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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 Central

    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. 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

  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. 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

  7. Structure-activity relationship of Garcinia xanthones analogues: Potent Hsp90 inhibitors with cytotoxicity and antiangiogenesis activity.

    PubMed

    Xu, Xiaoli; Wu, Yue; Hu, Mingyang; Li, Xiang; Gu, Congying; You, Qidong; Zhang, Xiaojin

    2016-10-01

    Hsp90 has long been recognized as an attractive and crucial molecular target for cancer therapy. Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported as a natural inhibitor of Hsp90. Here, we present the structure-activity relationship of Garcinia xanthones analogues as Hsp90 inhibitors and identify that compound 25, with a simplified skeleton, had an improved inhibitory effect toward Hsp90. Compound 25 inhibited the ATPase activity of Hsp90 with an IC50 value of 3.68±0.18μM. It also exhibited potent antiproliferative activities in some solid tumor cells. In SK-BR-3 cells with high Hsp90 expression, compound 25 induced the degradation of Hsp90 client proteins including Akt and Erk1/2 without causing the heat shock response. Additionally, compound 25 inhibited angiogenesis in HUVEC cells through Hsp90 regulation of the HIF-1α pathway. These results demonstrate that compound 25 as an Hsp90 inhibitor with a new structure could be further studied for the development of tumor therapy. PMID:27527413

  8. Constitutively-active androgen receptor variants function independently of the HSP90 chaperone but do not confer resistance to HSP90 inhibitors.

    PubMed

    Gillis, Joanna L; Selth, Luke A; Centenera, Margaret M; Townley, Scott L; Sun, Shihua; Plymate, Stephen R; Tilley, Wayne D; Butler, Lisa M

    2013-05-01

    The development of lethal, castration resistant prostate cancer is associated with adaptive changes to the androgen receptor (AR), including the emergence of mutant receptors and truncated, constitutively active AR variants. AR relies on the molecular chaperone HSP90 for its function in both normal and malignant prostate cells, but the requirement for HSP90 in environments with aberrant AR expression is largely unknown. Here, we investigated the efficacy of three HSP90 inhibitors, 17-AAG, HSP990 and AUY922, against clinically-relevant AR missense mutants and truncated variants. HSP90 inhibition effectively suppressed the signaling of wild-type AR and all AR missense mutants tested. By contrast, two truncated AR variants, AR-V7 and ARv567es, exhibited marked resistance to HSP90 inhibitors. Supporting this observation, nuclear localization of the truncated AR variants was not affected by HSP90 inhibition and AR variant:HSP90 complexes could not be detected in prostate cancer cells. Interestingly, HSP90 inhibition resulted in accumulation of AR-V7 and ARv567es in both cell lines and human tumor explants. Despite the apparent independence of AR variants from HSP90 and their treatment-associated induction, the growth of cell lines with endogenous or enforced expression of AR-V7 or ARv567es remained highly sensitive to AUY922. This study demonstrates that functional AR variant signaling does not confer resistance to HSP90 inhibition, yields insight into the interaction between AR and HSP90 and provides further impetus for the clinical application of HSP90 inhibitors in advanced prostate cancer.

  9. 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

  10. Dunaliella salina Hsp90 is halotolerant.

    PubMed

    Chen, Xiang-Jun; Wu, Ming-Jie; Jiang, Yan; Yang, Yi; Yan, Yong-Bin

    2015-04-01

    Dunaliella salina is a unicellular green alga with exceptional halotolerance. Although the D. salina cells are capable to proliferate in hypersaline medium, the intracellular salt concentrations are maintained at a low level. Thus the extracellular but not intracellular Dunaliella proteins are expected to be highly halotolerant. In this research, we compared the salt-dependence of the activity and stability of Hsp90s from the halotolerant alga D. salina (dsHsp90) and the mesophilic alga Chlamydomonas reinhardtii (crHsp90). We found that the ATPase activity of crHsp90 could be enhanced about six-fold by 2M NaCl, while the activity of dsHsp90 showed a much weaker dependence on salinity. When denatured by urea, both crHsp90 and dsHsp90 exhibited an apparent three-state unfolding with the population of an unfolding intermediate. High salinity significantly decreased the Gibbs free energy change of crHsp90 but not dsHsp90 for the transition from the native state to the intermediate. The little dependence of dsHsp90 activity and folding on salinity suggests that dsHsp90 is halotolerant though it is an intracellular protein. We propose that the halotolerance of intracellular Dunaliella proteins might play a role in fighting against the transient intracellular salt fluctuations during hyperosmotic or hypoosmotic shock.

  11. 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

  12. 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.

  13. Targeting Hsp90 in Non-Cancerous Maladies.

    PubMed

    Woodford, Mark R; Dunn, Diana M; Ciciarelli, Joseph G; Beebe, Kristin; Neckers, Len; Mollapour, Mehdi

    2016-01-01

    Heat shock protein-90 (Hsp90) is a molecular chaperone critical to the folding, stability and activity of over 200 client proteins including many responsible for tumor initiation, progression and metastasis. Hsp90 chaperone function is linked to its ATPase activity and Hsp90 inhibitors interfere with this activity, thereby making Hsp90 an attractive target for cancer therapy. Also post-translational modification (PTM) and co-chaperone proteins modulate Hsp90 function, providing additional targets for secondary inhibition. Recent reports have shown that pathogens utilize both their own Hsp90 and that of their host for the propagation of infectious elements. In this review we will summarize our current knowledge of Hsp90 structure and function in both the pathogen and the host. We will focus on the role of Hsp90 in viral and parasitic diseases and the potential beneficial application of Hsp90 inhibitors alone and in combination with disease-specific inhibitors.

  14. Synthesis and Structure activity relationships of EGCG Analogues, A Recently Identified Hsp90 Inhibitor

    PubMed Central

    Khandelwal, Anuj; Hall, Jessica

    2014-01-01

    Epigallocatechin-3-gallate (EGCG), the principal polyphenol isolated from green tea, was recently shown to inhibit Hsp90, however structure-activity relationships for this natural product have not yet been produced. Herein, we report the synthesis and biological evaluation of EGCG analogues to establish structure-activity relationships between EGCG and Hsp90. All four rings as well as the linker connecting the C- and the D-rings were systematically investigated, which led to the discovery of compounds that inhibit Hs90 and display improvement in efficacy over EGCG. Anti-proliferative activity of all the analogues was determined against MCF-7 and SKBr3 cell lines and Hsp90 inhibitory activity of four most potent analogues was further evaluated by western blot analyses and degradation of Hsp90-dependent client proteins. Prenyl substituted aryl ester of 3,5-dihydroxychroman-3-ol ring system was identified as novel scaffold that exhibit Hsp90 inhibitory activity. PMID:23834230

  15. Synthesis and structure-activity relationships of EGCG analogues, a recently identified Hsp90 inhibitor.

    PubMed

    Khandelwal, Anuj; Hall, Jessica A; Blagg, Brian S J

    2013-08-16

    Epigallocatechin-3-gallate (EGCG), the principal polyphenol isolated from green tea, was recently shown to inhibit Hsp90; however, structure-activity relationships for this natural product have not yet been produced. Herein, we report the synthesis and biological evaluation of EGCG analogues to establish structure-activity relationships between EGCG and Hsp90. All four rings as well as the linker connecting the C- and the D-rings were systematically investigated, which led to the discovery of compounds that inhibit Hs90 and display improvement in efficacy over EGCG. Antiproliferative activity of all the analogues was determined against MCF-7 and SKBr3 cell lines and Hsp90 inhibitory activity of the four most potent analogues was further evaluated by Western blot analyses and degradation of Hsp90-dependent client proteins. The prenyl-substituted aryl ester of 3,5-dihydroxychroman-3-ol ring system was identified as a novel scaffold that exhibits Hsp90 inhibitory activity. PMID:23834230

  16. Terazosin activated Pgk1 and Hsp90 to promote stress resistance

    PubMed Central

    Chen, Xinping; Zhao, Chunyue; Li, Xiaolong; Wang, Tao; Li, Yizhou; Cao, Cheng; Ding, Yuehe; Dong, Mengqiu; Finci, Lorenzo; Wang, Jia-huai; Li, Xiaoyu; Liu, Lei

    2015-01-01

    Drugs that can protect against organ damage are urgently needed, especially for diseases such as sepsis and brain stroke. We have discovered that terazosin (TZ), a widely marketed alpha1-adrenergic receptor agonist, alleviated organ damage and improved survival in rodent models of stroke and sepsis. Through combined studies of enzymology and X-ray crystallography, we have discovered that TZ binds to a novel target, phosphoglycerate kinase 1 (Pgk1) and activates its enzymatic activity, probably through 1,3-diamino-6,7-dimethoxyisoquinoline's ability to promote ATP release from Pgk1. Mechanistically, the ATP generated from Pgk1 may enhance the chaperone activity of Hsp90, an ATPase known to associate with Pgk1. Upon activation, Hsp90 promotes multi-stress resistance. Our studies have demonstrated that TZ has a novel protein target, Pgk1, and has revealed its corresponding biological effect. As a clinical drug, TZ may be quickly translated into treatment of devastating diseases including stroke and sepsis. PMID:25383758

  17. 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.

  18. 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

  19. TNF-induced recruitment and activation of the IKK complex require Cdc37 and Hsp90.

    PubMed

    Chen, Guoqing; Cao, Ping; Goeddel, David V

    2002-02-01

    The IKK complex, containing two catalytic subunits IKKalpha and IKKbeta and a regulatory subunit NEMO, plays central roles in signal-dependent activation of NF-kappaB. We identify Cdc37 and Hsp90 as two additional components of the IKK complex. IKKalpha/IKKbeta/NEMO and Cdc37/Hsp90 form an approximately 900 kDa heterocomplex, which is assembled via direct interactions of Cdc37 with Hsp90 and with the kinase domain of IKKalpha/IKKbeta. Geldanamycin (GA), an antitumor agent that disrupts the formation of this heterocomplex, prevents TNF-induced activation of IKK and NF-kappaB. GA treatment reduces the size of the IKK complex and abolishes TNF-dependent recruitment of the IKK complex to TNF receptor 1 (TNF-R1). Therefore, heterocomplex formation with Cdc37/Hsp90 is a prerequisite for TNF-induced activation and trafficking of IKK from the cytoplasm to the membrane.

  20. 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

  1. 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

  2. Conformational dynamics of the molecular chaperone Hsp90

    PubMed Central

    Krukenberg, Kristin A.; Street, Timothy O.; Lavery, Laura A.; Agard, David A.

    2016-01-01

    The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1–2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organism-dependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90’s conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90’s conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics. PMID:21414251

  3. 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

  4. Gedunin, a Novel Hsp90 Inhibitor: Semisynthesis of Derivatives and Preliminary Structure–Activity Relationships

    PubMed Central

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

    2010-01-01

    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.

  6. The universe of Hsp90.

    PubMed

    Stankiewicz, Marta; Mayer, Matthias P

    2012-02-01

    Abstract Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Among the ATP-dependent chaperones, heat shock proteins (Hsp90) proteins play a special role. While Hsp90s can interact with unfolded and misfolded proteins, their main (and in eukaryotic cells essential) function appears to involve interactions with a limited number of protein clients at late steps of maturation or in "alter-native" conformations for regulating their stability and activity. Because Hsp90 clients are hubs of diverse signaling networks and participate in nearly every cellular function, Hsp90s interconnect many regulatory circuits and link them to environmental impacts. The availability and activity of Hsp90 may thus influence complex physiological and pathophysiological processes, such as differentiation, development, aging, cancer, neurodegeneration, and infectious diseases. Furthermore, through homeostatic effects on differentiation and development, Hsp90s act as capacitors of phenotypic evolution. In this review, we discuss recent insights in the structure and chaperone cycle of Hsp90s, the mechanisms underlying Hsp90 binding to clients, and potential reasons why client proteins specifically require the assistance of Hsp90s. Moreover, the current views on Hsp90-cochaperone interactions and regulation of Hsp90 proteins via posttranslational modifications are summarized. The second half of this article is devoted to the role of Hsp90 proteins in health and disease, aging, and evolution.

  7. 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

  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.

  9. Hsp90 phosphorylation, Wee1 and the cell cycle.

    PubMed

    Mollapour, Mehdi; Tsutsumi, Shinji; Neckers, Len

    2010-06-15

    Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotic cells, and it maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival, and metastasis. It is possible to selectively inhibit Hsp90 using natural products such as geldanamycin (GA) or radicicol (RD), which have served as prototypes for development of synthetic Hsp90 inhibitors. These compounds bind within the ADP/ATP-binding site of the Hsp90 N-terminal domain to inhibit its ATPase activity. As numerous N-terminal domain inhibitors are currently undergoing extensive clinical evaluation, it is important to understand the factors that may modulate in vivo susceptibility to these drugs. We recently reported that Wee1Swe1-mediated, cell cycle-dependent, tyrosine phosphorylation of Hsp90 affects GA binding and impacts cancer cell sensitivity to Hsp90 inhibition. This phosphorylation also affects Hsp90 ATPase activity and its ability to chaperone a selected group of clients, comprised primarily of protein kinases. Wee1 regulates the G2/M transition. Here we present additional data demonstrating that tyrosine phosphorylation of Hsp90 by Wee1Swe1 is important for Wee1Swe1 association with Hsp90 and for Wee1Swe1 stability. Yeast expressing non-phosphorylatable yHsp90-Y24F, like swe1∆ yeast, undergo premature nuclear division that is insensitive to G2/M checkpoint arrest. These findings demonstrate the importance of Hsp90 phosphorylation for proper cell cycle regulation. PMID:20519952

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

    PubMed

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

    2015-08-18

    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 Ser(1177), 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 Ser(1177) 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. 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

  12. Cross monomer substrate contacts reposition the Hsp90 N-terminal domain and prime the chaperone activity

    PubMed Central

    Street, Timothy O.; Lavery, Laura A.; Verba, Kliment; Lee, Chung-Tien; Mayer, Matthias P.; Agard, David A.

    2012-01-01

    The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate catalyzed Hsp90 conformational changes are unknown. Here we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drive an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity. PMID:22063096

  13. Cytotoxic activity to acute myeloid leukemia cells by Antp-TPR hybrid peptide targeting Hsp90.

    PubMed

    Horibe, Tomohisa; Kawamoto, Megumi; Kohno, Masayuki; Kawakami, Koji

    2012-07-01

    We previously reported that Antp-TPR hybrid peptide inhibited the interaction of Hsp90 with TPR2A and had selective cytotoxic activity discriminating between normal and cancer cells to induce cancer cell death. In this study, we investigated the cytotoxic activity of Antp-TPR peptide toward acute myeloid leukemia (AML) cells. It was demonstrated that Antp-TPR peptide induced AML cell death in cell lines such as U937, K562, THP-1, and HL-60 via activation of caspases 3 and 7, and disruption of mitochondrial membrane potential. Conversely, Antp-TPR peptide did not reduce the viability of normal cells including peripheral blood mononuclear cells (PBMCs), although both geldanamycin and 17-AAG, small-molecule inhibitors of Hsp90, mediated cytotoxicity to these normal cells at low concentrations. In addition, mutation analysis of TPR peptide demonstrated that the highly conserved amino acids Lys and Arg were critical to the cytotoxic activity. These results indicated that Antp-TPR hybrid peptide would provide potent and selective therapeutic options in the treatment of AML.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Heat shock protein 90 as a drug target against protozoan infections: biochemical characterization of HSP90 from Plasmodium falciparum and Trypanosoma evansi and evaluation of its inhibitor as a candidate drug.

    PubMed

    Pallavi, Rani; Roy, Nainita; Nageshan, Rishi Kumar; Talukdar, Pinaki; Pavithra, Soundara Raghavan; Reddy, Raghunath; Venketesh, S; Kumar, Rajender; Gupta, Ashok Kumar; Singh, Raj Kumar; Yadav, Suresh Chandra; Tatu, Utpal

    2010-12-01

    Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

  1. 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

  2. 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

  3. 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

  4. Role of the HSP90-associated cochaperone p23 in enhancing activity of the androgen receptor and significance for prostate cancer.

    PubMed

    Reebye, Vikash; Querol Cano, Laia; Lavery, Derek N; Brooke, Greg N; Powell, Sue M; Chotai, Deepa; Walker, Marjorie M; Whitaker, Hayley C; Wait, Robin; Hurst, Helen C; Bevan, Charlotte L

    2012-10-01

    Prostate tumor growth initially depends on androgens, which act via the androgen receptor (AR). Despite androgen ablation therapy, tumors eventually progress to a castrate-resistant stage in which the AR remains active. The mechanisms are poorly understood but it may be that changes in levels or activity of AR coregulators affect trafficking and activation of the receptor. A key stage in AR signaling occurs in the cytoplasm, where unliganded receptor is associated with the heat shock protein (HSP)90 foldosome complex. p23, a key component of this complex, is best characterized as a cochaperone for HSP90 but also has HSP90-independent activity and has been reported as having differential effects on the activity of different steroid receptors. Here we report that p23 increases activity of the AR, and this appears to involve steps both in the cytoplasm (increasing ligand-binding capacity, possibly via direct interaction with AR) and the nucleus (enhancing AR occupancy at target promoters). We show, for the first time, that AR and p23 can interact, perhaps directly, when HSP90 is not present in the same complex. The effects of p23 on AR activity are at least partly HSP90 independent because a mutant form of p23, unable to bind HSP90, nevertheless increases AR activity. In human prostate tumors, nuclear p23 was higher in malignant prostate cells compared with benign/normal cells, supporting the utility of p23 as a therapeutic target in prostate cancer.

  5. Plasticity of the Hsp90 chaperone machine in divergent eukaryotic organisms

    PubMed Central

    Brown, Celeste

    2008-01-01

    Hsp90 is critical for the regulation and activation of numerous client proteins critical for diverse functions such as cell growth, differentiation, and reproduction. Cytosolic Hsp90 function is dependent on a battery of co-chaperone proteins that regulate the ATPase activity of Hsp90 function or direct Hsp90 to interact with specific client proteins. Little is known about how Hsp90 complexes vary between different organisms and how this affects the scope of clients that are activated by Hsp90. This study determined whether ten distinct Hsp90 co-chaperones were encoded by genes in 19 disparate eukaryotic organisms. Surprisingly, none of the co-chaperones were present in all organisms. The co-chaperone Hop/Sti1 was most widely dispersed (18 out of 19 species), while orthologs of Cdc37, which is critical for the stability and activation of diverse protein kinases in yeast and mammals, were identified in only nine out of 19 species examined. The organism with the smallest proteome, Encephalitozoon cuniculi, contained only three of these co-chaperones, suggesting a correlation between client diversity and the complexity of the Hsp90 co-chaperone machine. Our results suggest co-chaperones are critical for cytosolic Hsp90 function in vivo, but that the composition of Hsp90 complexes varies depending on the specialized protein folding requirements of divergent species. Electronic supplementary material The online version of this article (doi:10.1007/s12192-008-0058-9) contains supplementary material, which is available to authorized users. PMID:18636345

  6. Inhibition of HSP90 by AUY922 Preferentially Kills Mutant KRAS Colon Cancer Cells by Activating Bim through ER Stress.

    PubMed

    Wang, Chun Yan; Guo, Su Tang; Wang, Jia Yu; Liu, Fen; Zhang, Yuan Yuan; Yari, Hamed; Yan, Xu Guang; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

    2016-03-01

    Oncogenic mutations of KRAS pose a great challenge in the treatment of colorectal cancer. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the HSP90 inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with mutant KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3-only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. Remarkably, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and the agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy.

  7. 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

  8. 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

  9. 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

  10. 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

  11. Inhibition of HSP90 Promotes Neural Stem Cell Survival from Oxidative Stress through Attenuating NF-κB/p65 Activation

    PubMed Central

    Jiang, Wenkai; Zhou, Lin

    2016-01-01

    Stem cell survival after transplantation determines the efficiency of stem cell treatment, which develops as a novel potential therapy for several central nervous system (CNS) diseases in recent decades. The engrafted stem cells face the damage of oxidative stress, inflammation, and immune response at the lesion point in host. Among the damaging pathologies, oxidative stress directs stem cells to apoptosis and even death through several signalling pathways and DNA damage. However, the in-detail mechanism of stem cell survival from oxidative stress has not been revealed clearly. Here, in this study, we used hydrogen peroxide (H2O2) to induce the oxidative damage on neural stem cells (NSCs). The damage was in consequence demonstrated involving the activation of heat shock protein 90 (HSP90) and NF-κB/p65 signalling pathways. Further application of the pharmacological inhibitors, respectively, targeting at each signalling indicated an upper-stream role of HSP90 upon NF-κB/p65 on NSCs survival. Preinhibition of HSP90 with the specific inhibitor displayed a significant protection on NSCs against oxidative stress. In conclusion, inhibition of HSP90 would attenuate NF-κB/p65 activation by oxidative induction and promote NSCs survival from oxidative damage. The HSP90/NF-κB mechanism provides a new evidence on rescuing NSCs from oxidative stress and also promotes the stem cell application on CNS pathologies.

  12. 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

  13. 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

  14. Nitration of Hsp90 on Tyrosine 33 Regulates Mitochondrial Metabolism*

    PubMed Central

    Franco, Maria C.; Ricart, Karina C.; Gonzalez, Analía S.; Dennys, Cassandra N.; Nelson, Pascal A.; Janes, Michael S.; Mehl, Ryan A.; Landar, Aimee; Estévez, Alvaro G.

