI homolog. Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28–32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24–32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α. Although DNA methylation and mRNA levels of L1 retrotransposon were largely unchanged in the Hsp90α mutant testes, the L1-encoded protein was increased, suggesting the presence of post-transcriptional regulation. This study revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the development of male germ cells in mammals. PMID:25262350

  1. Combination of JAK2 and HSP90 inhibitors: an effective therapeutic option in drug-resistant chronic myelogenous leukemia.

    PubMed

    Chakraborty, Sandip N; Leng, Xiaohong; Perazzona, Bastianella; Sun, Xiaoping; Lin, Yu-Hsi; Arlinghaus, Ralph B

    2016-05-01

    Recent studies suggest that JAK2 serves as a novel therapeutic target in Bcr-Abl+ chronic myelogenous leukemia (CML). We have reported the existence of an HSP90- associated high molecular weight network complex (HMWNC) that is composed of HSP90 client proteins BCR-ABL, JAK2, and STAT3 in wild type Bcr-Abl+ leukemic cells. Here we showed that the HSP90-HMWNC is present in leukemia cells from CML patients in blast stage, and in Imatinib (IM)-resistant 32Dp210 (T315I) leukemia cells. We found that the HSP90-HMWNC could be disassembled by depleting JAK2 with either Jak2-specific shRNA or treatment with JAK2 inhibitors (TG101209 or Ruxolitinib) and HSP90 inhibitor (AUY922). Combinational treatment with JAK2 and HSP90 inhibitors diminished the activation of BCR-ABL, JAK2 and its downstream targets. As a result, the IM-resistant 32Dp210 T315I cells underwent apoptosis. When administered in mice bearing 32Dp210 T315I leukemia, combinational therapy using Ruxolitinib and AUY922 prolonged the survival significantly. Thus, a combination of JAK2 and HSP90 inhibitors could be a powerful strategy for the treatment of CML, especially in IM-resistant patients. PMID:27551334

  2. Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism

    PubMed Central

    Donet, M; Brassart-Pasco, S; Salesse, S; Maquart, F-X; Brassart, B

    2014-01-01

    Background: The elastin-derived peptides (EDPs) exert protumoural activities by potentiating the secretion of matrix metalloproteinases (MMP) and the plasminogen–plasmin activating system. In the present paper, we studied heat-shock protein 90 (Hsp90) involvement in this mechanism. Methods: HT-1080 fibrosarcoma cell migration and invasion were studied in artificial wound assay and modified Boyden chamber assay, respectively. Heat-shock protein 90 was studied by western blot and immunofluorescence. Matrix metalloproteinase–2 and urokinase plasminogen activator (uPA) were studied by gelatin±plasminogen zymography and immunofluorescence. Heat-shock protein 90 partners were studied by immunoprecipitation. Messenger RNA expression was studied using real-time PCR. Small interfering RNAs were used to confirm the essential role of Hsp90. Results: We showed that kappa-elastin and VGVAPG elastin hexapeptide stimulated Hsp90, pro-MMP-2 and uPA secretion within 6 h, whereas AGVPGLGVG and GRKRK peptides had no effect. No increase of mRNA level was observed. Heat-shock protein 90-specific inhibitors inhibit EDP-stimulated HT-1080 cell-invasive capacity and restrained EDP-stimulated pro-MMP-2 and uPA secretions. The inhibitory effect was reproduced by using Hsp90-blocking antibody or Hsp90 knockdown by siRNA. Heat-shock protein 90 interacted with and stabilised uPA and pro-MMP-2 in conditioned culture media of HT-1080 fibrosarcoma cells. Conclusions: Taken together, our results demonstrate that EDPs exert protumoural activities through an Hsp90-dependent mechanism involving pro-MMP-2 and uPA. PMID:24874477

  3. Hsp90 Inhibitors as New Leads To Target Parasitic Diarrheal Diseases

    PubMed Central

    Shahinas, Dea; Bryant, Clifford; Hirata, Ken; Miyamoto, Yukiko; Hwang, Grace; Gut, Jiri; Renslo, Adam R.; Pillai, Dylan R.; Eckmann, Lars; Reed, Sharon L.; McKerrow, James H.

    2014-01-01

    Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis. PMID:24820073

  4. Overexpression of GmHsp90s, a heat shock protein 90 (Hsp90) gene family cloning from soybean, decrease damage of abiotic stresses in Arabidopsis thaliana.

    PubMed

    Xu, Jinyan; Xue, Chenchen; Xue, Dong; Zhao, Jinming; Gai, Junyi; Guo, Na; Xing, Han

    2013-01-01

    Hsp90 is one of the most conserved and abundant molecular chaperones and is an essential component of the protective stress response; however, its roles in abiotic stress responses in soybean (Glycine max) remain obscure. Here, 12 GmHsp90 genes from soybean were identified and found to be expressed and to function differentially under abiotic stresses. The 12 GmHsp90 genes were isolated and named GmHsp90A1-GmHsp90A6, GmHsp90B1, GmHsp90B2, GmHsp90C1.1, GmHsp90C1.2, GmHsp90C2.1 and GmHsp90C2.2 based on their characteristics and high homology to other Hsp90s according to a new nomenclature system. Quantitative real-time PCR expression data revealed that all the genes exhibited higher transcript levels in leaves and could be strongly induced under heat, osmotic and salt stress but not cold stress. Overexpression of five typical genes (GmHsp90A2, GmHsp90A4, GmHsp90B1, GmHsp90C1.1 and GmHsp90C2.1) in Arabidopsis thaliana provided useful evidences that GmHsp90 genes can decrease damage of abiotic stresses. In addition, an abnormal accumulation of proline was detected in some transgenic Arabidopsis plants suggested overexpressing GmHsp90s may affect the synthesis and response system of proline. Our work represents a systematic determination of soybean genes encoding Hsp90s, and provides useful evidence that GmHsp90 genes function differently in response to abiotic stresses and may affect the synthesis and response system of proline. PMID:23936107

  5. Modulation of α2C adrenergic receptor temperature-sensitive trafficking by HSP90

    PubMed Central

    Filipeanu, Catalin M.; de Vries, René; Danser, A.H. Jan; Kapusta, Daniel R.

    2010-01-01

    Decreasing the temperature to 30°C is accompanied by significant enhancement of α2C-AR plasma membrane levels in several cell lines with fibroblast phenotype, as demonstrated by radioligand binding in intact cells or isolated membranes. No changes were observed on the effects of low-temperature after blocking receptor internalization in α2C-AR transfected HEK293T cells. In contrast, two pharmacological chaperones, dimethyl sulfoxide and glycerol, increased the cell surface receptor levels at 37°C, but not at 30°C. Further, at 37°C α2C-AR is co-localized with endoplasmic reticulum markers, but not with the lysosomal markers. Treatment with three distinct HSP90 inhibitors, radicicol, macbecin and 17-DMAG significantly enhanced α2C-AR cell surface levels at 37°C, but these inhibitors had no effect at 30°C. Similar results were obtained after decreasing the HSP90 cellular levels using specific siRNA. Co-immunoprecipitation experiments demonstrated that α2C-AR interacts with HSP90 and this interaction is decreased at 30°C. The contractile response to endogenous α2C-AR stimulation in rat tail artery was also enhanced at reduced temperature. Similar to HEK293T cells, HSP90 inhibition increased the α2C-AR contractile effects only at 37°C. Moreover, exposure to low-temperature of vascular smooth muscle cells from rat tail artery decreased the cellular levels of HSP90, but did not change HSP70 levels. These data demonstrate that exposure to low-temperature augments the α2C-AR transport to the plasma membrane by releasing the inhibitory activity of HSP90 on the receptor traffic, findings which may have clinical relevance for the diagnostic and treatment of Raynaud Phenomenon. PMID:21145921

  6. Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators.

    PubMed

    Hieronymus, Haley; Lamb, Justin; Ross, Kenneth N; Peng, Xiao P; Clement, Cristina; Rodina, Anna; Nieto, Maria; Du, Jinyan; Stegmaier, Kimberly; Raj, Srilakshmi M; Maloney, Katherine N; Clardy, Jon; Hahn, William C; Chiosis, Gabriela; Golub, Todd R

    2006-10-01

    Although androgen receptor (AR)-mediated signaling is central to prostate cancer, the ability to modulate AR signaling states is limited. Here we establish a chemical genomic approach for discovery and target prediction of modulators of cancer phenotypes, as exemplified by AR signaling. We first identify AR activation inhibitors, including a group of structurally related compounds comprising celastrol, gedunin, and derivatives. To develop an in silico approach for target pathway identification, we apply a gene expression-based analysis that classifies HSP90 inhibitors as having similar activity to celastrol and gedunin. Validating this prediction, we demonstrate that celastrol and gedunin inhibit HSP90 activity and HSP90 clients, including AR. Broadly, this work identifies new modes of HSP90 modulation through a gene expression-based strategy. PMID:17010675

  7. Mutations Y493G and K546D in human HSP90 disrupt binding of celastrol and reduce interaction with Cdc37.

    PubMed

    Peng, Bin; Gu, Yi-Jun; Wang, Ying; Cao, Fan-Fan; Zhang, Xue; Zhang, Deng-Hai; Hou, Jian