    2015-01-01

    Peroxynitrite production and tyrosine nitration are present in several pathological conditions, including neurodegeneration, stroke, aging, and cancer. Nitration of the pro-survival chaperone heat shock protein 90 (Hsp90) in position 33 and 56 induces motor neuron death through a toxic gain-of-function. Here we show that nitrated Hsp90 regulates mitochondrial metabolism independently of the induction of cell death. In PC12 cells, a small fraction of nitrated Hsp90 was located on the mitochondrial outer membrane and down-regulated mitochondrial membrane potential, oxygen consumption, and ATP production. Neither endogenous Hsp90 present in the homogenate nor unmodified and fully active recombinant Hsp90 was able to compete with the nitrated protein for the binding to mitochondria. Moreover, endogenous or recombinant Hsp90 did not prevent the decrease in mitochondrial activity but supported nitrated Hsp90 mitochondrial gain-of-function. Nitrotyrosine in position 33, but not in any of the other four tyrosine residues prone to nitration in Hsp90, was sufficient to down-regulate mitochondrial activity. Thus, in addition to induction of cell death, nitrated Hsp90 can also regulate mitochondrial metabolism, suggesting that depending on the cell type, distinct Hsp90 nitration states regulate different aspects of cellular metabolism. This regulation of mitochondrial homeostasis by nitrated Hsp90 could be of particular relevance in cancer cells. PMID:26085096

  15. DsHsp90 is involved in the early response of Dunaliella salina to environmental stress.

    PubMed

    Wang, Si-Jia; Wu, Ming-Jie; Chen, Xiang-Jun; Jiang, Yan; Yan, Yong-Bin

    2012-01-01

    Heat shock protein 90 (Hsp90) is a molecular chaperone highly conserved across the species from prokaryotes to eukaryotes. Hsp90 is essential for cell viability under all growth conditions and is proposed to act as a hub of the signaling network and protein homeostasis of the eukaryotic cells. By interacting with various client proteins, Hsp90 is involved in diverse physiological processes such as signal transduction, cell mobility, heat shock response and osmotic stress response. In this research, we cloned the dshsp90 gene encoding a polypeptide composed of 696 amino acids from the halotolerant unicellular green algae Dunaliella salina. Sequence alignment indicated that DsHsp90 belonged to the cytosolic Hsp90A family. Further biophysical and biochemical studies of the recombinant protein revealed that DsHsp90 possessed ATPase activity and existed as a dimer with similar percentages of secondary structures to those well-studied Hsp90As. Analysis of the nucleotide sequence of the cloned genomic DNA fragment indicated that dshsp90 contained 21 exons interrupted by 20 introns, which is much more complicated than the other plant hsp90 genes. The promoter region of dshsp90 contained putative cis-acting stress responsive elements and binding sites of transcriptional factors that respond to heat shock and salt stress. Further experimental research confirmed that dshsp90 was upregulated quickly by heat and salt shock in the D. salina cells. These findings suggested that dshsp90 might serve as a component of the early response system of the D. salina cells against environmental stresses.

  16. Spatiotemporal Regulation of Hsp90-Ligand Complex Leads to Immune Activation.

    PubMed

    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

  17. 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

  18. 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.

  19. Molecular docking study, synthesis and biological evaluation of Schiff bases as Hsp90 inhibitors.

    PubMed

    Dutta Gupta, Sayan; Snigdha, D; Mazaira, Gisela I; Galigniana, Mario D; Subrahmanyam, C V S; Gowrishankar, N L; Raghavendra, N M

    2014-04-01

    Heat shock protein 90 (Hsp90) is an emerging attractive target for the discovery of novel cancer therapeutic agents. Docking methods are powerful in silico tools for lead generation and optimization. In our mission to rationally develop novel effective small molecules against Hsp90, we predicted the potency of our designed compounds by Sybyl surflex Geom X docking method. The results of the above studies revealed that Schiff bases derived from 2,4-dihydroxy benzaldehyde/5-chloro-2,4-dihydroxy benzaldehyde demonstrated effective binding with the protein. Subsequently, a few of them were synthesized (1-10) and characterized by IR, (1)HNMR and mass spectral analysis. The synthesized molecules were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The anticancer studies were performed by 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The software generated results was in satisfactory agreement with the evaluated biological activity.

  20. STAT3 interacts directly with Hsp90.

    PubMed

    Prinsloo, Earl; Kramer, Adam H; Edkins, Adrienne L; Blatch, Gregory L

    2012-03-01

    Heat shock protein 90 (Hsp90) functionally modulates signal transduction. The signal transducer and activator of transcription 3 (STAT3) mediates interleukin-6 family cytokine signaling. Aberrant activation and mutation of STAT3 is associated with oncogenesis and immune disorders, respectively. Hsp90 and STAT3 have previously been shown to colocalize and coimmunoprecipitate in common complexes. Surface plasmon resonance spectroscopy revealed a direct, high affinity specific interaction between recombinant Hsp90β and STAT3β in the presence and absence of adenosine triphosphate (ATP) in molar excess. Furthermore, comparative analysis using a phosphomimetic mutation at tyrosine 705 showed that the direct interaction appeared to favor neither unactivated nor activated STAT3. Destabilizing mutation of STAT3 at arginine residues 414/417 to alanine in the DNA-binding domain, previously shown to disrupt nuclear translocation in vivo, reduced interaction with a STAT3 DNA binding site oligonucleotide and Hsp90β in vitro, indicating that STAT3 requires a functional DNA-binding domain for full direct interaction with Hsp90. Site-directed mutagenesis of a mammalian STAT3-EGFP-N1 fusion construct at RR414/417 and subsequent transfection into human MCF7 epithelial breast cancer cells showed no impaired nuclear translocation when observed by confocal laser scanning microscopy. However, costaining for Hsp90α/β isoforms and colocalization analysis revealed a defined decrease in pixel-on-pixel colocalization compared with the wild-type confirming the requirement of the DNA-binding domain for high-affinity interaction. PMID:22271514

  1. Chapter 8: Hsp90 and Client Protein Maturation

    PubMed Central

    Wayne, Natalie; Mishra, Parul; Bolon, Daniel N.

    2016-01-01

    Heat shock protein 90 (Hsp90) is a molecular chaperone that assists in the maturation of a limited set of substrate proteins that are collectively referred to as clients. The majority of identified Hsp90 clients are involved in signal transduction including many steroid hormone receptors and kinases. A handful of Hsp90 clients can be classified as non-signal transduction proteins including telomerase, cystic fibrosis transmembrane conductance regulator (CFTR), and antigenic peptides destined for major histocompatibility complex (MHC). Because Hsp90 clients are causative agents in cancer and cystic fibrosis, research on Hsp90 has intensified in recent years. We review the historical path of Hsp90 research within each class of client (kinase, hormone receptor, and non-signal transduction clients) and highlight current areas of active investigation. PMID:21898225

  2. Anti-Candida-biofilm activity of micafungin is attenuated by voriconazole but restored by pharmacological inhibition of Hsp90-related stress responses.

    PubMed

    Kaneko, Yukihiro; Ohno, Hideaki; Fukazawa, Hidesuke; Murakami, Yuko; Imamura, Yoshifumi; Kohno, Shigeru; Miyazaki, Yoshitsugu

    2010-06-01

    We have conducted an in vitro evaluation of the efficacy of a voriconazole-micafungin combination against Candida albicans. When used alone, both micafungin and voriconazole decreased the metabolic activity of planktonic cells, but only micafungin displayed potent anti-biofilm activity. Their combination appeared to have an additive effect against planktonic cells. However, voriconazole significantly antagonized the fungicidal effect of micafungin against Candida biofilms. Time-lag experiments showed that pre-treatment with voriconazole induced resistance to micafungin in Candida biofilms. The micafungin-antagonizing effect of voriconazole persisted even when the biofilm was no longer exposed to voriconazole. In contrast, voriconazole addition after 24 h of micafungin treatment did not alter micafungin sensitivity. To investigate the mechanism of antagonism, we used inhibitors of Hsp90 and its effectors because Hsp90 seems to be implicated in the resistance to micafungin. These molecules reversed the voriconazole-induced resistance to micafungin which suggests that Hsp90-related stress responses are involved in the antagonism. Our results may provide clues as to the mechanism of increased drug resistance in Candida biofilms and raises concerns about the use of the voriconazole-micafungin combination in clinical settings. PMID:19958255

  3. Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

    PubMed Central

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

    2016-01-01

    Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding. PMID:26929380

  4. 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

  5. The role of Hsp90 in protein complex assembly.

    PubMed

    Makhnevych, Taras; Houry, Walid A

    2012-03-01

    Hsp90 is a ubiquitous and essential molecular chaperone that plays central roles in many signaling and other cellular pathways. The in vivo and in vitro activity of Hsp90 depends on its association with a wide variety of cochaperones and cofactors, which form large multi-protein complexes involved in folding client proteins. Based on our proteomic work mapping the molecular chaperone interaction networks in yeast, especially that of Hsp90, as well as, on experiments and results presented in the published literature, one major role of Hsp90 appears to be the promotion and maintenance of proper assembly of protein complexes. To highlight this role of Hsp90, the effect of the chaperone on the assembly of the following seven complexes is discussed in this review: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome. For some complexes, it is observed that Hsp90 mediates complex assembly by stabilizing an unstable protein subunit and facilitating its incorporation into the complex; for other complexes, Hsp90 promotes change in the composition of that complex. In all cases, Hsp90 does not appear to be part of the final assembled complex. This article is part of a Special Issue entitled:Heat Shock Protein 90 (HSP90). PMID:21945180

  6. A novel C-terminal homologue of Aha1 co-chaperone binds to heat shock protein 90 and stimulates its ATPase activity in Entamoeba histolytica.

    PubMed

    Singh, Meetali; Shah, Varun; Tatu, Utpal

    2014-04-17

    Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with Kd values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12×10(-4) min(-1) μM(-1). Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. 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.

  17. 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

  18. HSP90 inhibitors as therapy for multiple myeloma.

    PubMed

    Usmani, Saad Z; Chiosis, Gabriela

    2011-06-01

    The heat shock protein 90 (HSP90) family of proteins are ubiquitous molecular chaperones that are intricately involved in folding, activation, maturation, and assembly of many proteins that include essential mediators of signal transduction and cell cycle progression. They are abundant in eukaryotic cells and localized to the cytoplasm and mitochondria as well as the endoplasmic reticulum under normal conditions, making up 1% to 2% of all cellular proteins. HSP90 proteins have increased expression in a number of malignancies, including multiple myeloma. HSP90 inhibition can influence multiple oncogenic pathways and proteins involved in myeloma, therefore making it an attractive target for drug development in this disease. This article serves as an overview of the pre-clinical data and clinical trial data on HSP90 inhibitors in multiple myeloma.

  19. 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

  20. 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

    2015-01-19

    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.

  1. 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

  2. Design, Synthesis and Biological Evaluation of Biphenylamide Derivatives as Hsp90 C-terminal Inhibitors

    PubMed Central

    Zhao, Huiping; Garg, Gaurav; Zhao, Jinbo; Moroni, Elisabetta; Girgis, Antwan; Franco, Lucas S.; Singh, Swapnil; Colombo, Giorgio; Blagg, Brian S. J.

    2015-01-01

    Modulation of Hsp90 C-terminal function represents a promising therapeutic approach for the treatment of cancer and neurodegenerative diseases. Current drug discovery efforts toward Hsp90 C-terminal inhibition focus on novobiocin, an antibiotic that was transformed into an Hsp90 inhibitor. Based on structural information obtained during the development of novobiocin derivatives and molecular docking studies, scaffolds containing a biphenyl moiety in lieu of the coumarin ring present in novobiocin were identified as new Hsp90 C-terminal inhibitors. Structure-activity relationship studies produced new derivatives that inhibit the proliferation of breast cancer cell lines at nanomolar concentrations, which corresponded directly with Hsp90 inhibition. PMID:25462258

  3. HSP90 protects the human T-cell leukemia virus type 1 (HTLV-1) tax oncoprotein from proteasomal degradation to support NF-κB activation and HTLV-1 replication.

    PubMed

    Gao, Linlin; Harhaj, Edward William

    2013-12-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients.

  4. Hsp90 in Cancer: Transcriptional Roles in the Nucleus.

    PubMed

    Calderwood, Stuart K; Neckers, Len

    2016-01-01

    Hsp90 plays a key role in fostering metabolic pathways essential in tumorigenesis through its functions as a molecular chaperone. Multiple oncogenic factors in the membrane and cytoplasm are thus protected from degradation and destruction. Here, we have considered Hsp90's role in transcription in the nucleus. Hsp90 functions both in regulating the activity of sequence-specific transcription factors such as nuclear receptors and HSF1, as well as impacting more globally acting factors that act on chromatin and RNA polymerase II. Hsp90 influences transcription by modulating histone modification mediated by its clients SMYD3 and trithorax/MLL, as well as by regulating the processivity of RNA polymerase II through negative elongation factor. It is not currently clear how the transcriptional role of Hsp90 may be influenced by the cancer milieu although recently discovered posttranslational modification of the chaperone may be involved. Dysregulation of Hsp90 may thus influence malignant processes both by modulating the function of specific transcription factors and effects on more globally acting general components of the transcriptional machinery. PMID:26916002

  5. Heat Shock Protein 90 (Hsp90) Expression and Breast Cancer

    PubMed Central

    Zagouri, Flora; Bournakis, Evangelos; Koutsoukos, Konstantinos; Papadimitriou, Christos A.

    2012-01-01

    Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc.) are currently under evaluation. PMID:24280702

  6. Alternate Strategies of Hsp90 Modulation for the Treatment of Cancer and Other Diseases

    PubMed Central

    Brandt, Gary E. L.; Blagg, Brian S. J.

    2010-01-01

    The 90 kDa heat shock protein (Hsp90) has become a validated target for the development of anti-cancer agents. Several Hsp90 inhibitors are currently under clinical trial investigation for the treatment of cancer. All of these agents inhibit Hsp90’s protein folding activity by binding to the N-terminal ATP binding site of the Hsp90 molecular chaperone. Administration of these investigational drugs elicits induction of the heat shock response, or the overexpression of several Hsps, which exhibit antiapoptotic and pro-survival effects that may complicate the application of these inhibitors. To circumvent this issue, alternate mechanisms for Hsp90 inhibition that do not elicit the heat shock response have been identified and pursued. After providing background on the structure, function, and mechanism of the Hsp90 protein folding machinery, this review describes several mechanisms of Hsp90 modulation via small molecules that do not induce the heat shock response. PMID:19860731

  7. ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system.

    PubMed

    Polier, Sigrun; Samant, Rahul S; Clarke, Paul A; Workman, Paul; Prodromou, Chrisostomos; Pearl, Laurence H

    2013-05-01

    Protein kinase clients are recruited to the Hsp90 molecular chaperone system via Cdc37, which simultaneously binds Hsp90 and kinases and regulates the Hsp90 chaperone cycle. Pharmacological inhibition of Hsp90 in vivo results in degradation of kinase clients, with a therapeutic effect in dependent tumors. We show here that Cdc37 directly antagonizes ATP binding to client kinases, suggesting a role for the Hsp90-Cdc37 complex in controlling kinase activity. Unexpectedly, we find that Cdc37 binding to protein kinases is itself antagonized by ATP-competitive kinase inhibitors, including vemurafenib and lapatinib. In cancer cells, these inhibitors deprive oncogenic kinases such as B-Raf and ErbB2 of access to the Hsp90-Cdc37 complex, leading to their degradation. Our results suggest that at least part of the efficacy of ATP-competitive inhibitors of Hsp90-dependent kinases in tumor cells may be due to targeted chaperone deprivation.

  8. 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

  9. 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.

  10. Quantitative phosphoproteomics of tomato mounting a hypersensitive response reveals a swift suppression of photosynthetic activity and a differential role for hsp90 isoforms.

    PubMed

    Stulemeijer, Iris J E; Joosten, Matthieu H A J; Jensen, Ole N

    2009-03-01

    An important mechanism by which plants defend themselves against pathogens is the rapid execution of a hypersensitive response (HR). Tomato plants containing the Cf-4 resistance gene mount an HR that relies on the activation of phosphorylation cascades, when challenged with the Avr4 elicitor secreted by the pathogenic fungus Cladosporium fulvum. Phosphopeptides were isolated from tomato seedlings expressing both Cf-4 and Avr4 using titanium dioxide columns and LC-MS/MS analysis led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Phosphopeptides were quantified using a label-free approach based on the MS peak areas. We identified 12 phosphopeptides for which the abundance changed upon HR initiation, as compared to control seedlings. Our results suggest that photosynthetic activity is specifically suppressed in a phosphorylation-dependent way during the very early stages of HR development. In addition, phosphopeptides originating from four Hsp90 isoforms exhibited altered abundances in Cf-4/Avr4 seedlings compared to control seedlings, suggesting that the isoforms of this chaperone protein have a different function in defense signaling. We show that label-free relative quantification of the phosphoproteome of complex samples is feasible, allowing extension of our knowledge on the general physiology and defense signaling of plants mounting the HR. PMID:19178300

  11. 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

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

    PubMed Central

    Zhao, Rongmin; Kakihara, Yoshito; Gribun, Anna; Huen, Jennifer; Yang, Guocheng; Khanna, May; Costanzo, Michael; Brost, Renée L.; Boone, Charles; Hughes, Timothy R.; Yip, Christopher M.; Houry, Walid A.

    2008-01-01

    Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs. PMID:18268103

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 2,4-dihydroxy benzaldehyde derived Schiff bases as small molecule Hsp90 inhibitors: rational identification of a new anticancer lead.

    PubMed

    Dutta Gupta, Sayan; Revathi, B; Mazaira, Gisela I; Galigniana, Mario D; Subrahmanyam, C V S; Gowrishankar, N L; Raghavendra, N M

    2015-04-01

    Hsp90 is a molecular chaperone that heals diverse array of biomolecules ranging from multiple oncogenic proteins to the ones responsible for development of resistance to chemotherapeutic agents. Moreover they are over-expressed in cancer cells as a complex with co-chaperones and under-expressed in normal cells as a single free entity. Hence inhibitors of Hsp90 will be more effective and selective in destroying cancer cells with minimum chances of acquiring resistance to them. In continuation of our goal to rationally develop effective small molecule azomethines against Hsp90, we designed few more compounds belonging to the class of 2,4-dihydroxy benzaldehyde derived imines (1-13) with our validated docking protocol. The molecules exhibiting good docking score were synthesized and their structures were confirmed by IR, (1)H NMR and mass spectral analysis. Subsequently, they were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The antiproliferative effect of the molecules were examined on PC3 prostate cancer cell lines by adopting 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay methodology. Finally, schiff base 13 emerged as the lead molecule for future design and development of Hsp90 inhibitors as anticancer agents.

  1. The assembly and intermolecular properties of the Hsp70-Tomm34-Hsp90 molecular chaperone complex.

    PubMed

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

    2014-04-01

    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.

  2. 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

  3. Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers

    PubMed Central

    Jhaveri, Komal; Taldone, Tony; Modi, Shanu; Chiosis, Gabriela

    2011-01-01

    Hsp90 is an ATP dependent molecular chaperone protein which integrates multiple oncogenic pathways. As such, Hsp90 inhibition is a promising anti-cancer strategy. Several inhibitors that act on Hsp90 by binding to its N-terminal ATP pocket have entered clinical evaluation. Robust pre-clinical data suggested anti-tumor activity in multiple cancer types. Clinically, encouraging results have been demonstrated in melanoma, acute myeloid leukemia, castrate refractory prostate cancer, non-small cell lung carcinoma and multiple myeloma. In breast cancer, proof-of-concept was demonstrated by first generation Hsp90 inhibitors in combination with trastuzumab mainly in human epidermal growth factor receptor 2 (HER2) + metastatic breast cancer. There are a multitude of second generation Hsp90 inhibitors currently under investigation. To date, however, there is no FDA approved Hsp90 inhibitor nor standardized assay to ascertain Hsp90 inhibition. This review summarizes the current status of both first and second generation Hsp90 inhibitors based on their chemical classification and stage of clinical development. It also discusses the pharmacodynamic assays currently implemented in clinic as well as other novel strategies aimed at enhancing the effectiveness of Hsp90 inhibitors. Ultimately, these efforts will aid in maximizing the full potential of this class of agents. PMID:22062686

  4. Inhibitor-κB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells

    PubMed Central

    Konopinski, Ryszard; Krishnan, Manickam; Roman, Linda; Bera, Alakesh; Hongying, Zheng; Habib, Samy L.; Mohan, Sumathy

    2015-01-01

    Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-κB kinase-β (IKKβ)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKKβ on Hsp90. Interestingly, IKKβ binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKKβ to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKKβ. The pathophysiological relevance of the IKKβ-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factor-mediated signaling and an Ins2Akita in vivo model. Our study further defines the preferential involvement of α- vs. β-isoforms of Hsp90 in the IKKβ-eNOS-Hsp90 interaction, even though both Hsp90α and Hsp90β stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKKβ within the cell system that regulates NO production, but they also confirm that the IKKβ-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications in diabetes. PMID:25652452

  5. 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

  6. 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

  7. 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.

  8. Hsp90 as a "Chaperone" of the Epigenome: Insights and Opportunities for Cancer Therapy.

    PubMed

    Isaacs, Jennifer S

    2016-01-01

    The cellular functions of Hsp90 have historically been attributed to its ability to chaperone client proteins involved in signal transduction. Although numerous stimuli and the signaling cascades they activate contribute to cancer progression, many of these pathways ultimately require transcriptional effectors to elicit tumor-promoting effects. Despite this obvious connection, the majority of studies evaluating Hsp90 function in malignancy have focused upon its regulation of cytosolic client proteins, and particularly members of receptor and/or kinase families. However, in recent years, Hsp90 has emerged as a pivotal orchestrator of nuclear events. Discovery of an expanding repertoire of Hsp90 clients has illuminated a vital role for Hsp90 in overseeing nuclear events and influencing gene transcription. Hence, this chapter will cast a spotlight upon several regulatory themes involving Hsp90-dependent nuclear functions. Highlighted topics include a summary of chaperone-dependent regulation of key transcription factors (TFs) and epigenetic effectors in malignancy, as well as a discussion of how the complex interplay among a subset of these TFs and epigenetic regulators may generate feed-forward loops that further support cancer progression. This chapter will also highlight less recognized indirect mechanisms whereby Hsp90-supported signaling may impinge upon epigenetic regulation. Finally, the relevance of these nuclear events is discussed within the framework of Hsp90's capacity to enable phenotypic variation and drug resistance. These newly acquired insights expanding our understanding of Hsp90 function support the collective notion that nuclear clients are major beneficiaries of Hsp90 action, and their impairment is likely responsible for many of the anticancer effects elicited by Hsp90-targeted approaches.

  9. Overexpression of Hsp90 from grass carp (Ctenopharyngodon idella) increases thermal protection against heat stress.

    PubMed

    Wu, Chu-Xin; Zhao, Feng-Yun; Zhang, Yuan; Zhu, Yu-Jiao; Ma, Mei-Sheng; Mao, Hui-Ling; Hu, Cheng-Yu

    2012-07-01

    With homologous DNA probes, we had screened a grass carp heat shock protein 90 gene (CiHsp90). The full sequence of CiHsp90 cDNA was 2793 bp, which could code a 798 amino acids peptide. The phylogenetic analysis demonstrated that CiHsp90 shared the high homology with Zebrafish Grp94. Quantitative RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34 °C or cold stress at 4 °C. To understand the function of CiHsp90 involving in thermal protection, an expression vector containing coding region cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37 °C to 42 °C, these cells that accumulated CiHsp90 peptides displayed greater thermoresistance than the control cells. While incubated at 4°C for different periods, it could also improve the cell viability. After transient transfected recombinant plasmid pcDNA3.1/CiHsp90 into mouse myeloma cell line SP2/0, we found that CiHsp90 could contribute to protecting cells against both thermal and cold extremes. On the contrary, the mutant construct ΔN-CiHsp90 (256-798aa) could abolish the protection activity both in prokaryotic cells and eukaryotic cells. Additionally, both CiHsp90 and ΔN-CiHsp90 peptides could reduce the level of citrate synthase aggregation at the high temperature.