    2016-07-01

    Celastrol, a natural compound derived from the Chinese herb Tripterygium wilfordii Hook F, has been proven to inhibit heat shock protein 90 (HSP90) activity and has attracted much attention because of its promising effects in cancer treatment and in ameliorating degenerative neuron diseases. However, the HSP90 structure involved in celastrol interaction is not known. Here, we report a novel celastrol-binding pocket in the HSP90 dimer, predicted by molecular docking. Mutation of the two key binding pocket amino acids (Lys546 and Tyr493) disrupted the binding of celastrol to HSP90 dimers, as detected by isothermal titration calorimetry (ITC). Interestingly, such mutations also reduced binding between HSP90 and the cochaperone Cdc37, thus providing a new explanation for reported findings that celastrol shows more obvious effects in disrupting binding between HSP90 and Cdc37 than between HSP90 and other cochaperones. In short, our work discloses a novel binding pocket in HSP90 dimer for celastrol and provides an explanation as to why celastrol has a strong effect on HSP90 and Cdc37 binding. PMID:27398312

  8. Molecular mechanism of cytotoxicity induced by Hsp90-targeted Antp-TPR hybrid peptide in glioblastoma cells

    PubMed Central

    2012-01-01

    Background Heat-shock protein 90 (Hsp90) is vital to cell survival under conditions of stress, and binds client proteins to assist in protein stabilization, translocation of polypeptides across cell membranes, and recovery of proteins from aggregates. Therefore, Hsp90 has emerged as an important target for the treatment of cancer. We previously reported that novel Antp-TPR hybrid peptide, which can inhibit the interaction of Hsp90 with the TPR2A domain of Hop, induces selective cytotoxic activity to discriminate between normal and cancer cells both in vitro and in vivo. Results In this study, we investigated the functional cancer-cell killing mechanism of Antp-TPR hybrid peptide in glioblastoma (GB) cell lines. It was demonstrated that Antp-TPR peptide induced effective cytotoxic activity in GB cells through the loss of Hsp90 client proteins such as p53, Akt, CDK4, and cRaf. Antp-TPR also did not induce the up-regulation of Hsp70 and Hsp90 proteins, although a small-molecule inhibitor of Hsp90, 17-AAG, induced the up-regulation of these proteins. It was also found that Antp-TPR peptide increased the endoplasmic reticulum unfolded protein response, and the cytotoxic activity of this hybrid peptide to GB cells in the endoplasmic reticulum stress condition. Conclusion These results show that targeting of Hsp90 by Antp-TPR could be an attractive approach to selective cancer-cell killing because no other Hsp90-targeted compounds show selective cytotoxic activity. Antp-TPR might provide potent and selective therapeutic options for the treatment of cancer. PMID:22913813

  9. Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90.

    PubMed

    Averna, Monica; Stifanese, Roberto; De Tullio, Roberta; Salamino, Franca; Bertuccio, Mara; Pontremoli, Sandro; Melloni, Edon

    2007-12-01

    Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced. PMID:17970747

  10. Novel 1,6-naphthyridin-2(1H)-ones as potential anticancer agents targeting Hsp90.

    PubMed

    Montoir, David; Barillé-Nion, Sophie; Tonnerre, Alain; Juin, Philippe; Duflos, Muriel; Bazin, Marc-Antoine

    2016-08-25

    Hsp90 is an ATP-dependent chaperone known to be overexpressed in many cancers. This way, Hsp90 is an important target for drug discovery. Novobiocin, an aminocoumarin antibiotic, was reported to inhibit Hsp90 targeting C-terminal domain, and showed anti-proliferative properties, leading to the development of new and more active compounds. Consequently, a new set of novobiocin analogs derived from 1,6-naphthyridin-2(1H)-one scaffold was designed, synthesized and evaluated against two breast cancer cell lines. Subsequently, cell cycle progression and apoptosis were conducted on best candidates, finally Western Blot analysis was performed to measure their ability to induce degradation of Hsp90 client proteins. PMID:27153346

  11. A large animal model to evaluate the effects of Hsp90 inhibitors for the treatment of lung adenocarcinoma

    SciTech Connect

    Varela, Mariana; Golder, Matthew; Archer, Fabienne; Heras, Marcelo de las; Leroux, Caroline; Palmarini, Massimo

    2008-02-05

    Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The JSRV envelope glycoprotein (Env) functions as a dominant oncoprotein in vitro and in vivo. In order to develop the basis for the use of OPA as a lung cancer model, we screened a variety of signal transduction inhibitors for their ability to block transformation by the JSRV Env. Most inhibitors were not effective in blocking JSRV Env-induced transformation. On the contrary, various Hsp90 inhibitors efficiently blocked JSRV transformation. This phenomenon was at least partly due to Akt degradation, which is activated in JSRV-transformed cells. Hsp90 was found expressed in tumor cells of sheep with naturally occurring OPA. In addition, Hsp90 inhibitors specifically inhibited proliferation of immortalized and moreover primary cells derived from OPA tumors. Thus, OPA could be used as a large animal model for comprehensive studies investigating the effects of Hsp90 inhibitors in lung adenocarcinoma.

  12. Modeling Signal Propagation Mechanisms and Ligand-Based Conformational Dynamics of the Hsp90 Molecular Chaperone Full-Length Dimer

    PubMed Central

    Morra, Giulia; Verkhivker, Gennady; Colombo, Giorgio

    2009-01-01

    Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine “hot spots” involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a “conformational selection model” of the Hsp90 mechanism, whereby the protein may exist in a

  13. Second-Generation HSP90 Inhibitor Onalespib Blocks mRNA Splicing of Androgen Receptor Variant 7 in Prostate Cancer Cells.

    PubMed

    Ferraldeschi, Roberta; Welti, Jonathan; Powers, Marissa V; Yuan, Wei; Smyth, Tomoko; Seed, George; Riisnaes, Ruth; Hedayat, Somaieh; Wang, Hannah; Crespo, Mateus; Nava Rodrigues, Daniel; Figueiredo, Ines; Miranda, Susana; Carreira, Suzanne; Lyons, John F; Sharp, Swee; Plymate, Stephen R; Attard, Gerhardt; Wallis, Nicola; Workman, Paul; de Bono, Johann S

    2016-05-01

    Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731-42. ©2016 AACR. PMID:27197266

  14. A Repurposing Strategy for Hsp90 Inhibitors Demonstrates Their Potency against Filarial Nematodes

    PubMed Central

    Gillan, Victoria; O'Neill, Kerry; Maitland, Kirsty; Sverdrup, Francis M.; Devaney, Eileen

    2014-01-01

    Novel drugs are required for the elimination of infections caused by filarial worms, as most commonly used drugs largely target the microfilariae or first stage larvae of these infections. Previous studies, conducted in vitro, have shown that inhibition of Hsp90 kills adult Brugia pahangi. As numerous small molecule inhibitors of Hsp90 have been developed for use in cancer chemotherapy, we tested the activity of several novel Hsp90 inhibitors in a fluorescence polarization assay and against microfilariae and adult worms of Brugia in vitro. The results from all three assays correlated reasonably well and one particular compound, NVP-AUY922, was shown to be particularly active, inhibiting Mf output from female worms at concentrations as low as 5.0 nanomolar after 6 days exposure to drug. NVP-AUY922 was also active on adult worms after a short 24 h exposure to drug. Based on these in vitro data, NVP-AUY922 was tested in vivo in a mouse model and was shown to significantly reduce the recovery of both adult worms and microfilariae. These studies provide proof of principle that the repurposing of currently available Hsp90 inhibitors may have potential for the development of novel agents with macrofilaricidal properties. PMID:24551261

  15. Current Understanding of HSP90 as a Novel Therapeutic Target: An Emerging Approach for the Treatment of Cancer.

    PubMed

    Haque, Absarul; Alam, Qamre; Alam, Mohammad Zubair; Azhar, Esam I; Sait, Khalid Hussain Wali; Anfinan, Nisrin; Mushtaq, Gohar; Kamal, Mohammad Amjad; Rasool, Mahmood

    2016-01-01

    Heat Shock Protein 90 (HSP90) is a ubiquitous molecular chaperone that is considered to be the most abundantly expressed protein in various human cancers such as breast, lung, colon, prostate, leukemia and skin. The master regulator, HSP90 plays a pivotal role in the conformational stabilization, maturation and activity of its various labile oncogenic client proteins such as p53, ErbB2, Bcr-Abl, Akt, Her-2, Cdk4, Cdk6, Raf-1 and v-Src in altered cells. Hence, making a guaranteed attempt to inhibit such a master regulator for cancer therapy appears to be a potential approach for combinatorial inhibition of numerous oncogenic signaling pathways simultaneously. Considerable efforts are being under way to develop novel molecular targets and its inhibitors that may block key signaling pathways involved in the process of tumorigenesis and metastasis. In this regards, HSP90 has acquired immense interest as a potent anticancer drug-target due to its key functional link with multiple signaling pathways involved in the process of cell proliferation and cell survival. Notably, geldanamycin and its derivatives (17-AAG, 17-DMAG) have shown quite encouraging results in inhibiting HSP90 function in several cancers and currently almost 17 drug candidates known to be target HSP90 are being under clinical trials either as single agents or combinatorial therapy. Hence, this review is an attempt to get new insight into novel drug target therapy by focusing on recent advances made in understanding HSP90 chaperone structure-function relationships, identification of new HSP90 client proteins and, more importantly, on the advancements of HSP90 targeted therapy based on various existing and emerging classical inhibitors. PMID:27013225

  16. Hsp90aa1: a novel target gene of miR-1 in cardiac ischemia/reperfusion injury

    PubMed Central

    Zhu, Wen Si; Guo, Wei; Zhu, Jie Ning; Tang, Chun Mei; Fu, Yong Heng; Lin, Qiu Xiong; Tan, Ning; Shan, Zhi Xin

    2016-01-01

    The role of microRNA-1 (miR-1) in ischemia/reperfusion (I/R)-induced injury is not well illustrated. The present study aimed to investigate the expression and potential target of miR-1 in the myocardium of a rat model of I/R. The apoptosis of cardiomyocytes in the ischemic rat myocardium increased on day 1, then attenuated on day 3 and day 7 post-I/R. Heat shot protein 90 (Hsp90) aa1 mRNA expression was decreased post-I/R, and Hsp90aa1 protein level was decreased on day1 post-I/R, but was reversed on day 3 and day 7 post-I/R. MiR-1 was downregulated post-I/R, and repression of miR-1 in cultured neonatal rat ventricular cells (NRVCs) led to an increase of Bcl-2 and decreases of Bax and active caspase-3. Dual luciferase reporter assays revealed that miR-1 interacted with the 310–315 nt site at the 3′UTR of Hsp90aa1, and miR-1 was verified to inhibit Hsp90aa1 expression at the posttranscriptional level. Over-expression of Hsp90aa1 could attenuate oxygen-glucose deprivation (OGD)-induced apoptosis of NRVCs. Additionally, miR-1 mimic, in parallel to Hsp90aa1 siRNA, could enhance OGD-induced apoptosis of NRVCs. Taken together, our results reveal that Hsp90aa1 is a novel target of miR-1, and repression of miR-1 may contribute to the recovery of Hsp90aa1 during myocardial I/R. PMID:27076094

  17. Ascorbic acid inhibition of Candida albicans Hsp90-mediated morphogenesis occurs via the transcriptional regulator Upc2.