  10. Hsp90: A New Player in DNA Repair?

    PubMed Central

    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

  11. Why is this effective HSP90 inhibitor not being developed in HER2+ breast cancer?

    PubMed

    Arteaga, Carlos L

    2011-08-01

    Inhibition of the HSP90 chaperone leads to degradation of the HER2 receptor. The HSP90 inhibitor tanespimycin in combination with trastuzumab is active in patients with HER2-overexpressing metastatic breast cancer. This combination is one of several HER2-targeted therapies that will significantly improve the outcome of patients with this subtype of breast cancer.

  12. High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase.

    PubMed

    Galam, Lakshmi; Hadden, M Kyle; Ma, Zeqiang; Ye, Qi-Zhuang; Yun, Bo-Geon; Blagg, Brian S J; Matts, Robert L

    2007-03-01

    Previously, we have demonstrated that the renaturation of heat denatured firefly luciferase is dependent upon the activity of Hsp90 in rabbit reticulocyte lysate. Here, we demonstrate that this assay may identify inhibitors that obstruct the chaperone activity of Hsp90 either by direct binding to its N-terminal or C-terminal nucleotide binding sites or by interference with the ability of the chaperone to switch conformations. The assay was adapted and optimized for high-throughput screening. Greater than 20,000 compounds were screened to demonstrate the feasibility of using this assay on a large scale. The assay was reproducible (av Z-factor=0.62) and identified 120 compounds that inhibited luciferase renaturation by greater than 70% at a concentration of 12.5 microg/mL. IC50 values for twenty compounds with varying structures were determined for inhibition of luciferase refolding and in cell-based assays for Hsp90 inhibition. Several compounds had IC50 values <10 microM and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors.

  13. 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

  14. 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.

  15. Analysis of Hsp90 cochaperone interactions reveals a novel mechanism for TPR protein recognition.

    PubMed

    Chadli, Ahmed; Bruinsma, Elizabeth S; Stensgard, Bridget; Toft, David

    2008-03-01

    The chaperone Hsp90 is required for the appropriate regulation of numerous key signaling molecules, including the progesterone receptor (PR). Many important cochaperones bind Hsp90 through their tetratricopeptide repeat (TPR) domains. Two such proteins, GCUNC45 and FKBP52, assist PR chaperoning and are thought to interact sequentially with PR-Hsp90 complexes. TPR proteins bind to the C-terminal MEEVD sequence of Hsp90, but GCUNC45 has been shown also to bind to a novel site near the N-terminus. We now show that FKBP52 is also able to bind to this site, and that these two cochaperones act competitively, through Hsp90, to modulate PR activity. The N-terminal site involves noncontiguous amino acids within or near the ATP binding pocket of Hsp90. TPR interactions at this site are thus strongly regulated by nucleotide binding and Hsp90 conformation. We propose an expanded model for client chaperoning in which the coordinated use of TPR recognition sites at both N- and C-terminal ends of Hsp90 enhances its ability to coordinate interactions with multiple TPR partners.

  16. 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

  17. 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.

  18. Expression of Heat Shock Protein (Hsp90) Paralogues Is Regulated by Amino Acids in Skeletal Muscle of Atlantic Salmon

    PubMed Central

    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

  19. 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

  20. 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

    2014-04-30

    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.

  1. Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors.

    PubMed

    Immormino, Robert M; Kang, Yanlong; Chiosis, Gabriela; Gewirth, Daniel T

    2006-08-10

    Hsp90 chaperones play a critical role in modulating the activity of many cell signaling proteins and are an attractive target for anti-cancer therapeutics. We report here the structures of the water soluble 8-aryl-sulfanyl adenine class Hsp90 inhibitors, 1 (PU-H71) and 2 (PU-H64), in complex with the N-terminal domain of human Hsp90alpha. The conformation of 1 when bound to Hsp90 differs from previously reported 8-aryl adenine Hsp90 inhibitors including 3 (PU24FCl). While the binding mode for 3 places the 2'-halide of the 8-aryl group on top of the adenine ring, for 1 and 2, we show that the 2'-halide is rotated approximately 180 degrees away. This difference explains the opposing trends in Hsp90 inhibitory activity for the 2'-halo derivatives of the 3',4',5'-trimethoxy series where Cl > Br > I compared to the 4',5'-methylenedioxy series where I > Br > Cl. We also present quantum chemical calculations of 2 and its analogues that illuminate their basis for Hsp90 inhibition. The calculated conformation of 2 agreed well with the crystallographically observed conformations of 1 and 2. The predictive nature of the calculations has allowed the exploration of additional derivatives based on the 8-aryl adenine scaffold.

  2. 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

  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. The role of HSP90 in evolution.

    PubMed

    Yahara, I

    1999-07-01

    A mechanism by which morphological mutations are stored without expressing phenotypes was unraveled by Rutherford & Lindquist (1998) through genetic studies of Hsp83 (HSP90) in Drosophila. Cryptic mutations are essentially neutral and therefore evolve in the absence of selective constraint. A shift from neutral mutations to selective mutations is induced when flies are exposed to environmental stress. This is a step toward understanding macroevolution in molecular terms.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. The Hsp90-Dependent Proteome Is Conserved and Enriched for Hub Proteins with High Levels of Protein–Protein Connectivity

    PubMed Central

    Swamy, Krishna B.S.; Yu, Jau-Song; Schuyler, Scott C.; Leu, Jun-Yi

    2014-01-01

    Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein–protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the “stable isotope labeling by amino acids in cell culture” method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes. PMID:25316598

  10. Crosstalk between Hsp90 and Hsp70 chaperones and heat stress transcription factors in tomato.

    PubMed

    Hahn, Alexander; Bublak, Daniela; Schleiff, Enrico; Scharf, Klaus-Dieter

    2011-02-01

    Heat stress transcription factors (Hsfs) regulate gene expression in response to environmental stress. The Hsf network in plants is controlled at the transcriptional level by cooperation of distinct Hsf members and by interaction with chaperones. We found two general mechanisms of Hsf regulation by chaperones while analyzing the three major Hsfs, A1, A2, and B1, in tomato (Solanum lycopersicum). First, Hsp70 and Hsp90 regulate Hsf function by direct interactions. Hsp70 represses the activity of HsfA1, including its DNA binding, and the coactivator function of HsfB1 in the complex with HsfA2, while the DNA binding activity of HsfB1 is stimulated by Hsp90. Second, Hsp90 affects the abundance of HsfA2 and HsfB1 by modulating hsfA2 transcript degradation involved in regulation of the timing of HsfA2 synthesis. By contrast, HsfB1 binding to Hsp90 and to DNA are prerequisites for targeting this Hsf for proteasomal degradation, which also depends on a sequence element in its carboxyl-terminal domain. Thus, HsfB1 represents an Hsp90 client protein that, by interacting with the chaperone, is targeted for, rather than protected from, degradation. Based on these findings, we propose a versatile regulatory regime involving Hsp90, Hsp70, and the three Hsfs in the control of heat stress response. PMID:21307284

  11. The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop

    PubMed Central

    Schmid, Andreas B; Lagleder, Stephan; Gräwert, Melissa Ann; Röhl, Alina; Hagn, Franz; Wandinger, Sebastian K; Cox, Marc B; Demmer, Oliver; Richter, Klaus; Groll, Michael; Kessler, Horst; Buchner, Johannes

    2012-01-01

    Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A–TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1–DP1 module may serve as an Hsp70–client delivery system for the TPR2A–TPR2B–DP2 segment, which is required for client activation in vivo. PMID:22227520

  12. 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

  13. 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

  14. Binding mechanism between Hsp90 and Sgt1 explored by homology modeling and molecular dynamics simulations in rice.

    PubMed

    Yan, Jun-jie; Zhang, Yu-bo; Ding, Yi

    2012-10-01

    The Hsp90 (for heat shock protein90) and the Sgt1 (for suppressor of the G2 allele of skp1) are widely distributed in animals, yeast, and plants. The former functions as molecular chaperon activating a series of client proteins, the latter functions as an adaptor protein participating in multiple biological processes such as immunity response through interactions with different protein complexes. In the present study, we have constructed a homology model of Hsp90-Sgt1 complex in rice based on a recently resolved structure from barley and Arabidopsis to explore its binding mechanisms and to understand the detailed interaction profile. A total of 20 ns explicit solvent molecular dynamics simulations combined with MM-GBSA computations and virtual alanine scanning were performed for the modeled complex. In the final structure, three strong salt bridges were found between OsHsp90 and OsSgt1, D217(OsHsp90)-K186(OsSgt1), D218(OsHsp90)-K237(OsSgt1) and K161(OsHsp90)-E239(OsSgt1). Besides, residue Y173 of OsSgt1 played a vital role in the interactions with OsHsp90, the detailed interactions were discussed. These results would help us understand the critical features determining the Hsp90-Sgt1 binding process. PMID:22653607

  15. A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation

    PubMed Central

    Echeverría, Pablo C.; Briand, Pierre-André

    2016-01-01

    Hsp90 is the ATP-consuming core component of a very abundant molecular chaperone machine that handles a substantial portion of the cytosolic proteome. Rather than one machine, it is in fact an ensemble of molecular machines, since most mammalian cells express two cytosolic isoforms of Hsp90 and a subset of up to 40 to 50 cochaperones and regulate their interactions and functions by a variety of posttranslational modifications. We demonstrate that the Hsp90 ensemble is fundamentally remodeled during muscle differentiation and that this remodeling is not just a consequence of muscle differentiation but possibly one of the drivers to accompany and to match the vast proteomic changes associated with this process. As myoblasts differentiate into myotubes, Hsp90α disappears and only Hsp90β remains, which is the only isoform capable of interacting with the novel muscle-specific Hsp90 cochaperone Aarsd1L. Artificially maintaining Hsp90α or knocking down Aarsd1L expression interferes with the differentiation of C2C12 myotubes. During muscle differentiation, Aarsd1L replaces the more ubiquitous cochaperone p23 and in doing so dampens the activity of the glucocorticoid receptor, one of the Hsp90 clients relevant to muscle functions. This cochaperone switch protects muscle cells against the inhibitory effects of glucocorticoids and may contribute to preventing muscle wasting induced by excess glucocorticoids. PMID:26884463

  16. 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.

  17. Experimental and Structural Testing Module to Analyze Paralogue-Specificity and Affinity in the Hsp90 Inhibitors Series

    PubMed Central

    Taldone, Tony; Patel, Pallav D.; Patel, Maulik; Patel, Hardik J.; Evans, Christopher E.; Rodina, Anna; Ochiana, Stefan; Shah, Smit K.; Uddin, Mohammad; Gewirth, Daniel; Chiosis, Gabriela

    2014-01-01

    We here describe the first reported comprehensive analysis of Hsp90 paralogue affinity and selectivity in the clinical Hsp90 inhibitor chemotypes. This has been possible through the development of a versatile experimental assay based on a new FP-probe (16a) that we both describe here. The assay can test rapidly and accurately the binding affinity of all major Hsp90 chemotypes and has a testing range that spans low nanomolar to millimolar binding affinities. We couple this assay with a computational analysis that allows for rationalization of paralogue selectivity and defines not only the major binding modes that relay pan-paralogue binding or, conversely, paralogue selectivity, but also identifies molecular characteristics that impart such features. The methods developed here provide a blueprint for parsing out the contribution of the four Hsp90 paralogues to the perceived biological activity with the current Hsp90 chemotypes and set the ground for the development of paralogue selective inhibitors. PMID:23965125

  18. Hsp90 modulates CAG repeat instability in human cells

    PubMed Central

    Mittelman, David; Sykoudis, Kristen; Hersh, Megan; Lin, Yunfu

    2010-01-01

    The Hsp90 molecular chaperone has been implicated as a contributor to evolution in several organisms by revealing cryptic variation that can yield dramatic phenotypes when the chaperone is diverted from its normal functions by environmental stress. In addition, as a cancer drug target, Hsp90 inhibition has been documented to sensitize cells to DNA-damaging agents, suggesting a function for Hsp90 in DNA repair. Here we explore the potential role of Hsp90 in modulating the stability of nucleotide repeats, which in a number of species, including humans, exert subtle and quantitative consequences for protein function, morphological and behavioral traits, and disease. We report that impairment of Hsp90 in human cells induces contractions of CAG repeat tracks by tenfold. Inhibition of the recombinase Rad51, a downstream target of Hsp90, induces a comparable increase in repeat instability, suggesting that Hsp90-enabled homologous recombination normally functions to stabilize CAG repeat tracts. By contrast, Hsp90 inhibition does not increase the rate of gene-inactivating point mutations. The capacity of Hsp90 to modulate repeat-tract lengths suggests that the chaperone, in addition to exposing cryptic variation, might facilitate the expression of new phenotypes through induction of novel genetic variation. PMID:20373063

  19. Hsp90-Dependent Assembly of the DBC2/RhoBTB2-Cullin3 E3-Ligase Complex

    PubMed Central

    Manjarrez, Jacob R.; Sun, Liang; Prince, Thomas; Matts, Robert L.

    2014-01-01

    The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes. PMID:24608665

  20. Secreted Hsp90 Is a Novel Regulator of the Epithelial to Mesenchymal Transition (EMT) in Prostate Cancer*

    PubMed Central

    Hance, Michael W.; Dole, Krystal; Gopal, Udhayakumar; Bohonowych, Jessica E.; Jezierska-Drutel, Agnieszka; Neumann, Carola A.; Liu, Haibo; Garraway, Isla P.; Isaacs, Jennifer S.

    2012-01-01

    Prostate cancer (PCa) is the most frequently diagnosed malignancy in men, and the second highest contributor of male cancer related lethality. Disease mortality is due primarily to metastatic spread, highlighting the urgent need to identify factors involved in this progression. Activation of the genetic epithelial to mesenchymal transition (EMT) program is implicated as a major contributor of PCa progression. Initiation of EMT confers invasive and metastatic behavior in preclinical models and is correlated with poor clinical prognosis. Extracellular Hsp90 (eHsp90) promotes cell motility and invasion in cancer cells and metastasis in preclinical models, however, the mechanistic basis for its widespread tumorigenic function remains unclear. We have identified a novel and pivotal role for eHsp90 in driving EMT events in PCa. In support of this notion, more metastatic PCa lines exhibited increased eHsp90 expression relative to their lineage-related nonmetastatic counterparts. We demonstrate that eHsp90 promoted cell motility in an ERK and matrix metalloproteinase-2/9-dependent manner, and shifted cellular morphology toward a mesenchymal phenotype. Conversely, inhibition of eHsp90 attenuated pro-motility signaling, blocked PCa migration, and shifted cell morphology toward an epithelial phenotype. Last, we report that surface eHsp90 was found in primary PCa tumor specimens, and elevated eHsp90 expression was associated with increased levels of matrix metalloproteinase-2/9 transcripts. We conclude that eHsp90 serves as a driver of EMT events, providing a mechanistic basis for its ability to promote cancer progression and metastasis in preclinical models. Furthermore, its newly identified expression in PCa specimens, and potential regulation of pro-metastatic genes, supports a putative clinical role for eHsp90 in PCa progression. PMID:22989880

  1. Control of canalization and evolvability by Hsp90.

    PubMed

    Milton, Claire C; Ulane, Christina M; Rutherford, Suzannah

    2006-01-01

    Partial reduction of Hsp90 increases expression of morphological novelty in qualitative traits of Drosophila and Arabidopsis, but the extent to which the Hsp90 chaperone also controls smaller and more likely adaptive changes in natural quantitative traits has been unclear. To determine the effect of Hsp90 on quantitative trait variability we deconstructed genetic, stochastic and environmental components of variation in Drosophila wing and bristle traits of genetically matched flies, differing only by Hsp90 loss-of-function or wild-type alleles. Unexpectedly, Hsp90 buffering was remarkably specific to certain normally invariant and highly discrete quantitative traits. Like the qualitative trait phenotypes controlled by Hsp90, highly discrete quantitative traits such as scutellor and thoracic bristle number are threshold traits. When tested across genotypes sampled from a wild population or in laboratory strains, the sensitivity of these traits to many types of variation was coordinately controlled, while continuously variable bristle types and wing size, and critically invariant left-right wing asymmetry, remained relatively unaffected. Although increased environmental variation and developmental noise would impede many types of selection response, in replicate populations in which Hsp90 was specifically impaired, heritability and 'extrinsic evolvability', the expected response to selection, were also markedly increased. However, despite the overall buffering effect of Hsp90 on variation in populations, for any particular individual or genotype in which Hsp90 was impaired, the size and direction of its effects were unpredictable. The trait and genetic-background dependence of Hsp90 effects and its remarkable bias toward invariant or canalized traits support the idea that traits evolve independent and trait-specific mechanisms of canalization and evolvability through their evolution of non-linearity and thresholds. Highly non-linear responses would buffer variation

  2. Structural asymmetry in the closed state of mitochondrial Hsp90 (TRAP1) supports a two-step ATP hydrolysis mechanism

    PubMed Central

    Lavery, Laura A.; Partridge, James R.; Ramelot, Theresa A.; Elnatan, Daniel; Kennedy, Michael A.; Agard, David A.

    2014-01-01

    Summary While structural symmetry is a prevailing feature of homo-oligomeric proteins, asymmetry provides unique mechanistic opportunities. We present the crystal structure of full-length TRAP1, the mitochondrial Hsp90 molecular chaperone, in a catalytically active closed state. The TRAP1 homodimer adopts a distinct, asymmetric conformation, where one protomer is reconfigured via a helix swap at the Middle:C-terminal Domain (MD:CTD) interface. Importantly, this interface plays a critical role in client binding. Solution methods validate the asymmetry and show extension to Hsp90 homologs. Point mutations that disrupt unique contacts at each MD:CTD interface reduce catalytic activity, substrate binding, and demonstrate that each protomer needs access to both conformations. Crystallographic data on a dimeric NTD:MD fragment suggests that asymmetry arises from strain induced by simultaneous NTD and CTD dimerization. The observed asymmetry provides the potential for an additional step in the ATPase cycle, allowing sequential ATP hydrolysis steps to drive both client remodeling and client release. PMID:24462206

  3. 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

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

    PubMed

    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; Fröhling, Stefan; Scholl, Claudia

    2012-04-01

    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.

  5. 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

  6. The Hsp90 cochaperone, FKBP51, increases Tau stability and polymerizes microtubules.

    PubMed

    Jinwal, Umesh K; Koren, John; Borysov, Sergiy I; Schmid, Andreas B; Abisambra, Jose F; Blair, Laura J; Johnson, Amelia G; Jones, Jeffrey R; Shults, Cody L; O'Leary, John C; Jin, Ying; Buchner, Johannes; Cox, Marc B; Dickey, Chad A

    2010-01-13

    Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Our previous work demonstrated that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimer's disease. Here, we show that the Hsp90 cochaperone, FK506-binding protein 51 (FKBP51), which possesses both an Hsp90-interacting tetratricopeptide domain and a peptidyl-prolyl cis-trans isomerase (PPIase) domain, prevents tau clearance and regulates its phosphorylation status. Regulation of the latter is dependent on the PPIase activity of FKBP51. FKB51 enhances the association of tau with Hsp90, but the FKBP51/tau interaction is not dependent on Hsp90. In vitro FKBP51 stabilizes microtubules with tau in a reaction depending on the PPIase activity of FKBP51. Based on these new findings, we propose that FKBP51 can use the Hsp90 complex to isomerize tau, altering its phosphorylation pattern and stabilizing microtubules.

  7. Discovery of potent N-(isoxazol-5-yl)amides as HSP90 inhibitors.

    PubMed

    Chen, Danqi; Shen, Aijun; Li, Jian; Shi, Feng; Chen, Wuyan; Ren, Jing; Liu, Hongchun; Xu, Yechun; Wang, Xin; Yang, Xinying; Sun, Yiming; Yang, Min; He, Jianhua; Wang, Yueqin; Zhang, Liping; Huang, Min; Geng, Meiyu; Xiong, Bing; Shen, Jingkang

    2014-11-24

    HSP90 is ubiquitously overexpressed in a broad spectrum of human cancers and has been recognized as an attractive target for cancer treatment. Here, we described the fragment screening, synthesis and structure-activity relationship studies of small molecule inhibitors with 4,5-diarylisoxazole scaffold targeting HSP90. Among them, the compound N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-morpholinopiperidin-1-yl)methyl)phenyl)isoxazol-5-yl)cyclopropanecarboxamide (108) showed high affinity for binding to HSP90 (FP binding assay, IC50 = 0.030 μM) and inhibited the proliferation of various human cancer cell lines with averaging GI50 about 88 nM. Compound 108 exhibited its functional inhibition of HSP90 by depleting key signaling pathways and concomitantly elevating of HSP70 and HSP27 in U-87MG cells. Further in vivo studies showed that compound 108 strongly suppressed the tumor growth of human glioblastoma xenograft model U-87MG with T/C = 18.35% at 50 mg/kg q3w/2.5w. Moreover, compound 108 also exhibited good pharmacokinetic properties. Together, our study implicates that compound 108 is a promising candidate of HSP90 inhibitor and is currently advanced to preclinical study.

  8. 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

  9. Uncovering a region of Hsp90 important for client binding in E. coli and chaperone function in yeast

    PubMed Central

    Genest, Olivier; Reidy, Michael; Street, Timothy O.; Hoskins, Joel R.; Camberg, Jodi L.; Agard, David A.; Masison, Daniel C.; Wickner, Sue

    2012-01-01

    Summary The Hsp90 family of heat shock proteins is an abundantly expressed and highly conserved family of ATP-dependent molecular chaperones. Hsp90 facilitates remodeling and activation of hundreds of proteins. In this study we developed a screen to identify Hsp90 defective mutants in E. coli. The mutations obtained define a region incorporating residues from the middle and C-terminal domains of E. coli Hsp90. The mutant proteins are defective in chaperone activity and client binding in vitro. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90. PMID:23260660

  10. 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

  11. Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in Saccharomyces cerevisiae.