    PubMed

    Van Hauwenhuyse, Frédérique; Fiori, Alessandro; Van Dijck, Patrick

    2014-10-01

    Morphogenetic transitions of the opportunistic fungal pathogen Candida albicans are influenced by temperature changes, with induction of filamentation upon a shift from 30 to 37°C. Hsp90 was identified as a major repressor of an elongated cell morphology at low temperatures, as treatment with specific inhibitors of Hsp90 results in elongated growth forms at 30°C. Elongated growth resulting from a compromised Hsp90 is considered neither hyphal nor pseudohyphal growth. It has been reported that ascorbic acid (vitamin C) interferes with the yeast-to-hypha transition in C. albicans. In the present study, we show that ascorbic acid also antagonizes the morphogenetic change caused by hampered Hsp90 function. Further analysis revealed that Upc2, a transcriptional regulator of genes involved in ergosterol biosynthesis, and Erg11, the target of azole antifungals, whose expression is in turn regulated by Upc2, are required for this antagonism. Ergosterol levels correlate with elongated growth and are reduced in cells treated with the Hsp90 inhibitor geldanamycin (GdA) and restored by cotreatment with ascorbic acid. In addition, we show that Upc2 appears to be required for ascorbic acid-mediated inhibition of the antifungal activity of fluconazole. These results identify Upc2 as a major regulator of ascorbic acid-induced effects in C. albicans and suggest an association between ergosterol content and elongated growth upon Hsp90 compromise. PMID:25084864

  18. HSP90 and HSP70: Implication in Inflammation Processes and Therapeutic Approaches for Myeloproliferative Neoplasms

    PubMed Central

    Sevin, Margaux; Girodon, François; Garrido, Carmen; de Thonel, Aurélie

    2015-01-01

    Myeloproliferative neoplasms (MPN) are clonal stem cell disorders that lead to the excessive production of one or more blood cell lineages. It has been reported that, in most MPN, inflammatory cytokines are frequently increased, indicating that inflammation plays a crucial role in these disorders. Heat shock proteins (HSP) are induced in response to many stressful conditions from heat shock to hypoxia and inflammation. Besides their chaperone and cytoprotective functions, HSPs are key players during inflammation, hence the term “chaperokine.” Through their chaperone activity, HSP90, a stabilizer of many oncogenes (e.g., JAK2), and HSP70, a powerful antiapoptotic chaperone, tightly regulate Nuclear Factor-kappa B signalling, a critical pathway in mediating inflammatory responses. In light of this potential, several HSP90 inhibitors have been generated as anticancer agents able to degrade oncogenes. As it turns out, however, these drugs are also potent inhibitors of the inflammatory response in various diseases. Given the chaperone potential of HSP70 and the fact that HSP90 inhibitors induce HSP70, interest in HSP70 inhibitors is also increasing. Here, we focus on the implication of HSP90 and HSP70 in inflammatory responses and on the emergence of new therapeutic approaches in MPN based on HSP inhibitors. PMID:26549943

  19. HSP90 is a therapeutic target in JAK2-dependent myeloproliferative neoplasms in mice and humans

    PubMed Central

    Marubayashi, Sachie; Koppikar, Priya; Taldone, Tony; Abdel-Wahab, Omar; West, Nathan; Bhagwat, Neha; Caldas-Lopes, Eloisi; Ross, Kenneth N.; Gönen, Mithat; Gozman, Alex; Ahn, James H.; Rodina, Anna; Ouerfelli, Ouathek; Yang, Guangbin; Hedvat, Cyrus; Bradner, James E.; Chiosis, Gabriela; Levine, Ross L.

    2010-01-01

    JAK2 kinase inhibitors were developed for the treatment of myeloproliferative neoplasms (MPNs), following the discovery of activating JAK2 mutations in the majority of patients with MPN. However, to date JAK2 inhibitor treatment has shown limited efficacy and apparent toxicities in clinical trials. We report here that an HSP90 inhibitor, PU-H71, demonstrated efficacy in cell line and mouse models of the MPN polycythemia vera (PV) and essential thrombocytosis (ET) by disrupting JAK2 protein stability. JAK2 physically associated with both HSP90 and PU-H71 and was degraded by PU-H71 treatment in vitro and in vivo, demonstrating that JAK2 is an HSP90 chaperone client. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth and signaling in JAK2 mutant cell lines and in primary MPN patient samples. PU-H71 treatment of mice resulted in JAK2 degradation, inhibition of JAK-STAT signaling, normalization of peripheral blood counts, and improved survival in MPN models at doses that did not degrade JAK2 in normal tissues or cause substantial toxicity. Importantly, PU-H71 treatment also reduced the mutant allele burden in mice. These data establish what we believe to be a novel therapeutic rationale for HSP90 inhibition in the treatment of JAK2-dependent MPN. PMID:20852385

  20. Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70.

    PubMed

    Carrello, Amerigo; Allan, Rudi K; Morgan, Sarah L; Owen, Barbara A L; Mok, Danny; Ward, Bryan K; Minchin, Rodney F; Toft, David O; Ratajczak, Thomas

    2004-01-01

    triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function. PMID:15497503

  1. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma

    PubMed Central

    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D Ashley; Dome, Jeffrey S

    2015-01-01

    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of γH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p = 0.045, alvespimycin, p = 0.034; combined treatment, p = 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy. PMID:25920748

  2. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma.

    PubMed

    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D Ashley; Dome, Jeffrey S

    2015-01-01

    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of γH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p = 0.045, alvespimycin, p = 0.034; combined treatment, p = 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy. PMID:25920748

  3. Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation

    PubMed Central

    Hubert, David A.; He, Yijian; McNulty, Brian C.; Tornero, Pablo; Dangl, Jeffery L.

    2009-01-01

    Both plants and animals require the activity of proteins containing nucleotide binding (NB) domain and leucine-rich repeat (LRR) domains for proper immune system function. NB-LRR proteins in plants (NLR proteins in animals) also require conserved regulation via the proteins SGT1 and cytosolic HSP90. RAR1, a protein specifically required for plant innate immunity, interacts with SGT1 and HSP90 to maintain proper NB-LRR protein steady-state levels. Here, we present the identification and characterization of specific mutations in Arabidopsis HSP90.2 that suppress all known phenotypes of rar1. These mutations are unique with respect to the many mutant alleles of HSP90 identified in all systems in that they can bypass the requirement for a cochaperone and result in the recovery of client protein accumulation and function. Additionally, these mutations separate HSP90 ATP hydrolysis from HSP90 function in client protein folding and/or accumulation. By recapitulating the activity of RAR1, these novel hsp90 alleles allow us to propose that RAR1 regulates the physical open–close cycling of a known “lid structure” that is used as a dynamic regulatory HSP90 mechanism. Thus, in rar1, lid cycling is locked into a conformation favoring NB-LRR client degradation, likely via SGT1 and the proteasome. PMID:19487680

  4. The soluble recombinant Neisseria meningitidis adhesin NadA(Δ351-405) stimulates human monocytes by binding to extracellular Hsp90.

    PubMed

    Cecchini, Paola; Tavano, Regina; Polverino de Laureto, Patrizia; Franzoso, Susanna; Mazzon, Cristina; Montanari, Paolo; Papini, Emanuele

    2011-01-01

    The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadA(Δ351-405,) devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadA(Δ351-405) cellular effects in monocytes. We show that NadA(Δ351-405) (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadA(Δ351-405) cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadA(Δ351-405) /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadA(Δ351-405) and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadA(Δ351-405) determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadA(Δ351-405) alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by

  5. HSP90 regulates temperature-dependent seedling growth in Arabidopsis by stabilizing the auxin co-receptor F-box protein TIR1

    PubMed Central

    Wang, Renhou; Zhang, Yi; Kieffer, Martin; Yu, Hong; Kepinski, Stefan; Estelle, Mark

    2016-01-01

    Recent studies have revealed that a mild increase in environmental temperature stimulates the growth of Arabidopsis seedlings by promoting biosynthesis of the plant hormone auxin. However, little is known about the role of other factors in this process. In this report, we show that increased temperature promotes rapid accumulation of the TIR1 auxin co-receptor, an effect that is dependent on the molecular chaperone HSP90. In addition, we show that HSP90 and the co-chaperone SGT1 each interact with TIR1, confirming that TIR1 is an HSP90 client. Inhibition of HSP90 activity results in degradation of TIR1 and interestingly, defects in a range of auxin-mediated growth processes at lower as well as higher temperatures. Our results indicate that HSP90 and SGT1 integrate temperature and auxin signalling in order to regulate plant growth in a changing environment. PMID:26728313

  6. Hsp90 inhibitors reduce influenza virus replication in cell culture

    SciTech Connect

    Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin Brownlee, George

    2008-08-01

    The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses.