    PubMed

    Kumar, Navinder; Gaur, Deepika; Gupta, Arpit; Puri, Anuradhika; Sharma, Deepak

    2015-10-01

    The role of Hsp70 chaperones in yeast prion propagation is well established. Highly conserved Hsp90 chaperones participate in a number of cellular processes, such as client protein maturation, protein degradation, cellular signalling and apoptosis, but little is known about their role in propagation of infectious prion like aggregates. Here, we examine the influence of Hsp90 in the maintenance of yeast prion [URE3] which is a prion form of native protein Ure2, and reveal a previously unknown role of Hsp90 as an important regulator of [URE3] stability. We show that the C-terminal MEEVD pentapeptide motif, but not the client maturation activity of Hsp90, is essential for [URE3] prion stability. In testing deletions of various Hsp90 co-chaperones known to bind this motif, we find the immunophilin homolog Cpr7 is essential for [URE3] propagation. We show that Cpr7 interacts with Ure2 and enhances its fibrillation. The requirement of Cpr7 is specific for [URE3] as its deletion does not antagonize both strong and weak variant of another yeast prion [PSI+], suggesting a distinct role of the Hsp90 co-chaperone with different yeast prions. Our data show that, similar to the Hsp70 family, the Hsp90 chaperones also influence yeast prion maintenance, and that immunophilins could regulate protein multimerization independently of their activity as peptidyl-prolyl isomerases.

  12. Development of a high-throughput screening cancer cell-based luciferase refolding assay for identifying Hsp90 inhibitors.

    PubMed

    Sadikot, Takrima; Swink, Megan; Eskew, Jeffery D; Brown, Douglas; Zhao, Huiping; Kusuma, Bhaskar R; Rajewski, Roger A; Blagg, Brian S J; Matts, Robert L; Holzbeierlein, Jeffrey M; Vielhauer, George A

    2013-10-01

    The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu. PMID:24127661

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Synthesis and Biological Evaluation of Coumarin Replacements of Novobiocin as Hsp90 Inhibitors

    PubMed Central

    Kusuma, Bhaskar Reddy; Khandelwal, Anuj; Gu, Wen; Brown, Douglas; Liu, Weiya; Vielhauer, George; Holzbeierlein, Jeffrey; Blagg, Brian S. J.

    2014-01-01

    Since Hsp90 modulates all six hallmarks of cancer simultaneously, it has become an attractive target for the development of cancer chemotherapeutics. In an effort to develop more efficacious compounds for Hsp90 inhibition, novobiocin analogues were prepared by replacing the central coumarin core with naphthalene, quinolinone, and quinoline surrogates. These modifications allowed for modification of the 2-position, which was previously unexplored. Biological evaluation of these compounds suggests a hydrophobic pocket about the 2-position of novobiocin. Anti-proliferative activities of these analogues against multiple cancer cell lines identified 2-alkoxyquinoline derivatives to exhibit improved activity. PMID:24461493

  20. 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

  1. 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.

  2. Hsp90: A Global Regulator of the Genotype-to-Phenotype Map in Cancers.

    PubMed

    Jarosz, Daniel

    2016-01-01

    Cancer cells have the unusual capacity to limit the cost of the mutation load that they harbor and simultaneously harness its evolutionary potential. This property fuels drug resistance, a key failure mode in oncogene-directed therapy. However, the factors that regulate this capacity might also provide an Achilles' heel that could be exploited therapeutically. Recently, insight has come from a seemingly distant field: protein folding. It is now clear that protein homeostasis broadly supports malignancy and fuels the rapid evolution of drug resistance. Among protein homeostatic mechanisms that influence cancer biology, the essential ATP-driven molecular chaperone heat-shock protein 90 (Hsp90) is especially important. Hsp90 catalyzes folding of many proteins that regulate growth and development. These "client" kinases, transcription factors, and ubiquitin ligases often play critical roles in human disease, especially cancer. Studies in a wide range of systems-from single-celled organisms to human tumor samples-suggest that Hsp90 can broadly reshape the map between genotype and phenotype, acting as a "capacitor" and "potentiator" of genetic variation. Indeed, it has likely done so to such a degree that it has left an impress on diverse genome sequences. Hsp90 can constitute as much as 5% of total protein in transformed cells and increased levels of heat-shock activation correlate with poor prognosis in breast cancer. These findings and others have motivated a flurry of interest in Hsp90 inhibitors as cancer therapeutics, which have met with rather limited success as single agents, but may eventually prove invaluable in limiting the emergence of resistance to other chemotherapeutics, both genotoxic and molecularly targeted. Here, we provide an overview of Hsp90 function, review its relationship to genetic variation and the evolution of new traits, and discuss the importance of these findings for cancer biology and future efforts to drug this pathway.

  3. 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

  4. Design, Synthesis and Evaluation of Small Molecule Hsp90 Probes

    PubMed Central

    Taldone, Tony; Zatorska, Danuta; Patel, Pallav D.; Zong, Hongliang; Rodina, Anna; Ahn, James H.; Moulick, Kamalika; Guzman, Monica L.

    2011-01-01

    A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated amongst these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors. PMID:21459002

  5. 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

  6. 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.

  7. Targeting highly expressed extracellular HSP90 in breast cancer stem cells inhibits tumor growth in vitro and in vivo.

    PubMed

    Stivarou, Theodora; Stellas, Dimitris; Vartzi, Georgia; Thomaidou, Dimitra; Patsavoudi, Evangelia

    2016-08-01

    Breast cancer stem cells (BCSC) have been identified in breast carcinoma as CD44(+)/CD24(-/low) cells, which display tumorigenic activity and have the ability to self-renew, differentiate and metastasize. Previous studies showed that extracellular HSP90 (eHSP90) participates in the invasion and metastatic processes of various cancers including breast cancer. Here, we show for the first time that eHSP90 is over-expressed in mammosphere cultures that are derived from the MDA-MB-231, MDA-MB-453 and MCF-7 breast cancer cell lines. These mammospheres are highly enriched in cells of the CD44(+)/CD24(-/low) BCSC phenotype and additionally show high expression of the BCSC markers CD49f and Sox2. Thus our results indicate that eHSP90 represents a potential novel BCSC marker. Moreover, we present evidence that eHSP90 is functionally involved in BCSC activity in vitro and in vivo. Selective neutralization of eHSP90, using the monoclonal antibody mAb 4C5, has the capacity to inhibit stem cell activity in vitro because the formation of mammosphere-derived colonies is dramatically reduced in its presence. In vivo, the treatment of mice with mAb4C5 using a prophylactic protocol, significantly inhibited the primary growth of MDA-MB-231 and mammosphere-derived tumors. More importantly, administration of this antibody in a therapeutic protocol caused a statistically significant regression of established tumors derived from MDA-MB-231 originating mammospheres. Tumor regression was even greater when mAb 4C5 was administered in combination with paclitaxel. Overall, our findings implicate eHSP90 as a potential novel BCSC biomarker. Moreover they show that eHSP90 participates in BCSC-derived primary tumor growth. Finally, we provide additional support for the possible therapeutic value of mAb4C5 in the treatment of breast cancer. PMID:27259689

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. Effectively Delivering a Unique Hsp90 Inhibitor Using Star Polymers

    PubMed Central

    2013-01-01

    We report the synthesis of a novel heat shock protein 90 (hsp90) inhibitor conjugated to a star polymer. Using reversible addition–fragmentation chain-transfer (RAFT) polymerization, we prepared star polymers comprising PEG attached to a predesigned functional core. The stars were cross-linked using disulfide linkers, and a tagged version of our hsp90 inhibitor was conjugated to the polymer core to generate nanoparticles (14 nm). Dynamic light scattering showed that the nanoparticles were stable in cell growth media for 5 days, and high-performance liquid chromatography (HPLC) analysis of compound-release at 3 different pH values showed that release was pH dependent. Cell cytotoxicity studies and confocal microscopy verify that our hsp90 inhibitor was delivered to cells using this nanoparticle delivery system. Further, delivery of our hsp90 inhibitor using star polymer induces apoptosis by a caspase 3-dependent pathway. These studies show that we can deliver our hsp90 inhibitor effectively using star polymers and induce apoptosis by the same pathway as the parent compound. PMID:24379910

  14. Hsp90 binds and regulates Gcn2, the ligand-inducible kinase of the alpha subunit of eukaryotic translation initiation factor 2 [corrected].

    PubMed

    Donzé, O; Picard, D

    1999-12-01

    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

  15. 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

  16. 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.

  17. The Soluble Recombinant Neisseria meningitidis Adhesin NadAΔ351–405 Stimulates Human Monocytes by Binding to Extracellular Hsp90

    PubMed Central

    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 NadAΔ351–405, 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 NadAΔ351–405 cellular effects in monocytes. We show that NadAΔ351–405 (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 NadAΔ351–405 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 NadAΔ351–405 /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadAΔ351–405 and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadAΔ351–405 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 NadAΔ351–405 alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by

  18. 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

  19. 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."

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

    PubMed

    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

  1. 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

  2. [Changes in heat shock protein synthesis and thermotolerance of Arabidopsis thaliana seedlings as a result of inhibition of Hsp90 by geldanamycin].

    PubMed

    Kozeko, L G

    2014-01-01

    The influence of geldanamycin (GA), which is a specific inhibitor of heat shock protein Hsp90 activities, on synthesis of Hsp70 and Hsp90 and thermotolerance of Arabidopsis thaliana seedlings has been studied. Incubation of seedlings with GA was shown to induce synthesis of these stress proteins under normal conditions. Treatment of seeds with the Hsp90 inhibitor resulted in the elevated constitutive levels of Hsp70 and Hsp90 in seedlings as well as increased induction of their synthesis under heat shock, at that the effect of GA increased with its concentration. These up-regulation of Hsp promoted thermotolerance of seedlings. The obtained results are considered as evidence for autoregulation of heat shock protein synthesis and regulation of plant tolerance by Hsp90.

  3. P-glycoprotein (P-gp)-mediated efflux limits intestinal absorption of the Hsp90 inhibitor SNX-2112 in rats.

    PubMed

    Liu, Hongming; Sun, Hua; Wu, Zhufeng; Zhang, Xingwang; Wu, Baojian

    2014-08-01

    1. The promising anticancer agent SNX-2112 (a novel Hsp90 inhibitor) is poorly bioavailable after oral administration. Here, we aim to determine the role of P-glycoprotein (P-gp) in the intestinal absorption of SNX-2112. 2. We found that SNX-2112 significantly stimulated P-gp ATPase activity in in vitro ATPase assay with a small EC50 (the half-maximal effective concentration) value of 0.32 µM. 3. In the single-pass perfused rat intestine model, absorption of SNX-2112 was not favored in the small intestine with a [Formula: see text] (the wall permeability) value of 0.38-0.64. By contrast, the compound was well absorbed in the colon with a [Formula: see text] value of 1.19. The P-gp inhibitors cyclosporine and elacridar (i.e. GF120918A) markedly enhanced SNX-2112 absorption in all four intestinal segments (i.e. duodenum, jejunum, ileum and colon) and the fold change ranged from 3.1 to 14.1. Pharmacokinetic study revealed that cyclosporine increased the systemic exposure of SNX-2112 by a 2.5-fold after oral administration. 4. This is the first report that P-gp-mediated efflux is a limiting factor for intestinal absorption of SNX-2112 in rats.

  4. 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

  5. Genome-scale co-evolutionary inference identifies functions and clients of bacterial Hsp90.

    PubMed

    Press, Maximilian O; Li, Hui; Creanza, Nicole; Kramer, Günter; Queitsch, Christine; Sourjik, Victor; Borenstein, Elhanan

    2013-01-01

    The molecular chaperone Hsp90 is essential in eukaryotes, in which it facilitates the folding of developmental regulators and signal transduction proteins known as Hsp90 clients. In contrast, Hsp90 is not essential in bacteria, and a broad characterization of its molecular and organismal function is lacking. To enable such characterization, we used a genome-scale phylogenetic analysis to identify genes that co-evolve with bacterial Hsp90. We find that genes whose gain and loss were coordinated with Hsp90 throughout bacterial evolution tended to function in flagellar assembly, chemotaxis, and bacterial secretion, suggesting that Hsp90 may aid assembly of protein complexes. To add to the limited set of known bacterial Hsp90 clients, we further developed a statistical method to predict putative clients. We validated our predictions by demonstrating that the flagellar protein FliN and the chemotaxis kinase CheA behaved as Hsp90 clients in Escherichia coli, confirming the predicted role of Hsp90 in chemotaxis and flagellar assembly. Furthermore, normal Hsp90 function is important for wild-type motility and/or chemotaxis in E. coli. This novel function of bacterial Hsp90 agreed with our subsequent finding that Hsp90 is associated with a preference for multiple habitats and may therefore face a complex selection regime. Taken together, our results reveal previously unknown functions of bacterial Hsp90 and open avenues for future experimental exploration by implicating Hsp90 in the assembly of membrane protein complexes and adaptation to novel environments. PMID:23874229

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

    PubMed Central

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

    2016-01-01

    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

  7. 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.

  8. 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

  9. Co-targeting AR and HSP90 suppresses prostate cancer cell growth and prevents resistance mechanisms.

    PubMed

    Centenera, Margaret M; Carter, Sarah L; Gillis, Joanna L; Marrocco-Tallarigo, Deborah L; Grose, Randall H; Tilley, Wayne D; Butler, Lisa M

    2015-10-01

    Persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) underpins the urgent need for therapeutic strategies that better target this pathway. Combining classes of agents that target different components of AR signaling has the potential to delay resistance and improve patient outcomes. Many oncoproteins, including the AR, rely on the molecular chaperone heat shock protein 90 (Hsp90) for functional maturation and stability. In this study, enhanced anti-proliferative activity of the Hsp90 inhibitors 17-allylamino-demethoxygeldanamycin (17-AAG) and AUY922 in androgen-sensitive and CRPC cells was achieved when the agents were used in combination with AR antagonists bicalutamide or enzalutamide. Moreover, significant caspase-dependent cell death was achieved using sub-optimal agent doses that individually have no effect. Expression profiling demonstrated regulation of a broadened set of AR target genes with combined 17-AAG and bicalutamide compared with the respective single agent treatments. This enhanced inhibition of AR signaling was accompanied by impaired chromatin binding and nuclear localization of the AR. Importantly, expression of the AR variant AR-V7 that is implicated in resistance to AR antagonists was not induced by combination treatment. Likewise, the heat shock response that is typically elicited with therapeutic doses of Hsp90 inhibitors, and is a potential mediator of resistance to these agents, was significantly reduced by combination treatment. In summary, the co-targeting strategy in this study more effectively inhibits AR signaling than targeting AR or HSP90 alone and prevents induction of key resistance mechanisms in prostate cancer cells. These findings merit further evaluation of this therapeutic strategy to prevent CRPC growth.

  10. Hsp70/Hsp90 organising protein (hop): beyond interactions with chaperones and prion proteins.

    PubMed

    Baindur-Hudson, Swati; Edkins, Adrienne L; Blatch, Gregory L

    2015-01-01

    The Hsp70/Hsp90 organising protein (Hop), also known as stress-inducible protein 1 (STI1), has received considerable attention for diverse cellular functions in both healthy and diseased states. There is extensive evidence that intracellular Hop is a co-chaperone of the major chaperones Hsp70 and Hsp90, playing an important role in the productive folding of Hsp90 client proteins. Consequently, Hop is implicated in a number of key signalling pathways, including aberrant pathways leading to cancer. However, Hop is also secreted and it is now well established that Hop also serves as a receptor for the prion protein, PrP(C). The intracellular and extracellular forms of Hop most likely represent two different isoforms, although the molecular determinants of these divergent functions are yet to be identified. There is also a growing body of research that reports the involvement of Hop in cellular activities that appear independent of either chaperones or PrP(C). While Hop has been shown to have various cellular functions, its biological function remains elusive. However, recent knockout studies in mammals suggest that Hop has an important role in embryonic development. This review provides a critical overview of the latest molecular, cellular and biological research on Hop, critically evaluating its function in healthy systems and how this function is adapted in diseases states.

  11. 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

  12. A compound that inhibits the HOP-Hsp90 complex formation and has unique killing effects in breast cancer cell lines.

    PubMed

    Pimienta, Genaro; Herbert, Kristina M; Regan, Lynne

    2011-12-01

    The chaperone Hsp90 is required for the correct folding and maturation of certain "client proteins" within all cells. Hsp90-mediated folding is particularly important in cancer cells, because upregulated or mutant oncogenic proteins are often Hsp90 clients. Hsp90 inhibitors thus represent a route to anticancer agents that have the potential to be active against several different types of cancer. Currently, various Hsp90 inhibitors that bind to Hsp90 at its ATP-binding site are in preclinical and clinical trials. Some of the most promising Hsp90 ATP-binding site inhibitors are the well characterized geldanamycin derivative 17-AAG and the recently described compounds PU-H71 and NVP-AUY922. An undesirable characteristic of these compounds is the transcriptional upregulation of Hsp70 that has prosurvival effects. Here we characterize the activity of a new type of chaperone inhibitor, 1,6-dimethyl-3-propylpyrimido[5,4-e][1,2,4]triazine-5,7-dione (named C9 for simplicity). Using purified protein components in vitro, C9 prevents Hsp90 from interacting with the cochaperone HOP and is thus expected to impair the Hsp90-dependent folding pathway in vivo. We show that this compound is effective in killing various breast cancer cell lines including the highly metastatic MDA-MB-231. An important property of this compound is that it does not induce the transcriptional upregulation of Hsp70. Moreover, when cells are treated with a combination of C9 and either 17-AAG or NVP-AUY922, the overexpression of Hsp70 is counteracted considerably and C9's lethal-IC50 decreases compared to its value when added alone.

  13. 17AAG Treatment Accelerates Doxorubicin Induced Cellular Senescence: Hsp90 Interferes with Enforced Senescence of Tumor Cells

    PubMed Central

    Sarangi, Upasana; Paithankar, Khande Rao; Kumar, Jonnala Ujwal; Subramaniam, Vaidyanathan; Sreedhar, Amere Subbarao

    2012-01-01

    Hsp90 chaperone has been identified as an attractive pharmacological target to combat cancer. However, some metastatic tumors either fail to respond to Hsp90 inhibition or show recovery necessitating irreversible therapeutic strategies. In response to this enforced senescence has been proposed as an alternate strategy. Here, we demonstrate that inhibiting Hsp90 with 17AAG sensitizes human neuroblastoma to DNA damage response mediated cellular senescence. Among individual and combination drug treatments, 17AAG pre-treatment followed by doxorubicin treatment exhibited senescence-like characteristics such as increased nucleus to cytoplasm ratio, cell cycle arrest, SA-β-gal staining and the perpetual increase in SAHF. Doxorubicin induced senescence signaling was mediated by p53-p21CIP/WAF-1 and was accelerated in the absence of functional Hsp90. Sustained p16INK4a and H3K4me3 expressions correlating with unaffected telomerase activation annulled replicative senescence and appraised stress induced senescence. Despite increases in [(ROS)i] and [(Ca2+)i], a concomitant increase in cellular antioxidant defense system suggested oxidation independent senescence activation. Sustained activation of survival (Akt) and proliferative (ERK1/2) kinases fosters robustness of cells. Invigorating senescent cells with growth factor or snooping with mTOR or PI3 kinase inhibitors compromised cell survival but not senescence. Intriguingly, senescence-associated secretory factors from the senescence cells manifested established senescence in neuroblastoma, which offers clinical advantage to our approach. Our study discusses tumor selective functions of Hsp90 and discusses irrefutable strategies of Hsp90 inhibition in anticancer treatments. PMID:22915839

  14. Identification of in vivo HSP90-interacting proteins reveals modularity of HSP90 complexes is dependent on the environment in psychrophilic bacteria

    PubMed Central

    García-Descalzo, Laura; Alcazar, Alberto; Baquero, Fernando

    2010-01-01

    Heat shock protein 90 (HSP90) is a conserved molecular chaperone that functions as part of complexes in which different client proteins target it to diverse sets of substrates. In this paper, HSP90 complexes were investigated in γ-proteobacteria from mild (Shewanella oneidensis) and cold environments (Shewanella frigidimarina and Psychrobacter frigidicola), to determine changes in HSP90 interactions with client proteins in response to the adaptation to cold environments. HSP90 participation in cold adaptation was determined using the specific inhibitor 17-allylamino-geldanamycin. Then, HSP90 was immunoprecipitated from bacterial cultures, and the proteins in HSP90 complexes were analyzed by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. According to HSP90-associated protein analysis, only 15 common proteins were found in both species from the same genus, S. oneidensis and S. frigidimarina, whereas a significant higher number of common proteins were found in both psychrophilic species S. frigidimarina and P. frigidicola 21 (p < 0.001). Only two HSP90-interacting proteins, the chaperone proteins DnaK and GroEL, were common to the three species. Interestingly, some proteins related to energy metabolism (isocitrate lyase, succinyl-CoA synthetase, alcohol dehydrogenase, NAD(+) synthase, and malate dehydrogenase) and some translation factors only interacted with HSP90 in psychrophilic bacteria. We can conclude that HSP90 and HSP90-associated proteins might take part in the mechanism of adaptation to cold environments, and interestingly, organisms living in similar environments conserve similar potential HSP90 interactors in opposition to phylogenetically closely related organisms of the same genus but from different environments. PMID:20890740

  15. Lupus-specific kidney deposits of HSP90 are associated with altered IgG idiotypic interactions of anti-HSP90 autoantibodies

    PubMed Central

    KENDEROV, A; MINKOVA, V; MIHAILOVA, D; GILTIAY, N; KYURKCHIEV, S; KEHAYOV, I; KAZATCHKINE, M; KAVERI, S; PASHOV, A

    2002-01-01

    Previous studies have shown that autoantibodies to heat shock protein 90 (HSP90) are elevated in a significant proportion of patients with systemic lupus erythematosus (SLE) who are more likely to have renal disease and a low C3 level. Using samples from 24 patients, we searched for glomerular deposits of HSP90 in renal biopsy specimens from seven patients with lupus nephritis and 17 cases of glomerulonephritis from patients without SLE. Positive glomerular immunofluorescent staining for HSP90 was observed in six of seven cases of SLE and positive tubular staining in two of seven SLE patients. The staining for HSP90 was granular in nature and was located in subepithelial, subendothelial and mesangial areas. None of the non-SLE renal biopsies revealed positive staining for HSP90 deposition. Further we showed the presence of anti-HSP90 IgG autoantibodies in IgG from sera of patients with SLE as well as in normal human IgG (IVIg). In normal IgG this autoreactivity could be adsorbed almost completely on F(ab′)2 fragments from the same IgG preparation, coupled to Sepharose and could be inhibited by the effluent obtained after subjecting normal IgG to HSP90 affinity column. These findings indicate that anti-HSP90 natural autoantibodies are blocked by idiotypic interactions within the IgG repertoire. Unlike natural autoantibodies, anti-HSP90 IgG from SLE patients’ sera were only moderately adsorbed on F(ab′)2 fragments of normal IgG. These results demonstrate that immunopathogenesis of lupus nephritis is associated with HSP90 (as an autoantigen) and that the pathology is associated with altered idiotypic regulation of the anti-HSP90 IgG autoantibodies. PMID:12100037

  16. The HSP90 binding mode of a radicicol-like E-oxime from docking, binding free energy estimations, and NMR 15N chemical shifts

    PubMed Central

    Spichty, Martin; Taly, Antoine; Hagn, Franz; Kessler, Horst; Barluenga, Sofia; Winssinger, Nicolas; Karplus, Martin

    2009-01-01

    We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We sample the macrocyclic scaffold of the unbound ligand by parallel tempering simulations and dock the most populated conformations to yeast HSP90. Docking poses are then evaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of back-bone 15N provides an additional evaluation criteria. As a last test we check the binding modes against available structure-activity-relationships. We find that the most likely binding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol-HSP90 complex. PMID:19482409

  17. 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

  18. 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

  19. 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

  20. Blocking the survival of the nastiest by HSP90 inhibition.

    PubMed

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

    2016-01-26

    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.

  1. 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

  2. HDAC6 Regulates Androgen Receptor Hypersensitivity and Nuclear Localization via Modulating Hsp90 Acetylation in Castration-Resistant Prostate Cancer

    PubMed Central

    Ai, Junkui; Wang, Yujuan; Dar, Javid A.; Liu, June; Liu, Lingqi; Nelson, Joel B.; Wang, Zhou

    2009-01-01

    The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa. PMID:19855091

  3. HDAC6 regulates androgen receptor hypersensitivity and nuclear localization via modulating Hsp90 acetylation in castration-resistant prostate cancer.

    PubMed

    Ai, Junkui; Wang, Yujuan; Dar, Javid A; Liu, June; Liu, Lingqi; Nelson, Joel B; Wang, Zhou

    2009-12-01

    The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa.

  4. A Tetrahymena Hsp90 co-chaperone promotes siRNA loading by ATP-dependent and ATP-independent mechanisms.

    PubMed

    Woehrer, Sophie L; Aronica, Lucia; Suhren, Jan H; Busch, Clara Jana-Lui; Noto, Tomoko; Mochizuki, Kazufumi

    2015-02-12

    The loading of small interfering RNAs (siRNAs) and microRNAs into Argonaute proteins is enhanced by Hsp90 and ATP in diverse eukaryotes. However, whether this loading also occurs independently of Hsp90 and ATP remains unclear. We show that the Tetrahymena Hsp90 co-chaperone Coi12p promotes siRNA loading into the Argonaute protein Twi1p in both ATP-dependent and ATP-independent manners in vitro. The ATP-dependent activity requires Hsp90 and the tetratricopeptide repeat (TPR) domain of Coi12p, whereas these factors are dispensable for the ATP-independent activity. Both activities facilitate siRNA loading by counteracting the Twi1p-binding protein Giw1p, which is important to specifically sort the 26- to 32-nt siRNAs to Twi1p. Although Coi12p lacking its TPR domain does not bind to Hsp90, it can partially restore the siRNA loading and DNA elimination defects of COI12 knockout cells, suggesting that Hsp90- and ATP-independent loading of siRNA occurs in vivo and plays a physiological role in Tetrahymena.

  5. 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.

  6. 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

  7. The achilles heel of ErbB-2/HER2: regulation by the Hsp90 chaperone machine and potential for pharmacological intervention.

    PubMed

    Citri, Ami; Kochupurakkal, Bose S; Yarden, Yosef

    2004-01-01

    Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated cochaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.

  8. RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation.

    PubMed

    Walsh, Naomi; Larkin, AnneMarie; Swan, Niall; Conlon, Kevin; Dowling, Paul; McDermott, Ray; Clynes, Martin

    2011-07-28

    We previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered. In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours. Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

  9. Tomato plant cell death induced by inhibition of HSP90 is alleviated by Tomato yellow leaf curl virus infection.

    PubMed

    Moshe, Adi; Gorovits, Rena; Liu, Yule; Czosnek, Henryk

    2016-02-01

    To ensure a successful long-term infection cycle, begomoviruses must restrain their destructive effect on host cells and prevent drastic plant responses, at least in the early stages of infection. The monopartite begomovirus Tomato yellow leaf curl virus (TYLCV) does not induce a hypersensitive response and cell death on whitefly-mediated infection of virus-susceptible tomato plants until diseased tomatoes become senescent. The way in which begomoviruses evade plant defences and interfere with cell death pathways is still poorly understood. We show that the chaperone HSP90 (heat shock protein 90) and its co-chaperone SGT1 (suppressor of the G2 allele of Skp1) are involved in the establishment of TYLCV infection. Inactivation of HSP90, as well as silencing of the Hsp90 and Sgt1 genes, leads to the accumulation of damaged ubiquitinated proteins and to a cell death phenotype. These effects are relieved under TYLCV infection. HSP90-dependent inactivation of 26S proteasome degradation and the transcriptional activation of the heat shock transcription factors HsfA2 and HsfB1 and of the downstream genes Hsp17 and Apx1/2 are suppressed in TYLCV-infected tomatoes. Following suppression of the plant stress response, TYLCV can replicate and accumulate in a permissive environment. PMID:25962748

  10. A member of the HSP90 family from ovine Babesia in China: molecular characterization, phylogenetic analysis and antigenicity.

    PubMed

    Guan, Guiquan; Liu, Junlong; Liu, Aihong; Li, Youquan; Niu, Qingli; Gao, Jinliang; Luo, Jianxun; Chauvin, Alain; Yin, Hong; Moreau, Emmanuelle

    2015-09-01

    Heat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. A Hsp90 gene (BQHsp90) was cloned and characterized from Babesia sp. BQ1 (Lintan), an ovine Babesia isolate belonging to Babesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA of BQHsp90 is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics to Hsp90 of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that the Babesia genus is clearly separated from other apicomplexa genera. Five Chinese ovine Babesia isolates were divided into 2 phylogenetic clusters, namely Babesia sp. Xinjiang (previously designated a new species) cluster and B. motasi-like cluster which could be further divided into 2 subclusters (Babesia sp. BQ1 (Lintan)/Babesia sp. Tianzhu and Babesia sp. BQ1 (Ningxian)/Babesia sp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

  11. ATPase Activity of Pea Cotyledon Submitochondrial Particles

    PubMed Central

    Grubmeyer, Charles; Spencer, Mary

    1980-01-01

    Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO3, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO3−, but inhibited by Cl−, indicating that Cl− stimulation is a distinguishing property of the pea mitochondrial ATPase complex. PMID:16661174

  12. 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

  13. Chiral resolution and pharmacological characterization of the enantiomers of the Hsp90 inhibitor 2-amino-7-[4-fluoro-2-(3-pyridyl)phenyl]-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime.

    PubMed

    Amici, Raffaella; Bigogno, Chiara; Boggio, Roberto; Colombo, Andrea; Courtney, Stephen M; Dal Zuffo, Roberto; Dondio, Giulio; Fusar, Fulvia; Gagliardi, Stefania; Minucci, Saverio; Molteni, Marco; Montalbetti, Christian A G N; Mortoni, Annalisa; Varasi, Mario; Vultaggio, Stefania; Mercurio, Ciro

    2014-07-01

    Heat-shock protein 90 (Hsp90) is a molecular chaperone involved in the stabilization of key oncogenic signaling proteins, and therefore, inhibition of Hsp90 represents a new strategy in cancer therapy. 2-Amino-7-[4-fluoro-2-(3-pyridyl)phenyl]-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime is a racemic Hsp90 inhibitor that targets the N-terminal adenosine triphosphatase site. We developed a method to resolve the enantiomers and evaluated their inhibitory activity on Hsp90 and the consequent antitumor effects. The (S) stereoisomer emerged as a potent Hsp90 inhibitor in biochemical and cellular assays. In addition, this enantiomer exhibited high oral bioavailability in mice and excellent antitumor activity in two different human cancer xenograft models.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. A novel biomarker for marine environmental pollution of HSP90 from Mytilus coruscus.

    PubMed

    Liu, Huihui; Wu, Jiong; Xu, Mengshan; He, Jianyu

    2016-10-15

    Heat shock protein 90 (HSP90) is a conserved molecular chaperone contributing to cell cycle control, organism development and the proper regulation of cytosolic proteins. The full-length HSP90 cDNA of Mytilus coruscus (McHSP90, KT946644) was 2420bp, including an ORF of 2169bp encoding a polypeptide of 722 amino acids with predicted pI/MW 4.89/83.22kDa. BLASTp analysis and phylogenetic relationship strongly suggested McHSP90 was a member of HSP90 family, and it was highly conserved with other known HSP90, especially in the HSP90 family signatures, ATP/GTP-Binding sites and 'EEVD' motif. The mRNA of McHSP90 in haemolymph was upregulated in all treatments including Vibrio alginolyticus and Vibrio harveyi challenge, metals stresses (copper and cadmium) and 180 CST fuel exposure. All the results implied the expression of McHSP90 could be affected by Vibrio challenge and environmental stress, which might help us gain more insight into the molecular mechanism of HSP against adverse stresses in mollusca.

  19. Selection Transforms the Landscape of Genetic Variation Interacting with Hsp90

    PubMed Central

    Geiler-Samerotte, Kerry A.; Zhu, Yuan O.; Goulet, Benjamin E.; Hall, David W.; Siegal, Mark L.

    2016-01-01

    The protein-folding chaperone Hsp90 has been proposed to buffer the phenotypic effects of mutations. The potential for Hsp90 and other putative buffers to increase robustness to mutation has had major impact on disease models, quantitative genetics, and evolutionary theory. But Hsp90 sometimes contradicts expectations for a buffer by potentiating rapid phenotypic changes that would otherwise not occur. Here, we quantify Hsp90’s ability to buffer or potentiate (i.e., diminish or enhance) the effects of genetic variation on single-cell morphological features in budding yeast. We corroborate reports that Hsp90 tends to buffer the effects of standing genetic variation in natural populations. However, we demonstrate that Hsp90 tends to have the opposite effect on genetic variation that has experienced reduced selection pressure. Specifically, Hsp90 tends to enhance, rather than diminish, the effects of spontaneous mutations and recombinations. This result implies that Hsp90 does not make phenotypes more robust to the effects of genetic perturbation. Instead, natural selection preferentially allows buffered alleles to persist and thereby creates the false impression that Hsp90 confers greater robustness. PMID:27768682

  20. Hsp90 Maintains Proteostasis of the Galactose Utilization Pathway To Prevent Cell Lethality

    PubMed Central

    Gopinath, Rajaneesh Karimpurath

    2016-01-01

    Hsp90 is a molecular chaperone that aids in the folding of its metastable client proteins. Past studies have shown that it can exert a strong impact on some cellular pathways by controlling key regulators. However, it is unknown whether several components of a single pathway are collectively regulated by Hsp90. Here, we observe that Hsp90 influences the protein abundance of multiple Gal proteins and the efficiency of galactose utilization even after the galactose utilization pathway (GAL pathway) is fully induced. The effect of Hsp90 on Gal proteins is not at the transcriptional level. Moreover, Gal1 is found to physically interact with Hsp90, and its stability is reduced in low-Hsp90 cells. When Hsp90 is compromised, several Gal proteins form protein aggregates that colocalize with the disaggregase Hsp104. These results suggest that Gal1 and other Gal proteins are probably the clients of Hsp90. An unbalanced GAL pathway has been known to cause fatal growth arrest due to accumulation of toxic galactose metabolic intermediates. It is likely that Hsp90 chaperones multiple Gal proteins to maintain proteostasis and prevent cell lethality especially in a fluctuating environment. PMID:26951197

  1. Molecular Characterization and Phylogenetic Evaluation of the Hsp90 Gene from Selected Nematodes

    PubMed Central

    Skantar, Andrea M.; Carta, Lynn K.

    2004-01-01

    While multiple genes are optimal for corroborating nematode phylogenies, only a few are commonly used. Here we examine the phylogenetic potential of the nuclear Hsp90 chaperone gene. We used degenerate primers to obtain partial Hsp90 sequences from several plant-parasitic and free-living nematodes. Hsp90 was single-copy in Heterodera glycines and Meloidogyne javanica, similar to the situation for Caenorhabditis elegans. The full-length H. glycines Hsp90 protein sequence showed homology to sequences from C. elegans and Brugia pahangi and to other eukaryotes, and contains several functionally important regions common to cytoplasmic Hsp90 proteins. The Hsp90 amino acid phylogeny supported the Coelomata hypothesis for metazoan evolution. Phylogenetic trees, substitution scatter plots, and statistics for phylogenetic signal were made for Hsp90, 18S small subunit (SSU), and 28S large subunit (LSU) over a limited but broad sampling of nematode taxa. Only the LSU data set failed to recover any of the expected topology and showed extensive substitution saturation. In an intensive sampling of plant-parasitic nematode taxa, the Hsp90 tree topologies were generally congruent with rDNA results and alignments were unambiguous. Hsp90 sequences may help strengthen branch support or clarify tree topologies when other molecules show ambiguous alignments, greater branch-length heterogeneity, or codon bias in certain taxonomic groups. PMID:19262827

  2. A novel biomarker for marine environmental pollution of HSP90 from Mytilus coruscus.

    PubMed

    Liu, Huihui; Wu, Jiong; Xu, Mengshan; He, Jianyu

    2016-10-15

    Heat shock protein 90 (HSP90) is a conserved molecular chaperone contributing to cell cycle control, organism development and the proper regulation of cytosolic proteins. The full-length HSP90 cDNA of Mytilus coruscus (McHSP90, KT946644) was 2420bp, including an ORF of 2169bp encoding a polypeptide of 722 amino acids with predicted pI/MW 4.89/83.22kDa. BLASTp analysis and phylogenetic relationship strongly suggested McHSP90 was a member of HSP90 family, and it was highly conserved with other known HSP90, especially in the HSP90 family signatures, ATP/GTP-Binding sites and 'EEVD' motif. The mRNA of McHSP90 in haemolymph was upregulated in all treatments including Vibrio alginolyticus and Vibrio harveyi challenge, metals stresses (copper and cadmium) and 180 CST fuel exposure. All the results implied the expression of McHSP90 could be affected by Vibrio challenge and environmental stress, which might help us gain more insight into the molecular mechanism of HSP against adverse stresses in mollusca. PMID:27491367

  3. 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

  4. 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

  5. Leptin increases HER2 protein levels through a STAT3-mediated up-regulation of Hsp90 in breast cancer cells.

    PubMed

    Giordano, Cinzia; Vizza, Donatella; Panza, Salvatore; Barone, Ines; Bonofiglio, Daniela; Lanzino, Marilena; Sisci, Diego; De Amicis, Francesca; Fuqua, Suzanne A W; Catalano, Stefania; Andò, Sebastiano

    2013-06-01

    Obesity condition confers risks to breast cancer development and progression, and several reports indicate that the adipokine leptin, whose synthesis and plasma levels increase with obesity, might play an important role in modulating breast cancer cell phenotype. Functional crosstalk occurring between leptin and different signaling molecules contribute to breast carcinogenesis. In this study, we show, in different human breast cancer cell lines, that leptin enhanced the expression of a chaperone protein Hsp90 resulting in increased HER2 protein levels. Silencing of Hsp90 gene expression by RNA interference abrogated leptin-mediated HER2 up-regulation. Leptin effects were dependent on JAK2/STAT3 activation, since inhibition of this signaling cascade by AG490 or ectopic expression of a STAT3 dominant negative abrogated leptin-induced HER2 and Hsp90 expressions. Functional experiments showed that leptin treatment significantly up-regulated human Hsp90 promoter activity. This occurred through an enhanced STAT3 transcription factor binding to its specific responsive element located in the Hsp90 promoter region as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Analysis of HER2, Akt and MAPK phosphorylation levels revealed that leptin treatment amplified the responsiveness of breast cancer cells to growth factor stimulation. Furthermore, we found that long-term leptin exposure reduced sensitivity of breast cancer cells to the antiestrogen tamoxifen. In the same experimental conditions, the combined treatment of tamoxifen with the Hsp90 inhibitor 17-AAG completely abrogated leptin-induced anchorage-independent breast cancer cell growth. In conclusion, our results highlight, for the first time, the ability of the adipocyte-secreted factor leptin to modulate Hsp90/HER2 expressions in breast cancer cells providing novel insights into the molecular mechanism linking obesity to breast cancer growth and progression.

  6. HSP90 Inhibitor Encapsulated Photo-Theranostic Nanoparticles for Synergistic Combination Cancer Therapy.

    PubMed

    Lin, Tzu-Yin; Guo, Wenchang; Long, Qilai; Ma, Aihong; Liu, Qiangqiang; Zhang, Hongyong; Huang, Yee; Chandrasekaran, Siddarth; Pan, Chongxian; Lam, Kit S; Li, Yuanpei

    2016-01-01

    Photodynamic therapy (PDT) is a promising non-invasive therapeutic modality that has been proposed for treating prostate cancer, but the procedure is associated with limited efficacy, tumor recurrence and photo-toxicity. In the present study, we proposed to develop a novel multifunctional nano-platform for targeted delivery of heat, reactive oxygen species (ROS) and heat shock protein 90 (Hsp90) inhibitor simultaneously for combination therapy against prostate cancer. This new nano-platform combines two newly developed entities: 1) a unique organic and biocompatible nanoporphyrin-based drug delivery system that can generate efficient heat and ROS simultaneously with light activation at the tumor sites for dual-modal photothermal- and photodynamic- therapy (PTT/PDT), and 2) new nano-formulations of Hsp90 inhibitors that can decrease the levels of pro-survival and angiogenic signaling molecules induced by phototherapy, therefore, further sensitizing cancer cells to phototherapy. Furthermore, the nanoparticles have activatable near infrared (NIR) fluorescence for optical imaging to conveniently monitor the real-time drug delivery in both subcutaneous and orthotopic mouse models bearing prostate cancer xenograft. This novel multifunctional nano-platform has great potential to improve the care of prostate cancer patients through targeted combination therapy. PMID:27375782

  7. Ansamycin inhibitors of Hsp90: nature's prototype for anti-chaperone therapy.

    PubMed

    Porter, James R; Ge, Jie; Lee, John; Normant, Emmanuel; West, Kip

    2009-01-01

    The ansamycin class of natural products is well known for its anti-tumor effects and has been extensively studied by cancer researchers for nearly four decades. The first description of geldanamycin in the scientific literature appeared in 1970 and nearly thirty years later the semi-synthetic derivative 17-AAG, or tanespimycin, entered Phase 1 clinical trials. In the subsequent years, three additional geldanamycin derivatives have entered clinical evaluation. Kosan Biosciences developed 17-DMAG or alvespimycin hydrochloride for clinical evaluation as both an intravenous and oral product. Infinity Pharmaceuticals is developing IPI-504 or retaspimycin hydrochloride as an intravenous product, which is in several Phase 2 clinical trials; IPI-504 is the hydroquinone hydrochloride salt of 17-AAG. More recently, Infinity Pharmaceuticals initiated a Phase 1 clinical trial with an oral formulation of 17-AG (IPI-493), the major metabolite of 17-AAG and IPI-504. Since a vast amount of scientific literature exists regarding the ansamycin field and Hsp90 inhibition, this review will survey key milestones in the development of the natural product class as anti-cancer drugs including discovery of the compounds and their anti-tumor effects, identification of Hsp90 as their biological target, the structure-activity relationships that have been identified in this interesting class of compounds, and development of clinical candidates for the treatment of cancer patients. A brief overview of important pre-clinical development data from each clinical lead is also provided.

  8. HSP90 Inhibitor Encapsulated Photo-Theranostic Nanoparticles for Synergistic Combination Cancer Therapy

    PubMed Central

    Lin, Tzu-yin; Guo, Wenchang; Long, Qilai; Ma, Aihong; Liu, Qiangqiang; Zhang, Hongyong; Huang, Yee; Chandrasekaran, Siddarth; Pan, Chongxian; Lam, Kit S.; Li, Yuanpei

    2016-01-01

    Photodynamic therapy (PDT) is a promising non-invasive therapeutic modality that has been proposed for treating prostate cancer, but the procedure is associated with limited efficacy, tumor recurrence and photo-toxicity. In the present study, we proposed to develop a novel multifunctional nano-platform for targeted delivery of heat, reactive oxygen species (ROS) and heat shock protein 90 (Hsp90) inhibitor simultaneously for combination therapy against prostate cancer. This new nano-platform combines two newly developed entities: 1) a unique organic and biocompatible nanoporphyrin-based drug delivery system that can generate efficient heat and ROS simultaneously with light activation at the tumor sites for dual-modal photothermal- and photodynamic- therapy (PTT/PDT), and 2) new nano-formulations of Hsp90 inhibitors that can decrease the levels of pro-survival and angiogenic signaling molecules induced by phototherapy, therefore, further sensitizing cancer cells to phototherapy. Furthermore, the nanoparticles have activatable near infrared (NIR) fluorescence for optical imaging to conveniently monitor the real-time drug delivery in both subcutaneous and orthotopic mouse models bearing prostate cancer xenograft. This novel multifunctional nano-platform has great potential to improve the care of prostate cancer patients through targeted combination therapy. PMID:27375782

  9. Developmental expression of Hsp90, Hsp70 and HSF during morphogenesis in the vetigastropod Haliotis asinina.

    PubMed

    Gunter, Helen M; Degnan, Bernard M

    2007-08-01

    Heat shock proteins (Hsps) have dual functions, participating in both the stress response and a broad range of developmental processes. At physiological temperatures, it has been demonstrated in deuterostomes (vertebrates) and ecdysozoans (insects) that Hsps are expressed in tissues that are undergoing differentiation and morphogenesis. Here we investigate the developmental expression of Hsp70, Hsp90 and their regulatory transcription factor heat shock transcription factor (HSF) in the marine gastropod Haliotis asinina, a representative of the 3rd major lineage of bilaterian animals, the Lophotrochozoa. HasHsp70, HasHsp90 and HasHSF are maternally expressed in H. asinina and are progressively restricted to the micromere lineage during cleavage. During larval morphogenesis, they are expressed in unique and overlapping patterns in the prototroch, foot, and mantle. Hsp expression peaked in these tissues during periods of cell differentiation and morphogenesis, returning to lower levels after morphogenesis was complete. These patterns of Hsp and HSF expression in H. asinina are akin to those observed in ecdysozoans and deuterostomes, with Hsps being activated in cells and tissues undergoing morphogenesis.