  7. Nematode Hsp90: highly conserved but functionally diverse.

    PubMed

    Gillan, Victoria; Devaney, Eileen

    2014-08-01

    Nematodes are amongst the most successful and abundant organisms on the planet with approximately 30 000 species described, although the actual number of species is estimated to be one million or more. Despite sharing a relatively simple and invariant body plan, there is considerable diversity within the phylum. Nematodes have evolved to colonize most ecological niches, and can be free-living or can parasitize plants or animals to the detriment of the host organism. In this review we consider the role of heat shock protein 90 (Hsp90) in the nematode life cycle. We describe studies on Hsp90 in the free-living nematode Caenorhabditis elegans and comparative work on the parasitic species Brugia pahangi, and consider whether a dependence upon Hsp90 can be exploited for the control of parasitic species. PMID:24721950

  8. Waddington's widget: Hsp90 and the inheritance of acquired characters.

    PubMed

    Ruden, Douglas M; Garfinkel, Mark D; Sollars, Vincent E; Lu, Xiangyi

    2003-10-01

    Conrad Waddington published an influential model for evolution in his 1942 paper, Canalization of Development and Inheritance of Acquired Characters. In this classic, albeit controversial, paper, he proposed that an unknown mechanism exists that conceals phenotypic variation until the organism is stressed. Recent studies have proposed that the highly conserved chaperone Hsp90 could function as a "capacitor," or an "adaptively inducible canalizer," that masks silent phenotypic variation of either genetic or epigenetic origin. This review will discuss evidence for, and arguments against, the role of Hsp90 as a capacitor for morphological evolution, and as a key component of what we call "Waddington's widget." PMID:14986860

  9. Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90.

    PubMed

    Sydor, Jens R; Normant, Emmanuel; Pien, Christine S; Porter, James R; Ge, Jie; Grenier, Louis; Pak, Roger H; Ali, Janid A; Dembski, Marlene S; Hudak, Jebecka; Patterson, Jon; Penders, Courtney; Pink, Melissa; Read, Margaret A; Sang, Jim; Woodward, Caroline; Zhang, Yilong; Grayzel, David S; Wright, Jim; Barrett, John A; Palombella, Vito J; Adams, Julian; Tong, Jeffrey K

    2006-11-14

    Heat shock protein 90 (Hsp90) is an emerging therapeutic target of interest for the treatment of cancer. Its role in protein homeostasis and the selective chaperoning of key signaling proteins in cancer survival and proliferation pathways has made it an attractive target of small molecule therapeutic intervention. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), the most studied agent directed against Hsp90, suffers from poor physical-chemical properties that limit its clinical potential. Therefore, there exists a need for novel, patient-friendly Hsp90-directed agents for clinical investigation. IPI-504, the highly soluble hydroquinone hydrochloride derivative of 17-AAG, was synthesized as an Hsp90 inhibitor with favorable pharmaceutical properties. Its biochemical and biological activity was profiled in an Hsp90-binding assay, as well as in cancer-cell assays. Furthermore, the metabolic profile of IPI-504 was compared with that of 17-AAG, a geldanamycin analog currently in clinical trials. The anti-tumor activity of IPI-504 was tested as both a single agent as well as in combination with bortezomib in myeloma cell lines and in vivo xenograft models, and the retention of IPI-504 in tumor tissue was determined. In conclusion, IPI-504, a potent inhibitor of Hsp90, is efficacious in cellular and animal models of myeloma. It is synergistically efficacious with the proteasome inhibitor bortezomib and is preferentially retained in tumor tissues relative to plasma. Importantly, it was observed that IPI-504 interconverts with the known agent 17-AAG in vitro and in vivo via an oxidation-reduction equilibrium, and we demonstrate that IPI-504 is the slightly more potent inhibitor of Hsp90. PMID:17090671

  10. Inhibition of HIV-1 reactivation by a telomerase-derived peptide in a HSP90-dependent manner

    PubMed Central

    Kim, Hong; Choi, Myung-Soo; Inn, Kyung-Soo; Kim, Bum-Joon

    2016-01-01

    A peptide vaccine designed to induce T-cell immunity to telomerase, GV1001, has been shown to modulate cellular signaling pathways and confer a direct anti-cancer effect through the interaction with heat shock protein (HSP) 90 and 70. Here, we have found that GV1001 can modulate transactivation protein-mediated human immunodeficiency virus (HIV)-1 transactivation in an HSP90-dependent manner. GV1001 treatment resulted in significant suppression of HIV-1 replication and rescue of infected cells from death by HIV-1. Transactivation of HIV-long terminal repeat (LTR) was inhibited by GV1001, indicating that GV1001 suppressed the transcription from proviral HIV DNA. The anti-HIV-1 activity of GV1001 was completely abrogated by an HSP90-neutralizing antibody, indicating that the antiviral activity depends on HSP90. Further mechanistic studies revealed that GV1001 suppresses basal NF-κB activation, which is required for HIV-1 LTR transactivation in an HSP90-dependent manner. Inhibition of LTR transactivation by GV1001 suggests its potential to suppress HIV-1 reactivation from latency. Indeed, PMA-mediated reactivation of HIV-1 from latent infected cells was suppressed by GV1001. The results suggest the potential therapeutic use of GV1001, a peptide proven to be safe for human use, as an anti-HIV-1 agent to suppress the reactivation from latently infected cells. PMID:27363520

  11. Inhibition of HIV-1 reactivation by a telomerase-derived peptide in a HSP90-dependent manner.

    PubMed

    Kim, Hong; Choi, Myung-Soo; Inn, Kyung-Soo; Kim, Bum-Joon

    2016-01-01

    A peptide vaccine designed to induce T-cell immunity to telomerase, GV1001, has been shown to modulate cellular signaling pathways and confer a direct anti-cancer effect through the interaction with heat shock protein (HSP) 90 and 70. Here, we have found that GV1001 can modulate transactivation protein-mediated human immunodeficiency virus (HIV)-1 transactivation in an HSP90-dependent manner. GV1001 treatment resulted in significant suppression of HIV-1 replication and rescue of infected cells from death by HIV-1. Transactivation of HIV-long terminal repeat (LTR) was inhibited by GV1001, indicating that GV1001 suppressed the transcription from proviral HIV DNA. The anti-HIV-1 activity of GV1001 was completely abrogated by an HSP90-neutralizing antibody, indicating that the antiviral activity depends on HSP90. Further mechanistic studies revealed that GV1001 suppresses basal NF-κB activation, which is required for HIV-1 LTR transactivation in an HSP90-dependent manner. Inhibition of LTR transactivation by GV1001 suggests its potential to suppress HIV-1 reactivation from latency. Indeed, PMA-mediated reactivation of HIV-1 from latent infected cells was suppressed by GV1001. The results suggest the potential therapeutic use of GV1001, a peptide proven to be safe for human use, as an anti-HIV-1 agent to suppress the reactivation from latently infected cells. PMID:27363520

  12. Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

    PubMed

    Iki, Taichiro; Yoshikawa, Manabu; Meshi, Tetsuo; Ishikawa, Masayuki

    2012-01-18

    Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants. PMID:22045333

  13. Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants

    PubMed Central

    Iki, Taichiro; Yoshikawa, Manabu; Meshi, Tetsuo; Ishikawa, Masayuki

    2012-01-01

    Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90–AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants. PMID:22045333

  14. Synthesis and Biological Evaluation of Novobiocin Core Analogues as Hsp90 Inhibitors.

    PubMed

    Byrd, Katherine M; Subramanian, Chitra; Sanchez, Jacqueline; Motiwala, Hashim F; Liu, Weiya; Cohen, Mark S; Holzbeierlein, Jeffrey; Blagg, Brian S J

    2016-05-10

    Development of heat shock protein 90 (Hsp90) C-terminal inhibitors has emerged as an exciting strategy for the treatment of cancer. Previous efforts have focused on modifications to the natural products novobiocin and coumermycin. Moreover, variations in both the sugar and amide moieties have been extensively studied, whereas replacements for the coumarin core have received less attention. Herein, 24 cores were synthesized with varying distances and angles between the sugar and amide moieties. Compounds that exhibited good anti-proliferative activity against multiple cancer cell lines and Hsp90 inhibitory activity, were those that placed the sugar and amide moieties between 7.7 and 12.1 Å apart along with angles of 180°. PMID:27037933

  15. BIIB021, a synthetic Hsp90 inhibitor, has broad application against tumors with acquired multidrug resistance.

    PubMed

    Zhang, Hong; Neely, Laura; Lundgren, Karen; Yang, Yong-Ching; Lough, Rachel; Timple, Noel; Burrows, Francis

    2010-03-01

    17-AAG, the first-generation clinical Hsp90 inhibitor, exhibits promising antitumor activity in clinical studies, but is limited by poor solubility and hepatotoxicity. To pursue compounds with better biopharmaceutical properties, we have developed a series of fully synthetic orally bioavailable inhibitors of Hsp90. Here, we report that 17-AAG and other ansamycin derivatives are inactive in P-gp and/or MRP-1 expressing cell lines and sensitivity could be restored by coadministration of P-gp or MRP inhibitors. In contrast, the synthetic Hsp90 inhibitor, BIIB021 was active in these models. Accordingly, BIIB021 was considerably more active than 17-AAG against adrenocortical carcinoma, a tumor that naturally expresses P-gp, both in vitro and in vivo. This efflux pump-mediated resistance is manifested in both cytotoxicity assays and measurements of target inhibition, such as client protein degradation. Other than this, the cytotoxic activity of BIIB021 was also not influenced by loss of NQO1 or Bcl-2 overexpression, molecular lesions that do not prevent client loss but are nonetheless associated with reduced cell killing by 17-AAG. Our results indicate that the activity of 17-AAG and other ansamycins may be curtailed in tumors that have upregulated efflux pumps or antiapoptotic proteins or other genetic alterations. These data indicate that the new generation of synthetic anti-Hsp90 drugs, exemplified by BIIB021 that is currently undergoing Phase II testing, may have broader application against tumors with acquired multidrug resistance or tumors located in organs protected by MDR proteins, such as the adrenal glands, brain and testis. PMID:19676042

  16. Hsp90 induces increased genomic instability toward DNA-damaging agents by tuning down RAD53 transcription.

    PubMed

    Khurana, Nidhi; Laskar, Shyamasree; Bhattacharyya, Mrinal K; Bhattacharyya, Sunanda

    2016-08-01

    It is well documented that elevated body temperature causes tumors to regress upon radiotherapy. However, how hyperthermia induces DNA damage sensitivity is not clear. We show that a transient heat shock and particularly the concomitant induction of Hsp90 lead to increased genomic instability under DNA-damaging conditions. Using Saccharomyces cerevisiae as a model eukaryote, we demonstrate that elevated levels of Hsp90 attenuate efficient DNA damage signaling and dictate preferential use of the potentially mutagenic double-strand break repair pathway. We show that under normal physiological conditions, Hsp90 negatively regulates RAD53 transcription to suppress DNA damage checkpoint activation. However, under DNA damaging conditions, RAD53 is derepressed, and the increased level of Rad53p triggers an efficient DNA damage response. A higher abundance of Hsp90 causes increased transcriptional repression on RAD53 in a dose-dependent manner, which could not be fully derepressed even in the presence of DNA damage. Accordingly, cells behave like a rad53 loss-of-function mutant and show reduced NHEJ efficiency, with a drastic failure to up-regulate RAD51 expression and manifestly faster accumulation of CLN1 and CLN2 in DNA-damaged G1, cells leading to premature release from checkpoint arrest. We further demonstrate that Rad53 overexpression is able to rescue all of the aforementioned deleterious effects caused by Hsp90 overproduction. PMID:27307581

  17. Expression of hsp90 mediates cytoprotective effects in the gastrodermis of planarians

    PubMed Central

    Conte, Maria; Isolani, Maria Emilia; Deri, Paolo; Mannini, Linda

    2010-01-01

    Heat shock proteins (HSPs) play a crucial role in the protection of cells. In the present study, we have identified an hsp90-related gene (Djhsp90) encoding a cytosolic form of HSP90 that is primarily expressed in gastrodermis of the planarian Dugesia japonica. Djhsp90 becomes significantly induced after traumatic amputation or other stress stimuli, such as exposure to X-ray or ultraviolet radiations, heat shock, or prolonged starvation. When Djhsp90 is silenced by ribonucleic acid interference (RNAi), planarians dramatically decrease in size, becoming unable to eat, and die in a few weeks. Our results indicate that this gene plays an essential cytoprotective role in the gastrodermis of planarians and suggest that this chaperone can be involved in autophagic processes that are activated by this tissue. PMID:20706815

  18. 83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases.

    PubMed Central

    Nadeau, K.; Sullivan, M. A.; Bradley, M.; Engman, D. M.; Walsh, C. T.

    1992-01-01

    A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids. PMID:1304385

  19. Treatment of Arabidopsis thaliana seeds with an HSP90 inhibitor increases plant resistance

    NASA Astrophysics Data System (ADS)

    Kozeko, Liudmyla

    2016-07-01

    Resistance of plants to unfavourable conditions is an important feature to use them as an autotrophic link of Life Support Systems in space exploration missions. It significantly depends on basic and stress-induced levels of heat shock proteins (HSP) in cells. It is known that HSP90 can bind and maintain heat shock transcription factors (HSF) as a monomer that lacks DNA binding activity and thereby regulate HSP expression. Modulation of activity of the HSP synthesis and resistance by HSP90 in plants is not well investigated. The objective of this study was to determine how treatment of seeds with an HSP90 inhibitor affects environmental responsiveness in Arabidopsis thaliana. Seed treatment with geldanamycin (GDA) was used to reduce HSP90 function. The affect of space flight stressors was simulated by gamma-irradiation and thermal upshift. Two series of experiments were carried out: 1) exposure of dry seeds to gamma-irradiation (1 kGy, ^{60}Co); 2) heat shock of seedlings. It was shown that GDA treatment of seeds stimulated the seedling growth after seed irradiation. It also increased both the basic thermotolerance (45°C for 45 min) and induced thermotolerance (45°C for 1,5-2,5 h after pretreatment at 37°C for 2 h) in seedlings. In addition, seed treatment with GDA had a prolonged effect on the HSP70 production in seedlings under normal and stressful conditions. It shows that the stimulatory effects of GDA may be caused by induction of HSP70 synthesis. The obtained data demonstrate that pre-treatment of seeds with GDA before planting allows inducing the stress resistance at least at early growth stages of plants.

  20. Comprehensive identification and expression analysis of Hsp90s gene family in Solanum lycopersicum.

    PubMed

    Zai, W S; Miao, L X; Xiong, Z L; Zhang, H L; Ma, Y R; Li, Y L; Chen, Y B; Ye, S G

    2015-01-01

    Heat shock protein 90 (Hsp90) is a protein produced by plants in response to adverse environmental stresses. In this study, we identified and analyzed Hsp90 gene family members using a bioinformatic method based on genomic data from tomato (Solanum lycopersicum L.). The results illustrated that tomato contains at least 7 Hsp90 genes distributed on 6 chromosomes; protein lengths ranged from 267-794 amino acids. Intron numbers ranged from 2-19 in the genes. The phylogenetic tree revealed that Hsp90 genes in tomato (Solanum lycopersicum L.), rice (Oryza sativa L.), and Arabidopsis (Arabidopsis thaliana L.) could be divided into 5 groups, which included 3 pairs of orthologous genes and 4 pairs of paralogous genes. Expression analysis of RNA-sequence data showed that the Hsp90-1 gene was specifically expressed in mature fruits, while Hsp90-5 and Hsp90-6 showed opposite expression patterns in various tissues of cultivated and wild tomatoes. The expression levels of the Hsp90-1, Hsp90-2, and Hsp90- 3 genes in various tissues of cultivated tomatoes were high, while both the expression levels of genes Hsp90-3 and Hsp90-4 were low. Additionally, quantitative real-time polymerase chain reaction showed that these genes were involved in the responses to yellow leaf curl virus in tomato plant leaves. Our results provide a foundation for identifying the function of the Hsp90 gene in tomato. PMID:26214462

  1. Chaperoning Steroidal Physiology: Lessons from Mouse Genetic Models of Hsp90 and its Cochaperones

    PubMed Central

    Sanchez, Edwin R.

    2012-01-01

    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 depend 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. PMID:22155719

  2. Ovine HSP90AA1 gene promoter: functional study and epigenetic modifications.

    PubMed

    Salces-Ortiz, Judit; González, Carmen; Bolado-Carrancio, Alfonso; Rodríguez-Rey, Jose Carlos; Calvo, Jorge H; Muñoz, Rubén; Serrano, M Magdalena

    2015-11-01

    When environmental temperatures exceed a certain threshold, the upregulation of the ovine HSP90AA1 gene is produced to cope with cellular injuries caused by heat stress. It has been previously pointed out that several polymorphisms located at the promoter region of this gene seem to be the main responsible for the differences in the heat stress response observed among alternative genotypes in terms of gene expression rate. The present study, focused on the functional study of those candidate polymorphisms by electrophoretic mobility shift assay (EMSA) and in vitro luciferase expression assays, has revealed that the observed differences in the transcriptional activity of the HSP90AA1 gene as response to heat stress are caused by the presence of a cytosine insertion (rs397514115) and a C to G transversion (rs397514116) at the promoter region. Next, we discovered the presence of epigenetic marks at the promoter and along the gene body founding an allele-specific methylation of the rs397514116 mutation in DNA extracted from blood samples. This regulatory mechanism interacts synergistically to modulate gene expression depending on environmental circumstances. Taking into account the results obtained, it is suggested that the transcription of the HSP90AA1 ovine gene is regulated by a cooperative action of transcription factors (TFs) whose binding sites are polymorphic and where the influence of epigenetic events should be also taken into account. PMID:26253285

  3. Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus

    PubMed Central

    Juvvadi, Praveen R.; Soderblom, Erik J.; Moseley, M. Arthur; Steinbach, William J.

    2015-01-01

    Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus. PMID:26014950

  4. Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus.

    PubMed

    Lamoth, Frédéric; Juvvadi, Praveen R; Soderblom, Erik J; Moseley, M Arthur; Steinbach, William J

    2015-08-01

    Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus. PMID:26014950

  5. Composition, assembly and activation of the avian progesterone receptor.

    PubMed

    Smith, D F; Toft, D O

    1992-03-01

    When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: (1) absence of progesterone, (2) elevated temperature (30 degrees C), (3) presence of ATP, and (4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP regenerating system. ATP depletion of lysate by dialysis or ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system. PMID:1562503

  6. Hsp90β is involved in the development of high salt-diet-induced nephropathy via interaction with various signalling proteins.