  10. BIIB021, a novel Hsp90 inhibitor, sensitizes esophageal squamous cell carcinoma to radiation

    SciTech Connect

    Wang, Xin-Tong; Bao, Ci-Hang; Jia, Yi-Bin; Wang, Nana; Ma, Wei; Liu, Fang; Wang, Cong; Wang, Jian-Bo; Song, Qing-Xu; Cheng, Yu-Feng

    2014-10-03

    Highlights: • BIIB021 downregulated radioresistant proteins in ESCC cell lines. • BIIB021 increased radiation-induced apoptotic cells. • BIIB021 enhanced G{sub 2} arrest in ESCC cell lines. • BIIB021 is a good candidate for radiosensitizer in radiotherapy of ESCC patients. - Abstract: BIIB021 is a novel, orally available inhibitor of heat shock protein 90 (Hsp90) that is currently in phase I/II clinical trials. BIIB021 induces the apoptosis of various types of tumor cells in vitro and in vivo. The aim of this study is to investigate the effect of BIIB021 on the radiosensitivity of esophageal squamous cell carcinoma (ESCC). The results indicated that BIIB021 exhibited strong antitumor activity in ESCC cell lines, either as a single agent or in combination with radiation. BIIB021 significantly downregulated radioresistant proteins including EGFR, Akt, Raf-1 of ESCC cell lines, increased apoptotic cells and enhanced G{sub 2} arrest that is more radiosensitive cell cycle phase. These results suggest that this synthetic Hsp90 inhibitor simultaneously affects multiple pathways involved in tumor development and progression in the ESCC setting and may represent a better strategy for the treatment of ESCC patients, either as a monotherapy or a radiosensitizer.

  11. Mechanism of Inhibition of Hsp90 Dimerization by Gyrase B Inhibitor Coumermycin A1 (C-A1) Revealed by Molecular Dynamics Simulations and Thermodynamic Calculations.

    PubMed

    Cele, Favourite N; Kumalo, Hezekiel; Soliman, Mahmoud E S

    2016-09-01

    Heat shock protein (Hsp) 90 an emerging and attracting target in the anti-HIV drug discovery process due to the key role it plays in the pathogenicity of HIV-1 virus. In this research study, long-range all-atom molecular dynamics simulations were engaged for the bound and the unbound proteins to enhance the understanding of the molecular mechanisms of the Hsp90 dimerization and inhibition. Results evidently showed that coumermycin A1 (C-A1), a recently discovered Hsp90 inhibitor, binds at the dimer's active site of the Hsp90 protein and leads to a substantial parting between dimeric opposed residues, which include Arg591.B, Lys594.A, Ser663.A, Thr653.B, Ala665.A, Thr649.B, Leu646.B and Asn669.A. Significant differences in magnitudes were observed in radius of gyration, root-mean-square deviation and root-mean-square fluctuation, which confirms a reasonably more flexible state in the apo conformation associated with it dimerization. In contrast, the bound conformer of Hsp90 showed less flexibility. This visibly highpoints the inhibition process resulting from the binding of the ligand. These findings were further validated by principal component analysis. We believe that the detailed dynamic analyses of Hsp90 presented in this study, would give an imperative insight and better understanding to the function and mechanisms of inhibition. Furthermore, information obtained from the binding mode of the inhibitor would be of great assistance in the design of more potent inhibitors against the HIV target Hsp90. PMID:27376828

  12. 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.

  13. Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance.

    PubMed

    Wang, Guan-Feng; Wei, Xuening; Fan, Renchun; Zhou, Huanbin; Wang, Xianping; Yu, Chunmei; Dong, Lingli; Dong, Zhenying; Wang, Xiaojie; Kang, Zhensheng; Ling, Hongqing; Shen, Qian-Hua; Wang, Daowen; Zhang, Xiangqi

    2011-07-01

    Heat shock protein 90 (Hsp90) molecular chaperones play important roles in plant growth and responses to environmental stimuli. However, little is known about the genes encoding Hsp90s in common wheat. Here, we report genetic and functional analysis of the genes specifying cytosolic Hsp90s in this species. Three groups of homoeologous genes (TaHsp90.1, TaHsp90.2 and TaHsp90.3), encoding three types of cytosolic Hsp90, were isolated. The loci containing TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes were assigned to groups 2, 7 and 5 chromosomes, respectively. TaHsp90.1 genes exhibited higher transcript levels in the stamen than in the leaf, root and culm. TaHsp90.2 and TaHsp90.3 genes were more ubiquitously transcribed in the vegetative and reproductive organs examined. Decreasing the expression of TaHsp90.1 genes through virus-induced gene silencing (VIGS) caused pronounced inhibition of wheat seedling growth, whereas the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the hypersensitive resistance response of the wheat variety Suwon 11 to stripe rust fungus. Our work represents the first systematic determination of wheat genes encoding cytosolic Hsp90s, and provides useful evidence for the functional involvement of cytosolic Hsp90s in the control of seedling growth and disease resistance in common wheat.

  14. hsp70 interacting protein Hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of BAG-1.

    PubMed

    Kanelakis, K C; Murphy, P J; Galigniana, M D; Morishima, Y; Takayama, S; Reed, J C; Toft, D O; Pratt, W B

    2000-11-21

    Reticulocyte lysate contains a chaperone system that assembles glucocorticoid receptor (GR).hsp90 heterocomplexes. Using purified proteins, we have prepared a five-protein heterocomplex assembly system consisting of two proteins essential for heterocomplex assembly-hsp90 and hsp70-and three proteins that act as co-chaperones to enhance assembly-Hop, hsp40, p23 [Morishima, Y., Kanelakis, K. C., Silverstein, A. M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900]. The hsp70 co-chaperone Hip has been recovered in receptor.hsp90 heterocomplexes at an intermediate stage of assembly in reticulocyte lysate, and Hip is also thought to be an intrinsic component of the assembly machinery. Here we show that immunodepletion of Hip from reticulocyte lysate or addition of high levels of Hip to the purified five-protein system does not affect GR.hsp90 heterocomplex assembly or the activation of steroid binding activity that occurs with assembly. Despite the fact that Hip does not affect assembly, it is recovered in GR.hsp90 heterocomplexes assembled by both systems. In the five-protein system, Hip prevents inhibition of assembly by the hsp70 co-chaperone BAG-1, and cotransfection of Hip with BAG-1 opposes BAG-1 reduction of steroid binding activity in COS cells. We conclude that Hip is not a component of the assembly machinery but that it could play a regulatory role in opposition to BAG-1.

  15. Substrate binding drives large-scale conformational changes in the Hsp90 molecular chaperone

    PubMed Central

    Street, Timothy O.; Lavery, Laura A.; Agard, David A.

    2011-01-01

    Summary Hsp90 is a ubiquitous molecular chaperone. Previous structural analysis demonstrated that Hsp90 can adopt a large number of structurally distinct conformations, however the functional role of this flexibility is not understood. Here we investigate the structural consequences of substrate binding with a model system in which Hsp90 interacts with a partially folded protein (Δ131Δ), a well-studied fragment of staphylococcal nuclease. SAXS measurements reveal that under apo conditions Hsp90 partially closes aroundΔ131Δ and in the presence of AMPPNP Δ131Δ binds with increased affinity to Hsp90’s fully closed state. FRET measurement show that Δ131Δ accelerates the nucleotide-driven open/closed transition and stimulates ATP hydrolysis by Hsp90. NMR measurements reveal that Hsp90 binds to a specific, highly structured, region of Δ131Δ. These results suggest that Hsp90 preferentially binds a locally structured region in a globally unfolded protein and this binding drives functional changes in the chaperone by lowering a rate-limiting conformational barrier. PMID:21474071

  16. Natural Product Inspired N-Terminal Hsp90 Inhibitors: From Bench to Bedside?

    PubMed

    Khandelwal, Anuj; Crowley, Vincent M; Blagg, Brian S J

    2016-01-01

    The 90 kDa heat shock proteins (Hsp90) are responsible for the conformational maturation of nascent polypeptides and the rematuration of denatured proteins. Proteins dependent upon Hsp90 are associated with all six hallmarks of cancer. Upon Hsp90 inhibition, protein substrates are degraded via the ubiquitin-proteasome pathway. Consequentially, inhibition of Hsp90 offers a therapeutic opportunity for the treatment of cancer. Natural product inhibitors of Hsp90 have been identified in vitro, which have served as leads for the development of more efficacious inhibitors and analogs that have entered clinical trials. This review highlights the development of natural product analogs, as well as the development of clinically important inhibitors that arose from natural products.

  17. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    PubMed Central

    Lawless, Nathan; Blacklock, Kristin; Berrigan, Elizabeth; Verkhivker, Gennady

    2013-01-01

    A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4) kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock) kinase from the system during client loading (release) stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery. PMID:24287464

  18. The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

    PubMed

    He, Qiuxia; Liu, Kechun; Tian, Zhenjun; Du, Shao Jun

    2015-01-01

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

  19. Marked enhancement of lysosomal targeting and efficacy of ErbB2-targeted drug delivery by HSP90 inhibition

    PubMed Central

    Mohapatra, Bhopal; Luan, Haitao; Soni, Kruti; Zhang, Jinjin; Storck, Matthew A.; Feng, Dan; Bielecki, Timothy A.; Band, Vimla; Cohen, Samuel M.; Bronich, Tatiana K.; Band, Hamid

    2016-01-01

    Targeted delivery of anticancer drugs to tumor cells using monoclonal antibodies against oncogenic cell surface receptors is an emerging therapeutic strategy. These strategies include drugs directly conjugated to monoclonal antibodies through chemical linkers (Antibody-Drug Conjugates, ADCs) or those encapsulated within nanoparticles that in turn are conjugated to targeting antibodies (Antibody-Nanoparticle Conjugates, ANPs). The recent FDA approval of the ADC Trastuzumab-TDM1 (Kadcyla®; Genentech; San Francisco) for the treatment of ErbB2-overexpressing metastatic breast cancer patients has validated the strong potential of these strategies. Even though the activity of ANPs and ADCs is dependent on lysosomal traffic, the roles of the endocytic route traversed by the targeted receptor and of cancer cell-specific alterations in receptor dynamics on the efficiency of drug delivery have not been considered in these new targeted therapies. For example, constitutive association with the molecular chaperone HSP90 is thought to either retard ErbB2 endocytosis or to promote its recycling, traits undesirable for targeted therapy with ANPs and ADCs. HSP90 inhibitors are known to promote ErbB2 ubiquitination, targeting to lysosome and degradation. We therefore hypothesized that ErbB2-targeted drug delivery using Trastuzumab-conjugated nanoparticles could be significantly improved by HSP90 inhibitor-promoted lysosomal traffic of ErbB2. Studies reported here validate this hypothesis and demonstrate, both in vitro and in vivo, that HSP90 inhibition facilitates the intracellular delivery of Trastuzumab-conjugated ANPs carrying a model chemotherapeutic agent, Doxorubicin, specifically into ErbB2-overexpressing breast cancer cells, resulting in improved antitumor activity. These novel findings highlight the need to consider oncogene-specific alterations in receptor traffic in the design of targeted drug delivery strategies. We suggest that combination of agents that enhance

  20. Marked enhancement of lysosomal targeting and efficacy of ErbB2-targeted drug delivery by HSP90 inhibition.

    PubMed

    Raja, Srikumar M; Desale, Swapnil S; Mohapatra, Bhopal; Luan, Haitao; Soni, Kruti; Zhang, Jinjin; Storck, Matthew A; Feng, Dan; Bielecki, Timothy A; Band, Vimla; Cohen, Samuel M; Bronich, Tatiana K; Band, Hamid

    2016-03-01

    Targeted delivery of anticancer drugs to tumor cells using monoclonal antibodies against oncogenic cell surface receptors is an emerging therapeutic strategy. These strategies include drugs directly conjugated to monoclonal antibodies through chemical linkers (Antibody-Drug Conjugates, ADCs) or those encapsulated within nanoparticles that in turn are conjugated to targeting antibodies (Antibody-Nanoparticle Conjugates, ANPs). The recent FDA approval of the ADC Trastuzumab-TDM1 (Kadcyla; Genentech; San Francisco) for the treatment of ErbB2-overexpressing metastatic breast cancer patients has validated the strong potential of these strategies. Even though the activity of ANPs and ADCs is dependent on lysosomal traffic, the roles of the endocytic route traversed by the targeted receptor and of cancer cell-specific alterations in receptor dynamics on the efficiency of drug delivery have not been considered in these new targeted therapies. For example, constitutive association with the molecular chaperone HSP90 is thought to either retard ErbB2 endocytosis or to promote its recycling, traits undesirable for targeted therapy with ANPs and ADCs. HSP90 inhibitors are known to promote ErbB2 ubiquitination, targeting to lysosome and degradation. We therefore hypothesized that ErbB2-targeted drug delivery using Trastuzumab-conjugated nanoparticles could be significantly improved by HSP90 inhibitor-promoted lysosomal traffic of ErbB2. Studies reported here validate this hypothesis and demonstrate, both in vitro and in vivo, that HSP90 inhibition facilitates the intracellular delivery of Trastuzumab-conjugated ANPs carrying a model chemotherapeutic agent, Doxorubicin, specifically into ErbB2-overexpressing breast cancer cells, resulting in improved antitumor activity. These novel findings highlight the need to consider oncogene-specific alterations in receptor traffic in the design of targeted drug delivery strategies. We suggest that combination of agents that enhance receptor

  1. Overcoming acquired BRAF inhibitor resistance in melanoma via targeted inhibition of Hsp90 with ganetespib.

    PubMed

    Acquaviva, Jaime; Smith, Donald L; Jimenez, John-Paul; Zhang, Chaohua; Sequeira, Manuel; He, Suqin; Sang, Jim; Bates, Richard C; Proia, David A

    2014-02-01

    Activating BRAF kinase mutations serve as oncogenic drivers in over half of all melanomas, a feature that has been exploited in the development of new molecularly targeted approaches to treat this disease. Selective BRAF(V600E) inhibitors, such as vemurafenib, typically induce initial, profound tumor regressions within this group of patients; however, durable responses have been hampered by the emergence of drug resistance. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90, in melanoma lines harboring the BRAF(V600E) mutation. Ganetespib exposure resulted in the loss of mutant BRAF expression and depletion of mitogen-activated protein kinase and AKT signaling, resulting in greater in vitro potency and antitumor efficacy compared with targeted BRAF and MAP-ERK kinase (MEK) inhibitors. Dual targeting of Hsp90 and BRAF(V600E) provided combinatorial benefit in vemurafenib-sensitive melanoma cells in vitro and in vivo. Importantly, ganetespib overcame mechanisms of intrinsic and acquired resistance to vemurafenib, the latter of which was characterized by reactivation of extracellular signal-regulated kinase (ERK) signaling. Continued suppression of BRAF(V600E) by vemurafenib potentiated sensitivity to MEK inhibitors after acquired resistance had been established. Ganetespib treatment reduced, but not abolished, elevations in steady-state ERK activity. Profiling studies revealed that the addition of a MEK inhibitor could completely abrogate ERK reactivation in the resistant phenotype, with ganetespib displaying superior combinatorial activity over vemurafenib. Moreover, ganetespib plus the MEK inhibitor TAK-733 induced tumor regressions in vemurafenib-resistant xenografts. Overall these data highlight the potential of ganetespib as a single-agent or combination treatment in BRAF(V600E)-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors. PMID:24398428

  2. The Molecular Chaperone Hsp90 Is Required for Cell Cycle Exit in Drosophila melanogaster

    PubMed Central

    Bandura, Jennifer L.; Jiang, Huaqi; Nickerson, Derek W.; Edgar, Bruce A.

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite+ reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. PMID:24086162

  3. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    PubMed

    Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. PMID:24086162

  4. The novel HSP90 inhibitor AT13387 potentiates radiation effects in squamous cell carcinoma and adenocarcinoma cells

    PubMed Central

    Spiegelberg, Diana; Dascalu, Adrian; Mortensen, Anja C.; Abramenkovs, Andris; Kuku, Gamze; Nestor, Marika; Stenerlöw, Bo

    2015-01-01

    Overexpression of heat shock protein 90 (HSP90) is associated with increased tumor cell survival and radioresistance. In this study we explored the efficacy of the novel HSP90 inhibitor AT13387 and examined its radiosensitizing effects in combination with gamma-radiation in 2D and 3D structures as well as mice-xenografts. AT13387 induced effective cytotoxic activity and radiosensitized cancer cells in monolayer and tumor spheroid models, where low drug doses triggered significant synergistic effects on cell survival together with radiation. Furthermore, AT13387 treatment resulted in G2/M-phase arrest and significantly reduced the migration capacity. The expression of selected client proteins involved in DNA repair, cell-signaling and cell growth was downregulated in vitro, though the expression of most investigated proteins recurred after 8–24 h. These results were confirmed in vivo where AT13387 treated tumors displayed effective downregulation of HSP90 and its oncogenic client proteins. In conclusion, our results demonstrate that AT13387 is a potent new cancer drug and effective radiosensitizer in vitro with an excellent in vivo efficacy. AT13387 treatment has the potential to improve external beam therapy and radionuclide therapy outcomes and restore treatment efficacy in cancers that are resistant to initial therapeutic regimes. PMID:26452257

  5. Hsf1 and Hsp90 orchestrate temperature-dependent global transcriptional remodelling and chromatin architecture in Candida albicans

    PubMed Central

    Leach, Michelle D.; Farrer, Rhys A.; Tan, Kaeling; Miao, Zhengqiang; Walker, Louise A.; Cuomo, Christina A.; Wheeler, Robert T.; Brown, Alistair J. P.; Wong, Koon Ho; Cowen, Leah E.

    2016-01-01

    Fever is a universal response to infection, and opportunistic pathogens such as Candida albicans have evolved complex circuitry to sense and respond to heat. Here we harness RNA-seq and ChIP-seq to discover that the heat shock transcription factor, Hsf1, binds distinct motifs in nucleosome-depleted promoter regions to regulate heat shock genes and genes involved in virulence in C. albicans. Consequently, heat shock increases C. albicans host cell adhesion, damage and virulence. Hsf1 activation depends upon the molecular chaperone Hsp90 under basal and heat shock conditions, but the effects are opposite and in part controlled at the level of Hsf1 expression and DNA binding. Finally, we demonstrate that Hsp90 regulates global transcription programs by modulating nucleosome levels at promoters of stress-responsive genes. Thus, we describe a mechanism by which C. albicans responds to temperature via Hsf1 and Hsp90 to orchestrate gene expression and chromatin architecture, thereby enabling thermal adaptation and virulence. PMID:27226156

  6. The novel HSP90 inhibitor AT13387 potentiates radiation effects in squamous cell carcinoma and adenocarcinoma cells.

    PubMed

    Spiegelberg, Diana; Dascalu, Adrian; Mortensen, Anja C; Abramenkovs, Andris; Kuku, Gamze; Nestor, Marika; Stenerlöw, Bo

    2015-11-01

    Overexpression of heat shock protein 90 (HSP90) is associated with increased tumor cell survival and radioresistance. In this study we explored the efficacy of the novel HSP90 inhibitor AT13387 and examined its radiosensitizing effects in combination with gamma-radiation in 2D and 3D structures as well as mice-xenografts. AT13387 induced effective cytotoxic activity and radiosensitized cancer cells in monolayer and tumor spheroid models, where low drug doses triggered significant synergistic effects on cell survival together with radiation. Furthermore, AT13387 treatment resulted in G2/M-phase arrest and significantly reduced the migration capacity. The expression of selected client proteins involved in DNA repair, cell-signaling and cell growth was downregulated in vitro, though the expression of most investigated proteins recurred after 8-24 h. These results were confirmed in vivo where AT13387 treated tumors displayed effective downregulation of HSP90 and its oncogenic client proteins.In conclusion, our results demonstrate that AT13387 is a potent new cancer drug and effective radiosensitizer in vitro with an excellent in vivo efficacy. AT13387 treatment has the potential to improve external beam therapy and radionuclide therapy outcomes and restore treatment efficacy in cancers that are resistant to initial therapeutic regimes. PMID:26452257

  7. [The role of heat shock proteins HSP90 in the response of immune cells to centimeter microwaves].

    PubMed

    Glushkova, O V; Novoselova, E G; Khrenov, M O; Novoselova, T V; Cherenkov, D A; Lunin, S M; Fesenko, E E

    2008-01-01

    The effects of low-level electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1 h) on the production of heat shock proteins, several cytokines, and nitric oxide in isolated mouse macrophages and lymphocytes were examined both under normal conditions and after the treatment of the cells with geldanamycin (GA), a depressor of activity of the heat shock protein 90 (Hsp90). The irradiation of cells without GA induced the production of Hsp70, nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-10 (IL-10), and the tumor necrosis factor -alpha (TNF-alpha). No changes in the production of Hsp90 in irradiated cells were observed, but intracellular locations of Hsp25 and Hsp70 altered. The preliminary treatment of cells with GA did not remove the effects of microwaves: in these conditions, the synthesis of all cytokines tested, nitric oxide, as well as total and membrane amount of Hsp70, and the amount of Hsp25 in the cytoplasm and cytoskeleton increased. Moreover, the exposure of cells incubated with GA resulted in the reduction of Hsp90-alpha production. PMID:18488507

  8. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

    PubMed Central

    Larsson, Andreas; Nordlund, Paer; Jansson, Anna; Anand, Ganesh S.