    PubMed

    Yan, Shi-Hai; Zhao, Ning-Wei; Jiang, Wei-Min; Wang, Xin-Tong; Zhang, Si-Qi; Zhu, Xuan-Xuan; Zhang, Chun-Bing; Gao, Yan-Hong; Gao, Feng; Liu, Fu-Ming; Fang, Zhu-Yuan

    2016-04-01

    A high-salt diet often leads to a local intrarenal increase in renal hypoxia and oxidative stress, which are responsible for an excess production of pathogenic substances. Here, Wistar Kyoto/spontaneous hypertensive (WKY/SHR) rats fed a high-salt diet developed severe proteinuria, resulting from pronounced renal inflammation, fibrosis and tubular epithelial cell apoptosis. All these were mainly non-pressure-related effects. Hsp90β, TGF-β, HIF-1α, TNF-α, IL-6 and MCP-1 were shown to be highly expressed in response to salt loading. Next, we found that Hsp90β might play the key role in non-pressure-related effects of salt loading through a series of cellular signalling events, including the NF-κB, p38 activation and Bcl-2 inactivation. Hsp90β was previously proven to regulate the upstream mediators in multiple cellular signalling cascades through stabilizing and maintaining their activities. In our study, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) or Hsp90β knockdown dramatically alleviated the high-salt-diet-induced proteinuria and renal damage without altering blood pressure significantly, when it reversed activations of NF-κB, mTOR and p38 signalling cascades. Meanwhile, Co-IP results demonstrated that Hsp90β could interact with and stabilize TAK1, AMPKα, IKKα/β, HIF-1α and Raptor, whereas Hsp90β inhibition disrupted this process. In addition, Hsp90β inhibition-mediated renal improvements also accompanied the reduction of renal oxidative stress. In conclusion, salt loading indeed exhibited non-pressure-related impacts on proteinuria and renal dysfunction in WKY/SHR rats. Hsp90β inhibition caused the destabilization of upstream mediators in various pathogenic signalling events, thereby effectively ameliorating this nephropathy owing to renal hypoxia and oxidative stress. PMID:27248656

  7. Hsp90β is involved in the development of high salt-diet-induced nephropathy via interaction with various signalling proteins

    PubMed Central

    Yan, Shi-hai; Zhao, Ning-wei; Jiang, Wei-min; Wang, Xin-tong; Zhang, Si-qi; Zhu, Xuan-xuan; Zhang, Chun-bing; Gao, Yan-hong; Gao, Feng; Liu, Fu-ming; Fang, Zhu-yuan

    2016-01-01

    A high-salt diet often leads to a local intrarenal increase in renal hypoxia and oxidative stress, which are responsible for an excess production of pathogenic substances. Here, Wistar Kyoto/spontaneous hypertensive (WKY/SHR) rats fed a high-salt diet developed severe proteinuria, resulting from pronounced renal inflammation, fibrosis and tubular epithelial cell apoptosis. All these were mainly non-pressure-related effects. Hsp90β, TGF-β, HIF-1α, TNF-α, IL-6 and MCP-1 were shown to be highly expressed in response to salt loading. Next, we found that Hsp90β might play the key role in non-pressure-related effects of salt loading through a series of cellular signalling events, including the NF-κB, p38 activation and Bcl-2 inactivation. Hsp90β was previously proven to regulate the upstream mediators in multiple cellular signalling cascades through stabilizing and maintaining their activities. In our study, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) or Hsp90β knockdown dramatically alleviated the high-salt-diet-induced proteinuria and renal damage without altering blood pressure significantly, when it reversed activations of NF-κB, mTOR and p38 signalling cascades. Meanwhile, Co-IP results demonstrated that Hsp90β could interact with and stabilize TAK1, AMPKα, IKKα/β, HIF-1α and Raptor, whereas Hsp90β inhibition disrupted this process. In addition, Hsp90β inhibition-mediated renal improvements also accompanied the reduction of renal oxidative stress. In conclusion, salt loading indeed exhibited non-pressure-related impacts on proteinuria and renal dysfunction in WKY/SHR rats. Hsp90β inhibition caused the destabilization of upstream mediators in various pathogenic signalling events, thereby effectively ameliorating this nephropathy owing to renal hypoxia and oxidative stress. PMID:27248656

  8. High-Throughput Screen of Natural Product Libraries for Hsp90 Inhibitors

    PubMed Central

    Davenport, Jason; Balch, Maurie; Galam, Lakshmi; Girgis, Antwan; Hall, Jessica; Blagg, Brian S. J.; Matts, Robert L.

    2014-01-01

    Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine. PMID:24833337

  9. Targeting Plasmodium falciparum Hsp90: Towards Reversing Antimalarial Resistance

    PubMed Central

    Shahinas, Dea; Folefoc, Asongna; Pillai, Dylan R.

    2013-01-01

    Malaria continues to exact a great human toll in tropical settings. Antimalarial resistance is rife and the parasite inexorably develops mechanisms to outwit our best drugs, including the now first-line choice, artesunate. Novel strategies to circumvent resistance are needed. Here we detail drug development focusing on heat shock protein 90 and its central role as a chaperone. A growing body of evidence supports the role for Hsp90 inhibitors as adjunctive drugs able to restore susceptibility to traditionally efficacious compounds like chloroquine. PMID:25436880

  10. Blocking the survival of the nastiest by HSP90 inhibition

    PubMed Central

    Workman, Paul; Clarke, Paul A.; Al-Lazikani, Bissan

    2016-01-01

    It is now recognised that genetic, epigenetic and phenotypic heterogeneity within individual human cancers is responsible for therapeutic resistance – knowledge that is having a profound impact on current thinking and experimentation. There has been concern that molecularly targeted therapy is doomed to failure, with resistant clones emerging in response to the Darwinian selective pressure of any drug treatment. However, two studies have shown that the evolution of drug resistance can be restrained by co-administration of a pharmacologic inhibitor of the HSP90 molecular chaperone. PMID:26820296

  11. Regulatory role of the 90-kDa-heat-shock protein (Hsp90) and associated factors on gene expression.

    PubMed

    Erlejman, Alejandra G; Lagadari, Mariana; Toneatto, Judith; Piwien-Pilipuk, Graciela; Galigniana, Mario D

    2014-02-01

    The term molecular chaperone was first used to describe the ability of nucleoplasmin to prevent the aggregation of histones with DNA during the assembly of nucleosomes. Subsequently, the name was extended to proteins that mediate the post-translational assembly of oligomeric complexes protecting them from denaturation and/or aggregation. Hsp90 is a 90-kDa molecular chaperone that represents the major soluble protein of the cell. In contrast to most conventional chaperones, Hsp90 functions as a refined sensor of protein function and its principal role in the cell is to facilitate biological activity to properly folded client proteins that already have a preserved tertiary structure. Consequently, Hsp90 is related to basic cell functions such as cytoplasmic transport of soluble proteins, translocation of client proteins to organelles, and regulation of the biological activity of key signaling factors such as protein kinases, ubiquitin ligases, steroid receptors, cell cycle regulators, and transcription factors. A growing amount of evidence links the protective action of this molecular chaperone to mechanisms related to posttranslational modifications of soluble nuclear factors as well as histones. In this article, we discuss some aspects of the regulatory action of Hsp90 on transcriptional regulation and how this effect could have impacted genetic assimilation mechanism in some organisms. PMID:24389346

  12. A novel function for the 90 kDa heat-shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits.

    PubMed Central

    Schlatter, Harald; Langer, Thomas; Rosmus, Susann; Onneken, Marie-Luise; Fasold, Hugo

    2002-01-01

    Ribosomal subunits are assembled in the nucleus, and mature 40 S and 60 S subunits are exported stoichiometrically into the cytoplasm. The nuclear export of ribosomal subunits is a unidirectional, saturable and energy-dependent process. An in vitro assay for the nuclear export of 60 S ribosomal subunits involves the use of resealed nuclear envelopes. The export of ribosomal subunits from resealed nuclear envelopes is enhanced by cytoplasmic proteins. Here we present evidence that the export-promoting activity was due to the cytoplasmic 90 kDa heat-shock protein (Hsp90). Isolated, purified Hsp90 vastly enhanced the export of 60 S ribosomal subunits from resealed nuclear envelopes, while inhibition of Hsp90 function, either with the Hsp90-binding drug geldanamycin or with anti-Hsp90 antibodies, resulted in reduced release of 60 S ribosomal subunits. To confirm these findings under in vivo conditions, corresponding experiments were performed with Xenopus oocytes using microinjection techniques; the results obtained confirmed the findings obtained with resealed nuclear envelopes. These findings suggest that Hsp90 facilitates the nuclear export of 60 S ribosomal subunits, probably by chaperoning protein interactions during the export process. PMID:11879195

  13. Hsp90 oligomerization process: How can p23 drive the chaperone machineries?

    PubMed

    Lepvrier, Eléonore; Nigen, Michaël; Moullintraffort, Laura; Chat, Sophie; Allegro, Diane; Barbier, Pascale; Thomas, Daniel; Nazabal, Alexis; Garnier, Cyrille

    2015-10-01

    The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer that is able to self-associate in the presence of divalent cations or under heat shock. In a previous work, we focused on the Mg2+-induced oligomerization process of Hsp90, and characterized the oligomers. Combining analytical ultracentrifugation, size-exclusion chromatography coupled to multi-angle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we studied the interaction of p23 with both Hsp90 dimer and oligomers. Even if p23 predominantly binds the Hsp90 dimer, we demonstrated, for the first time, that p23 is also able to interact with Hsp90 oligomers, shifting the Hsp90 dimer-oligomers equilibrium toward dimer. Our results showed that the Hsp90:p23 binding stoichiometry decreases with the Hsp90 oligomerization degree. Therefore, we propose a model in which p23 would act as a "protein wedge" regarding the Hsp90 dimer closure and the Hsp90 oligomerization process. PMID:26151834

  14. Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

    PubMed Central

    Liu, Zhengyi; Zhang, Lei; Pu, Yang; Liu, Zhaopu; Li, Zhiling; Zhao, Yushan; Qin, Song

    2014-01-01

    Heat shock protein 90 (HSP90), a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90) by combining homology cloning with rapid amplification of cDNA ends (RACE). Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA) in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C) for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity (‰) was almost fourfold of that at 25 in salinity (‰) for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes. PMID:24738061

  15. The Double-Edged Sword: Conserved Functions of Extracellular Hsp90 in Wound Healing and Cancer

    PubMed Central

    Hance, Michael W.; Nolan, Krystal D.; Isaacs, Jennifer S.