    2016-01-01

    A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD). PMID:27253209

  9. Harmine Is a Potent Antimalarial Targeting Hsp90 and Synergizes with Chloroquine and Artemisinin

    PubMed Central

    Shahinas, Dea; MacMullin, Gregory; Benedict, Christan; Crandall, Ian

    2012-01-01

    Previous studies have shown an antimalarial effect of total alkaloids extracted from leaves of Guiera senegalensis from Mali in West Africa. We independently observed that the beta-carboline alkaloid harmine obtained from a natural product library screen inhibited Plasmodium falciparum heat shock protein 90 (PfHsp90) ATP-binding domain. In this study, we confirmed harmine-PfHsp90-specific affinity using surface plasmon resonance analysis (dissociation constant [Kd] of 40 μM). In contrast, the related compound harmalol bound human Hsp90 (HsHsp90) (Kd of 224 μM) more tightly than PfHsp90 (Kd of 7,010 μM). Site-directed mutagenesis revealed that Arg98 in PfHsp90 is essential for harmine selectivity. In keeping with our model indicating that Hsp90 inhibition affords synergistic combinations with existing antimalarials, we demonstrated that harmine potentiates the effect of chloroquine and artemisinin in vitro and in the Plasmodium berghei mouse model. These findings have implications for the development of novel therapeutic combinations that are synergistic with existing antimalarials. PMID:22615284

  10. Human, vector and parasite Hsp90 proteins: A comparative bioinformatics analysis

    PubMed Central

    Faya, Ngonidzashe; Penkler, David L.; Tastan Bishop, Özlem

    2015-01-01

    The treatment of protozoan parasitic diseases is challenging, and thus identification and analysis of new drug targets is important. Parasites survive within host organisms, and some need intermediate hosts to complete their life cycle. Changing host environment puts stress on parasites, and often adaptation is accompanied by the expression of large amounts of heat shock proteins (Hsps). Among Hsps, Hsp90 proteins play an important role in stress environments. Yet, there has been little computational research on Hsp90 proteins to analyze them comparatively as potential parasitic drug targets. Here, an attempt was made to gain detailed insights into the differences between host, vector and parasitic Hsp90 proteins by large-scale bioinformatics analysis. A total of 104 Hsp90 sequences were divided into three groups based on their cellular localizations; namely cytosolic, mitochondrial and endoplasmic reticulum (ER). Further, the parasitic proteins were divided according to the type of parasite (protozoa, helminth and ectoparasite). Primary sequence analysis, phylogenetic tree calculations, motif analysis and physicochemical properties of Hsp90 proteins suggested that despite the overall structural conservation of these proteins, parasitic Hsp90 proteins have unique features which differentiate them from human ones, thus encouraging the idea that protozoan Hsp90 proteins should be further analyzed as potential drug targets. PMID:26793431

  11. Identification of residues on Hsp70 and Hsp90 ubiquitinated by the co-chaperone CHIP

    PubMed Central

    Kundrat, Lenka; Regan, Lynne

    2010-01-01

    Molecular chaperones Hsp70 and Hsp90 are in part responsible for maintaining the viability of cells by facilitating the folding and maturation process of many essential client proteins. The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) has been shown in vitro and in vivo to associate with Hsp70 and Hsp90 and ubiquitinate them; thus targeting them to the proteasome for degradation. Here, we study one facet of this CHIP-mediated turnover by determining the lysine residues on human Hsp70 and Hsp90 ubiquitinated by CHIP. We performed in vitro ubiquitination reactions of the chaperones using purified components and analyzed the samples by tandem mass spectrometry to identify modified lysine residues. Six such ubiquitination sites were identified on Hsp70 (K325, K451, K524, K526, K559, K561) and thirteen ubiquitinated lysine residues were found on Hsp90 (K107, K204, K219, K275, K284, K347, K399, K477, K481, K538, K550, K607, K623). We mapped the ubiquitination sites on homology models of almost full-length human Hsp70 and Hsp90, which were found to cluster in certain regions of the structures. Furthermore, we determined that CHIP forms poly-ubiquitin chains on Hsp70 and Hsp90 linked via K6, K11, K48, and K63. These findings clarify the mode of ubiquitination of Hsp70 and Hsp90 by CHIP which ultimately leads to their degradation. PMID:19913553

  12. Unusual Suspects in the Twilight Zone Between the Hsp90 Interactome and Carcinogenesis.

    PubMed

    Vartholomaiou, Evangelia; Echeverría, Pablo C; Picard, Didier

    2016-01-01

    The molecular chaperone Hsp90 has attracted a lot of interest in cancer research ever since cancer cells were found to be more sensitive to Hsp90 inhibition than normal cells. Why that is has remained a matter of debate and is still unclear. In addition to increased Hsp90 dependence for some mutant cancer proteins and modifications of the Hsp90 machinery itself, a number of other characteristics of cancer cells probably contribute to this phenomenon; these include aneuploidy and overall increased numbers and levels of defective and mutant proteins, which all contribute to perturbed proteostasis. Work over the last two decades has demonstrated that many cancer-related proteins are Hsp90 clients, and yet only few of them have been extensively investigated, selected either on the basis of their obvious function as cancer drivers or because they proved to be convenient biomarkers for monitoring the effects of Hsp90 inhibitors. The purpose of our review is to go beyond these "usual suspects." We established a workflow to select poorly studied proteins that are related to cancer processes and qualify as Hsp90 clients. By discussing and taking a fresh look at these "unusual suspects," we hope to stimulate others to revisit them as novel therapeutic targets or diagnostic markers.

  13. HSP90 canonical content organizes a molecular scaffold mechanism to progress flowering.

    PubMed

    Margaritopoulou, Theoni; Kryovrysanaki, Nikoleta; Megkoula, Panagiota; Prassinos, Constantinos; Samakovli, Despoina; Milioni, Dimitra; Hatzopoulos, Polydefkis

    2016-07-01

    Highly interactive signaling processes constitute a set of parameters intertwining in a continuum mode to shape body formation and development. A sophisticated gene network is required to integrate environmental and endogenous cues in order to modulate flowering. However, the molecular mechanisms that coordinate the circuitries of flowering genes remain unclear. Here using complemented experimental approaches, we uncover the decisive and essential role of HEAT SHOCK PROTEIN 90 (HSP90) in restraining developmental noise to an acceptable limit. Localized depletion of HSP90 mRNAs in the shoot apex resulted in low penetrance of vegetative-to-reproductive phase transition and completely abolished flower formation. Extreme variation in expression of flowering genes was also observed in HSP90 mRNA-depleted transformed plants. Transient heat-shock treatments moderately increased HSP90 mRNA levels and rescued flower arrest. The offspring had a low, nevertheless noticeable failure to promote transition from vegetative into the reproductive phase and showed flower morphological heterogeneity. In floral tissues a moderate variation in HSP90 transcript levels and in the expression of flowering genes was detected. Key flowering proteins comprised clientele of the molecular chaperone demonstrating that the HSP90 is essential during vegetative-to-reproductive phase transition and flower development. Our results uncover that HSP90 consolidates a molecular scaffold able to arrange and organize flowering gene network and protein circuitry, and effectively counterbalance the extent to which developmental noise perturbs phenotypic traits. PMID:27121421

  14. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  15. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae. PMID:16350755

  16. Functional inactivation of endogenous MDM2 and CHIP by HSP90 causes aberrant stabilization of mutant p53 in human cancer cells.

    PubMed

    Li, Dun; Marchenko, Natalia D; Schulz, Ramona; Fischer, Victoria; Velasco-Hernandez, Talia; Talos, Flaminia; Moll, Ute M

    2011-05-01

    The tight control of wild-type p53 by mainly MDM2 in normal cells is permanently lost in tumors harboring mutant p53, which exhibit dramatic constitutive p53 hyperstabilization that far exceeds that of wild-type p53 tumors. Importantly, mutant p53 hyperstabilization is critical for oncogenic gain of function of mutant p53 in vivo. Current insight into the mechanism of this dysregulation is fragmentary and largely derived from ectopically constructed cell systems. Importantly, mutant p53 knock-in mice established that normal mutant p53 tissues have sufficient enzymatic reserves in MDM2 and other E3 ligases to maintain full control of mutant p53. We find that in human cancer cells, endogenous mutant p53, despite its ability to interact with MDM2, suffers from a profound lack of ubiquitination as the root of its degradation defect. In contrast to wild-type p53, the many mutant p53 proteins which are conformationally aberrant are engaged in complexes with the HSP90 chaperone machinery to prevent its aggregation. In contrast to wild-type p53 cancer cells, we show that in mutant p53 cancer cells, this HSP90 interaction blocks the endogenous MDM2 and CHIP (carboxy-terminus of Hsp70-interacting protein) E3 ligase activity. Interference with HSP90 either by RNA interference against HSF1, the transcriptional regulator of the HSP90 pathway, or by direct knockdown of Hsp90 protein or by pharmacologic inhibition of Hsp90 activity with 17AAG (17-allylamino-17-demethoxygeldanamycin) destroys the complex, liberates mutant p53, and reactivates endogenous MDM2 and CHIP to degrade mutant p53. Of note, 17AAG induces a stronger viability loss in mutant p53 than in wild-type p53 cancer cells. Our data support the rationale that suppression of mutant p53 levels in vivo in established cancers might achieve clinically significant effects.

  17. The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64

    PubMed Central

    Qbadou, Soumya; Becker, Thomas; Mirus, Oliver; Tews, Ivo; Soll, Jürgen; Schleiff, Enrico

    2006-01-01

    Precursor protein targeting toward organellar surfaces is assisted by different cytosolic chaperones. We demonstrate that the chloroplast protein translocon subunit Toc64 is the docking site for Hsp90 affiliated preproteins. Thereby, Hsp90 is recognised by the clamp type TPR domain of Toc64. The subsequent transfer of the preprotein from Toc64 to the major receptor of the Toc complex, namely Toc34, is affinity driven and nucleotide dependent. We propose that Toc64 acts as an initial docking site for Hsp90 associated precursor proteins. We outline a mechanism in which chaperones are recruited for a specific targeting event by a membrane-inserted receptor. PMID:16619024

  18. Single nucleotide polymorphisms of HSP90AA1 gene influence response of SLE patients to glucocorticoids treatment.

    PubMed

    Zou, Yan-Feng; Xu, Jian-Hua; Gu, Yuan-Yuan; Pan, Fa-Ming; Tao, Jin-Hui; Wang, De-Guang; Xu, Sheng-Qian; Xiao, Hui; Chen, Pei-Ling; Liu, Shuang; Cai, Jing; Lian, Li; Liu, Sheng-Xiu; Liang, Chun-Mei; Tian, Guo; Ye, Qian-Ling; Pan, Hai-Feng; Su, Hong; Ye, Dong-Qing

    2016-01-01

    Heat shock protein 90 (HSP90) is an important glucocorticoid receptor (GR) chaperone protein, and is supposed to be the key factor in regulating glucocorticoids (GCs) effects. The aim of the present study was to explore whether single nucleotide polymorphisms (SNPs) within HSP90AA1 gene affect the response of systemic lupus erythematosus (SLE) patients to GCs treatment. Two hundred and forty-five SLE patients were treated with GCs (prednisone) for 12 weeks. SLE disease activity index (SLEDAI) was used to assess the response of SLE patients to GCs treatment, and patients were classified into sensitive group and insensitive group. HapMap database and Haploview software were used to select tag SNPs. Tag SNPs were genotyped by using multiplex SNaPshot method. Univariate and multivariate logistic regression analyses were used to discriminate the impact of SNPs of HSP90AA1 gene on the response of SLE patients to GCs treatment. Two hundred and thirty three SLE patients finished the 12-week follow-up. Of these patients, 128 patients were included in sensitive group, and 105 patients were included in insensitive group. Seven tag SNPs were selected within HSP90AA1 gene. We detected significant associations for rs7160651 (dominant model: crude OR 0.514, 95 % CI 0.297-0.890, P = 0.018; adjusted OR 0.518, 95 % CI 0.293-0.916, P = 0.024), rs10873531 (dominant model: crude OR 0.516, 95 % CI 0.305-0.876, P = 0.014; adjusted OR 0.522, 95 % CI 0.304-0.898, P = 0.019) and rs2298877 (dominant model: crude OR 0.543, 95 % CI 0.317-0.928, P = 0.026, adjusted OR 0.558, 95 % CI 0.323-0.967, P = 0.037) polymorphisms, but not for other polymorphisms (P > 0.05). The present study demonstrates that HSP90AA1 gene SNPs may affect the response of SLE patients to GCs treatment.

  19. Single nucleotide polymorphisms of HSP90AA1 gene influence response of SLE patients to glucocorticoids treatment.

    PubMed

    Zou, Yan-Feng; Xu, Jian-Hua; Gu, Yuan-Yuan; Pan, Fa-Ming; Tao, Jin-Hui; Wang, De-Guang; Xu, Sheng-Qian; Xiao, Hui; Chen, Pei-Ling; Liu, Shuang; Cai, Jing; Lian, Li; Liu, Sheng-Xiu; Liang, Chun-Mei; Tian, Guo; Ye, Qian-Ling; Pan, Hai-Feng; Su, Hong; Ye, Dong-Qing

    2016-01-01

    Heat shock protein 90 (HSP90) is an important glucocorticoid receptor (GR) chaperone protein, and is supposed to be the key factor in regulating glucocorticoids (GCs) effects. The aim of the present study was to explore whether single nucleotide polymorphisms (SNPs) within HSP90AA1 gene affect the response of systemic lupus erythematosus (SLE) patients to GCs treatment. Two hundred and forty-five SLE patients were treated with GCs (prednisone) for 12 weeks. SLE disease activity index (SLEDAI) was used to assess the response of SLE patients to GCs treatment, and patients were classified into sensitive group and insensitive group. HapMap database and Haploview software were used to select tag SNPs. Tag SNPs were genotyped by using multiplex SNaPshot method. Univariate and multivariate logistic regression analyses were used to discriminate the impact of SNPs of HSP90AA1 gene on the response of SLE patients to GCs treatment. Two hundred and thirty three SLE patients finished the 12-week follow-up. Of these patients, 128 patients were included in sensitive group, and 105 patients were included in insensitive group. Seven tag SNPs were selected within HSP90AA1 gene. We detected significant associations for rs7160651 (dominant model: crude OR 0.514, 95 % CI 0.297-0.890, P = 0.018; adjusted OR 0.518, 95 % CI 0.293-0.916, P = 0.024), rs10873531 (dominant model: crude OR 0.516, 95 % CI 0.305-0.876, P = 0.014; adjusted OR 0.522, 95 % CI 0.304-0.898, P = 0.019) and rs2298877 (dominant model: crude OR 0.543, 95 % CI 0.317-0.928, P = 0.026, adjusted OR 0.558, 95 % CI 0.323-0.967, P = 0.037) polymorphisms, but not for other polymorphisms (P > 0.05). The present study demonstrates that HSP90AA1 gene SNPs may affect the response of SLE patients to GCs treatment. PMID:27026916

  20. Chemical proteomics-driven discovery of oleocanthal as an Hsp90 inhibitor.

    PubMed

    Margarucci, Luigi; Monti, Maria Chiara; Cassiano, Chiara; Mozzicafreddo, Matteo; Angeletti, Mauro; Riccio, Raffaele; Tosco, Alessandra; Casapullo, Agostino

    2013-07-01

    Hsp90, a key target in cancer therapy, has been identified as the main partner of oleocanthal, an olive oil bioactive compound. A combination of chemical and biological assays disclosed its mechanism of action at the molecular level. PMID:23703283

  1. A review of recent patents on the protozoan parasite HSP90 as a drug target.

    PubMed

    Angel, Sergio O; Matrajt, Mariana; Echeverria, Pablo C

    2013-04-01

    Diseases caused by protozoan parasites are still an important health problem. These parasites can cause a wide spectrum of diseases, some of which are severe and have high morbidity or mortality if untreated. Since they are still uncontrolled, it is important to find novel drug targets and develop new therapies to decrease their remarkable social and economic impact on human societies. In the past years, human HSP90 has become an interesting drug target that has led to a large number of investigations both at state organizations and pharmaceutical companies, followed by clinical trials. The finding that HSP90 has important biological roles in some protozoan parasites like Plasmodium spp, Toxoplasma gondii and trypanosomatids has allowed the expansion of the results obtained in human cancer to these infections. This review summarizes the latest important findings showing protozoan HSP90 as a drug target and presents three patents targeting T. gondii, P. falciparum and trypanosomatids HSP90.

  2. Hsp90 inhibitor celastrol reinstates growth plate angiogenesis in thiram-induced tibial dyschondroplasia.

    PubMed

    Nabi, Fazul; Shahzad, Muhammad; Liu, Jingying; Li, Kun; Han, Zhaoqing; Zhang, Ding; Iqbal, Muhammad Kashif; Li, Jiakui

    2016-01-01

    Tibial dyschondroplasia (TD) is an important long bone defect of broiler chickens that disturbs the proximal growth plate and is characterized by non-vascularized cartilage, a distended growth plate and lameness. Celastrol, a medicinal root extract from the plant Tripterygium wilfordii, is reported widely as a well-known heat-shock protein 90 (Hsp90) inhibitor. Recently, Hsp90 inhibition in chondrocyte differentiation and growth-plate vascularization were effective in restoring the morphology of the growth plate. The present study was aimed at investigating Hsp90 inhibition in TD using celastrol. The broiler chicks were divided into three groups; Control; TD induced (40 mg/kg thiram) and celastrol treatment. Hsp90, vascular endothelial growth factor and Flk-1 expressions were evaluated by quantitative real-time polymerase chain reaction and the protein levels of Hsp90 were measured by Western blot analysis. Antioxidant enzymes were determined to assess the liver damage caused by thiram and the protective effects of the medicine were evaluated by levels of serum biomarkers. The expression levels of Hsp90 and vascular endothelial growth factor mRNA transcripts were increased while Flk-1 receptor was decreased in TD-affected chicks. Celastrol therapy inhibited Hsp90 mRNA and protein levels and up-regulated the expressions of receptor Flk-1 in TD-affected tibial growth plates significantly (P < 0.05) in addition to rectifying the damaging effects of thiram on the liver by decreasing the levels of aspartate aminotransferase, alanine aminotransferase and malondialdehyde and correcting the oxidative imbalance. In conclusion, administering celastrol to dyschondroplastic chicks prevented un-vascularized growth plate, lameness and reinstated angiogenesis. Celastrol may be efficacious for the treatment of TD through the inhibition of Hsp90 expression and limiting the liver damage caused by thiram in broiler chickens.

  3. Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes.

    PubMed

    Travers, Timothy S; Harlow, Lisa; Rosas, Ivan O; Gochuico, Bernadette R; Mikuls, Ted R; Bhattacharya, Sanjoy K; Camacho, Carlos J; Ascherman, Dana P

    2016-09-01

    Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90β/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90β]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context. PMID:27448590

  4. Cloning and characterization of salmon hsp90 cDNA: upregulation by thermal and hyperosmotic stress.

    PubMed

    Pan, F; Zarate, J M; Tremblay, G C; Bradley, T M

    2000-08-01

    Accumulating evidence suggests that glucocorticoids are essential for development of hypoosmoregulatory capacity in salmon during adaptation to seawater. Heat shock protein (hsp)90 has been reported to function in signal transduction and the maturation and affinity of glucocorticoid receptors. We sought to determine whether this hsp might be upregulated by thermal and hyperosmotic stress in salmon, a species that migrates between the freshwater and marine environments. A 2625-bp cDNA cloned from a salmon cDNA library was found to code for a protein of 722 amino acids exhibiting a high degree of identity with zebra fish (92%) and human (89%) hsp90beta. Accumulation of hsp90 mRNA was observed in isolated branchial lamellae incubated under hyperosmotic conditions and in branchial lamellae of salmon exposed to hyperosmotic stress in vivo. In contrast, exposure of kidney to hyperosmotic stress in vitro and in vivo failed to elicit an increase in the quantity of hsp90 mRNA. By way of comparison, accumulation of hsp90 mRNA was observed in both branchial lamellae and kidney tissue subjected to thermal stress in vitro and in vivo. Western blot analyses of proteins isolated from tissues under identical conditions in vitro revealed that the pool of hsp90 increased with thermal stress but not with osmotic stress. The results suggest that accumulation of hsp90 mRNA in response to osmotic stress is unrelated to cellular protein denaturation and that synthesis of hsp90 may be regulated at both the level of transcription and translation.

  5. Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes.

    PubMed

    Travers, Timothy S; Harlow, Lisa; Rosas, Ivan O; Gochuico, Bernadette R; Mikuls, Ted R; Bhattacharya, Sanjoy K; Camacho, Carlos J; Ascherman, Dana P

    2016-09-01

    Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90β/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90β]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.

  6. Heat shock protein Hsp90-2 expression in the Arabidopsis thaliana seedlings under clinorotation

    NASA Astrophysics Data System (ADS)

    Kozeko, Liudmyla

    Heat shock proteins 90 kDa (Hsp90) are abundant under normal conditions and induced by stress. This family is distinguished from other chaperones in that most of its substrates are signal transduction proteins. Previously, we determined some time-dependent increase in the Hsp90 level in pea seedlings in response to simulated microgravity that indicated a stress-reaction. However, expression of the individual members of the Hsp90 family have specific pattern. The purpose of this study was to investigate possible alterations in the gene expression pattern of cytosolic Hsp90-2 in Arabidopsis thaliana seedlings under 2D-clinorotation. To obtain detailed expression pattern of the HSP90-2 genes we used seeds that provides a resource of loss-of-function mutations gene expression patterns via translational fusions with the reporter gene, GUS (a line N 166718, NASC). There were two variants of the experiment: 1) seedlings grew under clinorotation for 10, 12, 14 d; 2) seedlings grew in the stationary conditions for 10 d followed by clinorotation for 3 h -at 22o C and 16h light cycle. The seedlings grown in the stationary conditions were used as a control. GUS staining showed that HSP90-2 expression was regulated during seedling development and affected by clinorotation in the heterozygous mutant plants. In the homozygous for the mutation plants, HSP90-2 expression was stable during seedling development and not affected by clinorotation. GUS staining was observed in cotyledons, leaves and hypocotyls of the seedlings (especially intense in vascular bundles), indicating intensive cellular processes with participation of this chaperone. Possible pathways of influence of clinorotation on HSP90-2 expression are discussed.

  7. Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

    SciTech Connect

    Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H. Prodromou, Chrisostomos

    2015-05-01

    A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90){sub 2}–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

  8. Structural and functional coupling of Hsp90- and Sgt1-centred multi-protein complexes.

    PubMed

    Zhang, Minghao; Botër, Marta; Li, Kuoyu; Kadota, Yasuhiro; Panaretou, Barry; Prodromou, Chrisostomos; Shirasu, Ken; Pearl, Laurence H

    2008-10-22

    Sgt1 is an adaptor protein implicated in a variety of processes, including formation of the kinetochore complex in yeast, and regulation of innate immunity systems in plants and animals. Sgt1 has been found to associate with SCF E3 ubiquitin ligases, the CBF3 kinetochore complex, plant R proteins and related animal Nod-like receptors, and with the Hsp90 molecular chaperone. We have determined the crystal structure of the core Hsp90-Sgt1 complex, revealing a distinct site of interaction on the Hsp90 N-terminal domain. Using the structure, we developed mutations in Sgt1 interfacial residues, which specifically abrogate interaction with Hsp90, and disrupt Sgt1-dependent functions in vivo, in plants and yeast. We show that Sgt1 bridges the Hsp90 molecular chaperone system to the substrate-specific arm of SCF ubiquitin ligase complexes, suggesting a role in SCF assembly and regulation, and providing multiple complementary routes for ubiquitination of Hsp90 client proteins. PMID:18818696

  9. The charged region of Hsp90 modulates the function of the N-terminal domain

    PubMed Central

    Scheibel, Thomas; Siegmund, Heiko Ingo; Jaenicke, Rainer; Ganz, Peter; Lilie, Hauke; Buchner, Johannes

    1999-01-01

    Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90. PMID:9990018

  10. Characterisation of the Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop).

    PubMed

    Gitau, Grace W; Mandal, Pradipta; Blatch, Gregory L; Przyborski, Jude; Shonhai, Addmore

    2012-03-01

    Malaria is caused by Plasmodium species, whose transmission to vertebrate hosts is facilitated by mosquito vectors. The transition from the cold blooded mosquito vector to the host represents physiological stress to the parasite, and additionally malaria blood stage infection is characterised by intense fever periods. In recent years, it has become clear that heat shock proteins play an essential role during the parasite's life cycle. Plasmodium falciparum expresses two prominent heat shock proteins: heat shock protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90). Both of these proteins have been implicated in the development and pathogenesis of malaria. In eukaryotes, Hsp70 and Hsp90 proteins are functionally linked by an essential adaptor protein known as the Hsp70-Hsp90 organising protein (Hop). In this study, recombinant P. falciparum Hop (PfHop) was heterologously produced in E. coli and purified by nickel affinity chromatography. Using specific anti-PfHop antisera, the expression and localisation of PfHop in P. falciparum was investigated. PfHop was shown to co-localise with PfHsp70 and PfHsp90 in parasites at the trophozoite stage. Gel filtration and co-immunoprecipitation experiments suggested that PfHop was present in a complex together with PfHsp70 and PfHsp90. The association of PfHop with both PfHsp70 and PfHsp90 suggests that this protein may mediate the functional interaction between the two chaperones.