    2014-01-01

    Heat shock proteins (Hsps) represent a diverse group of chaperones that play a vital role in the protection of cells against numerous environmental stresses. Although our understanding of chaperone biology has deepened over the last decade, the “atypical” extracellular functions of Hsps have remained somewhat enigmatic and comparatively understudied. The heat shock protein 90 (Hsp90) chaperone is a prototypic model for an Hsp family member exhibiting a duality of intracellular and extracellular functions. Intracellular Hsp90 is best known as a master regulator of protein folding. Cancers are particularly adept at exploiting this function of Hsp90, providing the impetus for the robust clinical development of small molecule Hsp90 inhibitors. However, in addition to its maintenance of protein homeostasis, Hsp90 has also been identified as an extracellular protein. Although early reports ascribed immunoregulatory functions to extracellular Hsp90 (eHsp90), recent studies have illuminated expanded functions for eHsp90 in wound healing and cancer. While the intended physiological role of eHsp90 remains enigmatic, its evolutionarily conserved functions in wound healing are easily co-opted during malignancy, a pathology sharing many properties of wounded tissue. This review will highlight the emerging functions of eHsp90 and shed light on its seemingly dichotomous roles as a benevolent facilitator of wound healing and as a sinister effector of tumor progression. PMID:24805867

  16. Molecular cloning and characterization of the full-length Hsp90 gene from Matricaria recutita.

    PubMed

    Ling, S P; Su, S S; Zhang, H M; Zhang, X S; Liu, X Y; Pan, G F; Yuan, Y

    2014-01-01

    Heat shock protein 90 (Hsp90) is one of the most abundant and conserved chaperone proteins and plays important roles in plant growth and responses to environmental stimuli. However, little is known regarding the sequence and function of Hsp90s in Matricaria recutita. In the present study, we cloned the full-length cDNA sequence of the hsp90 gene from this species. Using rapid amplification of cDNA ends technologies with 2 degenerate primers that were designed based on the hsp90 gene sequence from other members of Asteraceae, we isolated and characterized an Hsp90 homolog gene from M. recutita (Mr-Hsp90). The full-length Mr-hsp90 cDNA sequence, containing 2097 base pairs, encodes a protein of 698 amino acids. Based on amino acid sequence identity, Mr-Hsp90 showed high similarity to other cloned Hsp90 proteins. The Mr-Hsp90 protein was closely clustered with the Lactuca sativa in a phylogenetic tree. These results indicate that the cloned sequence of Mr-Hsp90 is a member of the Hsp90 family, which is reported for the first time in M. recutita. Next, we conducted a salt stress experiment to determine the protein's function under salt stress conditions. Survival of chamomile seedlings subjected to heat-shock pretreatment was significantly increased compared with groups that had not undergone heat-shock pretreatment in a salt stress environment. This indicates that Mr-Hsp90 plays an important role in the salt resistance of chamomile seedlings. PMID:25526220

  17. Functional Divergence of Hsp90 Genetic Interactions in Biofilm and Planktonic Cellular States

    PubMed Central

    Cowen, Leah E.

    2015-01-01

    Candida albicans is among the most prevalent opportunistic fungal pathogens. Its capacity to cause life-threatening bloodstream infections is associated with the ability to form biofilms, which are intrinsically drug resistant reservoirs for dispersal. A key regulator of biofilm drug resistance and dispersal is the molecular chaperone Hsp90, which stabilizes many signal transducers. We previously identified 226 C. albicans Hsp90 genetic interactors under planktonic conditions, of which 56 are involved in transcriptional regulation. Six of these transcriptional regulators have previously been implicated in biofilm formation, suggesting that Hsp90 genetic interactions identified in planktonic conditions may have functional significance in biofilms. Here, we explored the relationship between Hsp90 and five of these transcription factor genetic interactors: BCR1, MIG1, TEC1, TUP1, and UPC2. We deleted each transcription factor gene in an Hsp90 conditional expression strain, and assessed biofilm formation and morphogenesis. Strikingly, depletion of Hsp90 conferred no additional biofilm defect in the mutants. An interaction was observed in which deletion of BCR1 enhanced filamentation upon reduction of Hsp90 levels. Further, although Hsp90 modulates expression of TEC1, TUP1, and UPC2 in planktonic conditions, it has no impact in biofilms. Lastly, we probed for physical interactions between Hsp90 and Tup1, whose WD40 domain suggests that it might interact with Hsp90 directly. Hsp90 and Tup1 formed a stable complex, independent of temperature or developmental state. Our results illuminate a physical interaction between Hsp90 and a key transcriptional regulator of filamentation and biofilm formation, and suggest that Hsp90 has distinct genetic interactions in planktonic and biofilm cellular states. PMID:26367740

  18. Novel Hsp90 inhibitor NVP-AUY922 radiosensitizes prostate cancer cells

    PubMed Central

    Gandhi, Nishant; Wild, Aaron T.; Chettiar, Sivarajan T.; Aziz, Khaled; Kato, Yoshinori; Gajula, Rajendra P.; Williams, Russell D.; Cades, Jessica A.; Annadanam, Anvesh; Song, Danny; Zhang, Yonggang; Hales, Russell K.; Herman, Joseph M.; Armour, Elwood; DeWeese, Theodore L.; Schaeffer, Edward M.; Tran, Phuoc T.

    2013-01-01

    Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the “non-oncogene” addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded “client” proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4–1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies. PMID:23358469

  19. Hsp90 Inhibitors Are Efficacious against Kaposi Sarcoma by Enhancing the Degradation of the Essential Viral Gene LANA, of the Viral Co-Receptor EphA2 as well as Other Client Proteins

    PubMed Central

    Chen, Wuguo; Sin, Sang-Hoon; Wen, Kwun Wah; Damania, Blossom; Dittmer, Dirk P.

    2012-01-01

    Heat-shock protein 90 (Hsp90) inhibitors exhibit activity against human cancers. We evaluated a series of new, oral bioavailable, chemically diverse Hsp90 inhibitors (PU-H71, AUY922, BIIB021, NVP-BEP800) against Kaposi sarcoma (KS). All Hsp90 inhibitors exhibited nanomolar EC50 in culture and AUY922 reduced tumor burden in a xenograft model of KS. KS is associated with KS-associated herpesvirus (KSHV). We identified the viral latency associated nuclear antigen (LANA) as a novel client protein of Hsp90 and demonstrate that the Hsp90 inhibitors diminish the level of LANA through proteasomal degradation. These Hsp90 inhibitors also downregulated EphA2 and ephrin-B2 protein levels. LANA is essential for viral maintenance and EphA2 has recently been shown to facilitate KSHV infection; which in turn feeds latent persistence. Further, both molecules are required for KS tumor formation and both were downregulated in response to Hsp90 inhibitors. This provides a rationale for clinical testing of Hsp90 inhibitors in KSHV-associated cancers and in the eradication of latent KSHV reservoirs. PMID:23209418

  20. A Highly Selective Hsp90 Affinity Chromatography Resin with a Cleavable Linker

    PubMed Central

    Hughes, Philip F; Barrott, Jared J; Carlson, David A; Loiselle, David R; Speer, Brittany L; Bodoor, Khaldon; Rund, Lauretta A; Haystead, Timothy A J

    2012-01-01

    Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media. PMID:22520629

  1. Structure and regulation of the HSP90 gene from the pathogenic fungus Candida albicans.

    PubMed Central

    Swoboda, R K; Bertram, G; Budge, S; Gooday, G W; Gow, N A; Brown, A J

    1995-01-01

    Candida albicans HSP90 sequences were isolated by screening cDNA and genomic libraries with a probe derived from the Saccharomyces cerevisiae homolog, HSP82, which encodes a member of the heat shock protein 90 family of molecular chaperones. Identical sequences were obtained for the 2,197-bp overlap of the cDNA and gene sequences, which were derived from C. albicans 3153A and ATCC 10261, respectively. The C. albicans HSP90 gene contained no introns, and it showed strong homology (61 to 79% identity) to HSP90 sequences from other fungi, vertebrates, and plants. The C-terminal portion of the predicted Hsp90 amino acid sequence was identical to the 47-kDa protein which is thought to be immunoprotective during C. albicans infections (R. C. Matthews, J. Med. Microbiol. 36:367-370, 1992), confirming that this protein represents the C-terminal portion of the 81-kDa Hsp90 protein. Quantitative Northern (RNA) analyses revealed that C. albicans HSP90 mRNA was heat shock inducible and that its levels changed during batch growth, with its maximum levels being reached during the mid-exponential growth phase. HSP90 mRNA levels increased transiently during the yeast-to-hyphal transition but did not correlate directly with germ tube production per se. These data do not exclude a role for Hsp90 in the dimorphic transition. Southern blotting revealed only one HSP90 locus in the diploid C. albicans genome. Repeated attempts to disrupt both alleles and generate a homozygous C. albicans delta hsp90/delta hsp90 null mutant were unsuccessful. These observations suggest the existence of a single HSP90 locus which is essential for viability in C. albicans. PMID:7591093

  2. Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

    PubMed Central

    Kasioulis, Ioannis; Syred, Heather M.; Tate, Peri; Finch, Andrew; Shaw, Joseph; Seawright, Anne; Fuszard, Matt; Botting, Catherine H.; Shirran, Sally; Adams, Ian R.; Jackson, Ian J.; van Heyningen, Veronica; Yeyati, Patricia L.

    2014-01-01

    The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components. PMID:24554764

  3. Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis.

    PubMed

    Kasioulis, Ioannis; Syred, Heather M; Tate, Peri; Finch, Andrew; Shaw, Joseph; Seawright, Anne; Fuszard, Matt; Botting, Catherine H; Shirran, Sally; Adams, Ian R; Jackson, Ian J; van Heyningen, Veronica; Yeyati, Patricia L

    2014-04-01

    The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components. PMID:24554764

  4. Fused kinase is stabilized by Cdc37/Hsp90 and enhances Gli protein levels

    SciTech Connect

    Kise, Yoshiaki; Takenaka, Kei; Tezuka, Tohru; Yamamoto, Tadashi; Miki, Hiroaki . E-mail: miki@ims.u-tokyo.ac.jp

    2006-12-08

    Serine/threonine kinase Fused (Fu) is an essential component of Hedgehog (Hh) signaling in Drosophila, but the biochemical functions of Fu remain unclear. Here, we have investigated proteins co-precipitated with mammalian Fu and identified a kinase-specific chaperone complex, Cdc37/Hsp90, as a novel-binding partner of Fu. Inhibition of Hsp90 function by geldanamycin (GA) induces rapid degradation of Fu through a ubiquitin-proteasome pathway. We next show that co-expression of Fu with transcription factors Gli1 and Gli2 significantly increases their protein levels and luciferase reporter activities, which are blocked by GA. These increases can be ascribed to Fu-mediated stabilization of Gli because co-expression of Fu prolongs half-life of Gli1 and reduces polyubiquitination of Gli1. Finally, we show that GA inhibits proliferation of PC3, a Hh signaling-activated prostate cancer cell line. This growth inhibition is partially rescued by expression of ectopic Gli1, suggesting that Fu may contribute to enhance Hh signaling activity in cancer cells.

  5. Role of oxidative stress in Geldanamycin-induced cytotoxicity and disruption of Hsp90 signaling complex

    PubMed Central

    Clark, Christina B.; Rane, Madhavi J.; Mehdi, Delphine El; Miller, Cynthia J.; Sachleben, Leroy R.; Gozal, Evelyne

    2009-01-01

    Heat shock protein 90 (Hsp90) is a chaperone protein regulating PC-12 cell survival by binding and stabilizing Akt, Raf-1, and Cdc37. Hsp90 inhibitor Geldanamycin (GA) cytotoxicity has been attributed to disruption of Hsp90 binding, and the contribution of oxidative stress generated by its quinone group has not been studied in this context. Reactive oxygen species (ROS) and cell survival were assessed in PC-12 cells exposed to GA or Menadione (MEN), and Akt, Raf-1, and Cdc37 expression and binding to Hsp90 were determined. GA disrupted Hsp90 binding and increased ROS production starting at 1h, and cell death occurred at 6h, inhibited by N-acetyl cysteine (NAC) without preventing dissociation of proteins. At 24h, NAC prevented cytotoxicity and Hsp90 complex disruption. However MnTBAP antioxidant treatment failed to inhibit GA cytotoxicity, suggesting that NAC acts by restoring gluthathione. In contrast, 24h MEN induced cytotoxicity without disrupting Hsp90 binding. GA and MEN decreased Hsp90-binding proteins expression, and proteasomal inhibition prevented MEN, but not GA-induced degradation. In conclusion, while MEN cytotoxicity is mediated by ROS and proteasomal degradation, GA-induced cytotoxicity requires ROS but induces HSP90 complex dissociation and proteasome-independent protein degradation. These differences between MEN and GA-induced cytotoxicity, may allow more specific targeting of cancer cells. PMID:19703551

  6. Targeting Hsp90 and its co-chaperones to treat Alzheimer’s disease

    PubMed Central

    Blair, Laura J.; Sabbagh, Jonathan J.; Dickey, Chad A.

    2015-01-01

    Introduction Alzheimer’s disease (AD), characterized by the accumulation of hyperphosphorylated tau and beta amyloid (Aβ), currently lacks effective treatment. Chaperone proteins, such as the heat shock protein (Hsp) 90, form macromolecular complexes with co-chaperones, which can regulate tau metabolism and Aβ processing. While small molecule inhibitors of Hsp90 have been successful at ameliorating tau and Aβ burden, their development into drugs to treat disease has been slow due to the off- and on-target effects of this approach as well as challenges with the pharmacology of current scaffolds. Thus, other approaches are being developed to improve these compounds and to target co-chaperones of Hsp90 in an effort to limit these liabilities. Areas Covered This article discusses the most current developments in Hsp90 inhibitors including advances in blood-brain barrier permeability, decreased toxicity, and homolog-specific small molecule inhibitors. In addition, we discuss current strategies targeting Hsp90 co-chaperones rather than Hsp90 itself to reduce off-target effects. Expert Opinion While Hsp90 inhibitors have proven their efficacy at reducing tau pathology, they have yet to meet with success in the clinic. The development of Hsp90/tau complex specific inhibitors and further development of Hsp90 co-chaperone specific drugs should yield more potent, less toxic therapeutics. PMID:25069659

  7. Paralog specific Hsp90 Inhibitors – a brief history and a bright future

    PubMed Central

    Gewirth, Daniel T.

    2016-01-01

    Structured Abstract Background The high sequence and structural homology among the hsp90 paralogs – Hsp90α, Hsp90β, Grp94, and Trap-1 – has made the development of paralog-specific inhibitors a challenging proposition. Objective This review surveys the state of developments in structural analysis, compound screening, and structure-based design that have been brought to bear on this problem. Results First generation compounds that selectively bind to Hsp90, Grp94, or Trap-1 have been identified. Conclusion With the proof of principle firmly established, the prospects for further progress are bright. PMID:27072700

  8. Translational downregulation of HSP90 expression by iron chelators in neuroblastoma cells.

    PubMed

    Sidarovich, Viktoryia; Adami, Valentina; Gatto, Pamela; Greco, Valentina; Tebaldi, Toma; Tonini, Gian Paolo; Quattrone, Alessandro

    2015-01-01

    Iron is an essential cellular nutrient, being a critical cofactor of several proteins involved in cell growth and replication. Compared with normal cells, neoplastic cells have been shown to require a greater amount of iron, thus laying the basis for the promising anticancer activity of iron chelators. In this work, we evaluated the effects of molecules with iron chelation activity on neuroblastoma (NB) cell lines. Of the 17 iron chelators tested, six reduced cell viability of two NB cell lines with an inhibition of growth of 50% below 10 µM; four of the six molecules-ciclopirox olamine (CPX), piroctone, 8-hydroxyquinoline, and deferasirox-were also shown to efficiently chelate intracellular iron within minutes after addition. Effects on cell viability of one of the compounds, CPX, were indeed dependent on chelation of intracellular iron and mediated by both G0/G1 cell cycle block and induction of apoptosis. By combined transcriptome and translatome profiling we identified early translational downregulation of several members of the heat shock protein group as a specific effect of CPX treatment. We functionally confirmed iron-dependent depletion of HSP90 and its client proteins at pharmacologically achievable concentrations of CPX, and we extended this effect to piroctone, 8-hydroxyquinoline, and deferasirox. Given the documented sensitivity of NB cells to HSP90 inhibition, we propose CPX and other iron chelators as investigational antitumor agents in NB therapy. PMID:25564462

  9. Expression of genes encoding cytosolic and endoplasmic reticulum HSP90 proteins in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Pugliese, Luciana; Georg, Raphaela C; Fietto, Luciano G; Gomes, Suely L

    2008-03-31

    HSP90 proteins are important molecular chaperones involved in multiple cellular processes. This work reports the characterization of cDNAs encoding two distinct HSP90 proteins (named HSP90A and HSP90B) from the chytridiomycete Blastocladiella emersonii. Deduced amino acid sequences of HSP90A and HSP90B exhibit signatures of the cytosolic and endoplasmic reticulum (ER) HSP90 proteins, respectively. A genomic clone encoding HSP90A was also characterized indicating the presence of a single intron of 184 bp interrupting the coding region, located near the amino-terminus of the protein. Expression of both HSP90A and HSP90B genes increases significantly during heat shock at 38 degrees C, with highest induction ratios observed in cells stressed during germination of the fungus. Changes in the amount of HSP90A transcript were also evaluated during B. emersonii life cycle at physiological temperature (27 degrees C), and its levels were found to increase both during germination and sporulation of the fungus. HSP90A protein levels were analyzed during B. emersonii life cycle and significant changes were observed only during sporulation. Furthermore, during heat stress a large increase in the amount of HSP90A protein was observed. Induction of HSP90A and HSP90B genes during heat stress indicates the importance of both genes in the response to high temperature in B. emersonii. PMID:18281163

  10. Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation.

    PubMed

    Assimon, Victoria A; Southworth, Daniel R; Gestwicki, Jason E

    2015-12-01

    Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones. PMID:26565746