  11. Synthesis and evaluation of cell-permeable biotinylated PU-H71 derivatives as tumor Hsp90 probes

    PubMed Central

    DaGama Gomes, Erica M; Riolo, Matthew; Patel, Hardik J; Alonso-Sabadell, Raul; Zatorska, Danuta; Patel, Maulik R; Kishinevsky, Sarah

    2013-01-01

    Summary The attachment of biotin to a small molecule provides a powerful tool in biology. Here, we present a systematic approach to identify biotinylated analogues of the Hsp90 inhibitor PU-H71 that are capable of permeating cell membranes so as to enable the investigation of Hsp90 complexes in live cells. The identified derivative 2g can isolate Hsp90 through affinity purification and, as we show, represents a unique and useful tool to probe tumor Hsp90 biology in live cells by affinity capture, flow cytometry and confocal microscopy. To our knowledge, 2g is the only reported biotinylated Hsp90 probe to have such combined characteristics. PMID:23616796

  12. The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain.

    PubMed

    Chen, Kelan; Dobson, Renwick C J; Lucet, Isabelle S; Young, Samuel N; Pearce, F Grant; Blewitt, Marnie E; Murphy, James M

    2016-06-15

    Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level. PMID:27059856

  13. Molecular cloning and expression analysis of a pearl oyster (Pinctada martensii) heat shock protein 90 (HSP90).

    PubMed

    Liang, H Y; Wang, Z X; Lei, Q N; Huang, R L; Deng, Y W; Wang, Q H; Jiao, Y; Du, X D

    2015-01-01

    Heat shock protein 90 (HSP90) is an important molecular chaperone required for proper folding of cellular proteins, and thus, it plays an essential role in protecting cells from damage during stress. In this study, an HSP90 cDNA designated PmHSP90 was cloned from the mantle tissue of the pearl oyster Pinctada martensii using reverse transcription polymerase chain reaction (RT-PCR) coupled with the rapid amplification of cDNA ends (RACE) approach. PmHSP90 cDNA was 2584 bp in length, including an open reading frame of 2160 bp, which encodes a polypeptide of 719 amino acid residues, with predicted molecular mass and isoelectric point of 83.0 kDa and 4.87, respectively. Multiple-sequence alignment indicated that HSP90 is highly conserved among species, and PmHSP90 showed 89% sequence identity to Crassostrea gigas HSP90. Five conserved amino acid blocks defined as HSP90 protein family signatures were also observed in PmHSP90, indicating that PmHSP90 may be a cytosolic member of the HSP90 family. Expression levels of PmHSP90 were detected in various tissues of P. martensii and in hemocytes under three different stress conditions using quantitative real-time PCR (qPCR). The results demonstrate that PmHSP90 mRNA is constitutively expressed in all the tested tissues and may be involved in the immune response against thermal stress, lipopolysaccharide stimulation, and nucleus insertion operations. Studies on PmHSP90 are a valuable source to further explore the immune system in pearl oysters during the production of pearls, and may enhance our knowledge of molluscan innate immunity. PMID:26782528

  14. Proposal of Dual Inhibitor Targeting ATPase Domains of Topoisomerase II and Heat Shock Protein 90

    PubMed Central

    Jun, Kyu-Yeon; Kwon, Youngjoo

    2016-01-01

    There is a conserved ATPase domain in topoisomerase II (topo II) and heat shock protein 90 (Hsp90) which belong to the GHKL (gyrase, Hsp90, histidine kinase, and MutL) family. The inhibitors that target each of topo II and Hsp90 are intensively studied as anti-cancer drugs since they play very important roles in cell proliferation and survival. Therefore the development of dual targeting anti-cancer drugs for topo II and Hsp90 is suggested to be a promising area. The topo II and Hsp90 inhibitors, known to bind to their ATP binding site, were searched. All the inhibitors investigated were docked to both topo II and Hsp90. Four candidate compounds as possible dual inhibitors were selected by analyzing the molecular docking study. The pharmacophore model of dual inhibitors for topo II and Hsp90 were generated and the design of novel dual inhibitor was proposed. PMID:27582553

  15. Na+/K+-ATPase: Activity and inhibition

    NASA Astrophysics Data System (ADS)

    Čolović, M.; Krstić, D.; Krinulović, K.; Momić, T.; Savić, J.; Vujačić, A.; Vasić, V.

    2009-09-01

    The aim of the study was to give an overview of the mechanism of inhibition of Na+/K+-ATPase activity induced by some specific and non specific inhibitors. For this purpose, the effects of some ouabain like compounds (digoxin, gitoxin), noble metals complexes ([PtCl2DMSO2], [AuCl4]-, [PdCl4]2-, [PdCl(dien)]+, [PdCl(Me4dien)]+), transition metal ions (Cu2+, Zn2+, Fe2+, Co2+), and heavy metal ions (Hg2+, Pb2+, Cd2+) on the activity of Na+/K+-ATPase from rat synaptic plasma membranes (SPM), porcine cerebral cortex and human erythrocytes were discussed.

  16. The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans.

    PubMed

    Ni, Weiming; Hutagalung, Alex H; Li, Shumin; Epstein, Henry F

    2011-09-15

    The UNC-45 family of molecular chaperones is expressed in metazoan organisms from Caenorhabditis elegans to humans. The UNC-45 protein is essential in C. elegans for early body-wall muscle cell development and A-band assembly. We show that the myosin-binding UCS domain of UNC-45 alone is sufficient to rescue lethal unc-45 null mutants arrested in embryonic muscle development and temperature-sensitive loss-of-function unc-45 mutants defective in worm A-band assembly. Removal of the Hsp90-binding TPR domain of UNC-45 does not affect rescue. Similar results were obtained with overexpression of the same fragments in wild-type nematodes when assayed for diminution of myosin accumulation and assembly. Titration experiments show that, on a per molecule basis, UCS has greater activity in C. elegans muscle in vivo than full-length UNC-45 protein, suggesting that UNC-45 is inhibited by either the TPR domain or its interaction with the general chaperone Hsp90. In vitro experiments with purified recombinant C. elegans Hsp90 and UNC-45 proteins show that they compete for binding to C. elegans myosin. Our in vivo genetic and in vitro biochemical experiments are consistent with a novel inhibitory role for Hsp90 with respect to UNC-45 action.

  17. Casein Kinase 2 (CK2)-mediated Phosphorylation of Hsp90β as a Novel Mechanism of Rifampin-induced MDR1 Expression.

    PubMed

    Kim, So Won; Hasanuzzaman, Md; Cho, Munju; Heo, Ye Rang; Ryu, Min-Jung; Ha, Na-Young; Park, Hyun June; Park, Hyung-Yeon; Shin, Jae-Gook

    2015-07-01

    The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression. PMID:25995454

  18. Molecular cloning, mRNA expression, and characterization of HSP90 gene from Chinese mitten crab Eriocheir japonica sinensis.

    PubMed

    Li, Peng; Zha, Jie; Zhang, Zhenhua; Huang, Hua; Sun, Hongying; Song, Daxiang; Zhou, Kaiya

    2009-07-01

    HSP90 is a highly conserved molecular chaperone important in the maturation of a broad spectrum of proteins. Using expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques, an HSP90 gene designated as EjsHSP90 was cloned and characterized from the Chinese mitten crab Eriocheir japonica sinensis. The full-length cDNA of EjsHSP90 is 2,517 bp and contains an open reading frame of 2157 bp which encodes a 718 amino acid polypeptide (82.8 kDa) bearing characteristics of the HSP90 family and an ATP binding domain. Sequence alignment shows that EjsHSP90 shared 79%-96% identity with HSP90 sequences reported in other animals, and it shares identical structural features. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsHSP90 mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that EjsHSP90 is expressed throughout the three developmental stages but expression levels varied among different body parts of crabs. EjsHSP90 mRNA expression in the abdomen of the first crab stage is consistently higher than that of the other two stages, suggesting that EjsHSP90 gene is involved in the crabs' early developmental process, especially in the crab brachyurization process. Results from quantitative RT-PCR excluded the possibility that the expression of EjsHSP90 mRNA is induced primarily by osmotic stress. Phylogenetic analyses reveal that HSP90 gene is informative and complementary for reconstruction of arthropod phylogenetic relationships. PMID:19166961

  19. Transcriptomic and Proteomic Investigation of HSP90A as a Potential Biomarker for HCC

    PubMed Central

    Zhou, Yi; Deng, Xiaofang; Zang, Ning; Li, Hongtao; Li, Gang; Li, Cuiping; He, Min

    2015-01-01

    Background Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related death in adults. Despite recent advances in the clinical technologies, the screening and diagnostic efficacy for HCC remains poor. Discovering novel and reliable HCC biomarkers is urgently needed. Material/Methods We performed a transcriptome-proteome integrated assay to track the possible HCC biomarkers from the process of HCC-derived gene expression in malignant cells to its protein product released into serum. Results Our screening results demonstrated that heat shock protein 90A (HSP90A), which participates in the PI3K-Akt signaling pathway and many other cancer-related pathways, warrants further investigation. The expression of HSP90A was increased in the HCC cells, serum, and tissues. Immunohistochemistry analysis on 76 clinical tissue samples also suggested the relevance between HSP90A expression and HCC metastatic behavior. Conclusions These findings suggest a role for HSP90A in HCC pathogenesis and the potential use of HSP90A for the screening and diagnosis of this malignancy. PMID:26704341

  20. Conformational diversity in the TPR domain-mediated interaction of protein phosphatase 5 with Hsp90.

    PubMed

    Cliff, Matthew J; Harris, Richard; Barford, David; Ladbury, John E; Williams, Mark A

    2006-03-01

    Protein phosphatase 5 (Ppp5) is one of several proteins that bind to the Hsp90 chaperone via a tetratricopeptide repeat (TPR) domain. We report the solution structure of a complex of the TPR domain of Ppp5 with the C-terminal pentapeptide of Hsp90. This structure has the "two-carboxylate clamp" mechanism of peptide binding first seen in the Hop-TPR domain complexes with Hsp90 and Hsp70 peptides. However, NMR data reveal that the Ppp5 clamp is highly dynamic, and that there are multiple modes of peptide binding and mobility throughout the complex. Although this interaction is of very high affinity, relatively few persistent contacts are found between the peptide and the Ppp5-TPR domain, thus explaining its promiscuity in binding both Hsp70 and Hsp90 in vivo. We consider the possible implications of this dynamic structure for the mechanism of relief of autoinhibition in Ppp5 and for the mechanisms of TPR-mediated recognition of Hsp90 by other proteins.

  1. Gene expression profiles of cytosolic heat shock proteins Hsp70 and Hsp90 from symbiotic dinoflagellates in response to thermal stress: possible implications for coral bleaching.

    PubMed

    Rosic, Nedeljka N; Pernice, Mathieu; Dove, Sophie; Dunn, Simon; Hoegh-Guldberg, Ove

    2011-01-01

    Unicellular photosynthetic dinoflagellates of the genus Symbiodinium are the most common endosymbionts of reef-building scleractinian corals, living in a symbiotic partnership known to be highly susceptible to environmental changes such as hyperthermic stress. In this study, we identified members of two major heat shock proteins (HSPs) families, Hsp70 and Hsp90, in Symbiodinium sp. (clade C) with full-length sequences that showed the highest similarity and evolutionary relationship with other known HSPs from dinoflagellate protists. Regulation of HSPs gene expression was examined in samples of the scleractinian coral Acropora millepora subjected to elevated temperatures progressively over 18 h (fast) and 120 h (gradual thermal stress). Moderate to severe heat stress at 26°C and 29°C (+3°C and +6°C above average sea temperature) resulted in an increase in algal Hsp70 gene expression from 39% to 57%, while extreme heat stress (+9°C) reduced Hsp70 transcript abundance by 60% (after 18 h) and 70% (after 120 h). Elevated temperatures decreased an Hsp90 expression under both rapid and gradual heat stress scenarios. Comparable Hsp70 and Hsp90 gene expression patterns were observed in Symbiodinium cultures and in hospite, indicating their independent regulation from the host. Differential gene expression profiles observed for Hsp70 and Hsp90 suggests diverse roles of these molecular chaperones during heat stress response. Reduced expression of the Hsp90 gene under heat stress can indicate a reduced role in inhibiting the heat shock transcription factor which may lead to activation of heat-inducible genes and heat acclimation.

  2. Gene expression profiles of cytosolic heat shock proteins Hsp70 and Hsp90 from symbiotic dinoflagellates in response to thermal stress: possible implications for coral bleaching

    PubMed Central

    Pernice, Mathieu; Dove, Sophie; Dunn, Simon; Hoegh-Guldberg, Ove

    2010-01-01

    Unicellular photosynthetic dinoflagellates of the genus Symbiodinium are the most common endosymbionts of reef-building scleractinian corals, living in a symbiotic partnership known to be highly susceptible to environmental changes such as hyperthermic stress. In this study, we identified members of two major heat shock proteins (HSPs) families, Hsp70 and Hsp90, in Symbiodinium sp. (clade C) with full-length sequences that showed the highest similarity and evolutionary relationship with other known HSPs from dinoflagellate protists. Regulation of HSPs gene expression was examined in samples of the scleractinian coral Acropora millepora subjected to elevated temperatures progressively over 18 h (fast) and 120 h (gradual thermal stress). Moderate to severe heat stress at 26°C and 29°C (+3°C and +6°C above average sea temperature) resulted in an increase in algal Hsp70 gene expression from 39% to 57%, while extreme heat stress (+9°C) reduced Hsp70 transcript abundance by 60% (after 18 h) and 70% (after 120 h). Elevated temperatures decreased an Hsp90 expression under both rapid and gradual heat stress scenarios. Comparable Hsp70 and Hsp90 gene expression patterns were observed in Symbiodinium cultures and in hospite, indicating their independent regulation from the host. Differential gene expression profiles observed for Hsp70 and Hsp90 suggests diverse roles of these molecular chaperones during heat stress response. Reduced expression of the Hsp90 gene under heat stress can indicate a reduced role in inhibiting the heat shock transcription factor which may lead to activation of heat-inducible genes and heat acclimation. PMID:20821176

  3. Trypsin-induced ATPase Activity in Potato Mitochondria.

    PubMed

    Jung, D W; Laties, G G

    1976-04-01

    Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg(2+), 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg(2+)-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60 C for 2 minutes also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.

  4. Vibsanin B preferentially targets HSP90β, inhibits interstitial leukocyte migration, and ameliorates experimental autoimmune encephalomyelitis.

    PubMed

    Ye, Bai-Xin; Deng, Xu; Shao, Li-Dong; Lu, Ying; Xiao, Run; Liu, Yi-Jie; Jin, Yi; Xie, Yin-Yin; Zhao, Yan; Luo, Liu-Fei; Ma, Shun; Gao, Ming; Zhang, Lian-Ru; He, Juan; Zhang, Wei-Na; Chen, Yi; Xia, Cheng-Feng; Deng, Min; Liu, Ting-Xi; Zhao, Qin-Shi; Chen, Sai-Juan; Chen, Zhu

    2015-05-01

    Interstitial leukocyte migration plays a critical role in inflammation and offers a therapeutic target for treating inflammation-associated diseases such as multiple sclerosis. Identifying small molecules to inhibit undesired leukocyte migration provides promise for the treatment of these disorders. In this study, we identified vibsanin B, a novel macrocyclic diterpenoid isolated from Viburnum odoratissimum Ker-Gawl, that inhibited zebrafish interstitial leukocyte migration using a transgenic zebrafish line (TG:zlyz-enhanced GFP). We found that vibsanin B preferentially binds to heat shock protein (HSP)90β. At the molecular level, inactivation of HSP90 can mimic vibsanin B's effect of inhibiting interstitial leukocyte migration. Furthermore, we demonstrated that vibsanin B ameliorates experimental autoimmune encephalomyelitis in mice with pathological manifestation of decreased leukocyte infiltration into their CNS. In summary, vibsanin B is a novel lead compound that preferentially targets HSP90β and inhibits interstitial leukocyte migration, offering a promising drug lead for treating inflammation-associated diseases.

  5. Characterisation of the Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop).

    PubMed

    Gitau, Grace W; Mandal, Pradipta; Blatch, Gregory L; Przyborski, Jude; Shonhai, Addmore

    2012-03-01

    Malaria is caused by Plasmodium species, whose transmission to vertebrate hosts is facilitated by mosquito vectors. The transition from the cold blooded mosquito vector to the host represents physiological stress to the parasite, and additionally malaria blood stage infection is characterised by intense fever periods. In recent years, it has become clear that heat shock proteins play an essential role during the parasite's life cycle. Plasmodium falciparum expresses two prominent heat shock proteins: heat shock protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90). Both of these proteins have been implicated in the development and pathogenesis of malaria. In eukaryotes, Hsp70 and Hsp90 proteins are functionally linked by an essential adaptor protein known as the Hsp70-Hsp90 organising protein (Hop). In this study, recombinant P. falciparum Hop (PfHop) was heterologously produced in E. coli and purified by nickel affinity chromatography. Using specific anti-PfHop antisera, the expression and localisation of PfHop in P. falciparum was investigated. PfHop was shown to co-localise with PfHsp70 and PfHsp90 in parasites at the trophozoite stage. Gel filtration and co-immunoprecipitation experiments suggested that PfHop was present in a complex together with PfHsp70 and PfHsp90. The association of PfHop with both PfHsp70 and PfHsp90 suggests that this protein may mediate the functional interaction between the two chaperones. PMID:22005844

  6. Synthetic lethality of combined glutaminase and Hsp90 inhibition in mTORC1-driven tumor cells.

    PubMed

    Li, Jing; Csibi, Alfredo; Yang, Sun; Hoffman, Gregory R; Li, Chenggang; Zhang, Erik; Yu, Jane J; Blenis, John

    2015-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals from growth factors, nutrients, and cellular energy status to control a wide range of metabolic processes, including mRNA biogenesis; protein, nucleotide, and lipid synthesis; and autophagy. Deregulation of the mTORC1 pathway is found in cancer as well as genetic disorders such as tuberous sclerosis complex (TSC) and sporadic lymphangioleiomyomatosis. Recent studies have shown that the mTORC1 inhibitor rapamycin and its analogs generally suppress proliferation rather than induce apoptosis. Therefore, it is critical to use alternative strategies to induce death of cells with activated mTORC1. In this study, a small-molecule screen has revealed that the combination of glutaminase (GLS) and heat shock protein 90 (Hsp90) inhibitors selectively triggers death of TSC2-deficient cells. At a mechanistic level, high mTORC1-driven translation rates in TSC1/2-deficient cells, unlike wild-type cells, sensitizes these cells to endoplasmic reticulum (ER) stress. Thus, Hsp90 inhibition drives accumulation of unfolded protein and ER stress. When combining proteotoxic stress with oxidative stress by depletion of the intracellular antioxidant glutathione by GLS inhibition, acute cell death is observed in cells with activated mTORC1 signaling. This study suggests that this combination strategy may have the potential to be developed into a therapeutic use for the treatment of mTORC1-driven tumors. PMID:25524627

  7. Sequence and domain conservation of the coelacanth Hsp40 and Hsp90 chaperones suggests conservation of function.

    PubMed

    Bishop, Özlem Tastan; Edkins, Adrienne Lesley; Blatch, Gregory Lloyd

    2014-09-01

    Molecular chaperones and their associated co-chaperones play an important role in preserving and regulating the active conformational state of cellular proteins. The chaperone complement of the Indonesian Coelacanth, Latimeria menadoensis, was elucidated using transcriptomic sequences. Heat shock protein 90 (Hsp90) and heat shock protein 40 (Hsp40) chaperones, and associated co-chaperones were focused on, and homologous human sequences were used to search the sequence databases. Coelacanth homologs of the cytosolic, mitochondrial and endoplasmic reticulum (ER) homologs of human Hsp90 were identified, as well as all of the major co-chaperones of the cytosolic isoform. Most of the human Hsp40s were found to have coelacanth homologs, and the data suggested that all of the chaperone machinery for protein folding at the ribosome, protein translocation to cellular compartments such as the ER and protein degradation were conserved. Some interesting similarities and differences were identified when interrogating human, mouse, and zebrafish homologs. For example, DnaJB13 is predicted to be a non-functional Hsp40 in humans, mouse, and zebrafish due to a corrupted histidine-proline-aspartic acid (HPD) motif, while the coelacanth homolog has an intact HPD. These and other comparisons enabled important functional and evolutionary questions to be posed for future experimental studies.

  8. Hsp90 inhibition increases SOCS3 transcript and regulates migration and cell death in chronic lymphocytic leukemia

    PubMed Central

    Chen, Timothy L.; Gupta, Nikhil; Lehman, Amy; Ruppert, Amy S.; Yu, Lianbo; Oakes, Christopher C.; Claus, Rainer; Plass, Christoph; Maddocks, Kami J.; Andritsos, Leslie; Jones, Jeffery A.; Lucas, David M.; Johnson, Amy J.; Byrd, John C.; Hertlein, Erin

    2016-01-01

    Epigenetic or transcriptional silencing of important tumor suppressors has been described to contribute to cell survival and tumorigenesis in chronic lymphocytic leukemia (CLL). Using gene expression microarray analysis, we found that thousands of genes are repressed more than 2-fold in CLL compared to normal B cells; however therapeutic approaches to reverse this have been limited in CLL. Following treatment with the Hsp90 inhibitor 17-DMAG, a significant number of these repressed genes were significantly re-expressed. One of the genes significantly repressed in CLL and up-regulated by 17-DMAG was suppressor of cytokine signaling 3, (SOCS3). SOCS3 has been shown to be silenced in solid tumors as well as myeloid leukemia; however little is known about the regulation in CLL. We found that 17-DMAG induces expression of SOCS3 by via the activation of p38 signaling, and subsequently inhibits AKT and STAT3 phosphorylation resulting in downstream effects on cell migration and survival. We therefore suggest that SOCS3 is an important signaling protein in CLL, and Hsp90 inhibitors represent a novel approach to target transcriptional repression in B cell lymphoproliferative disorders which exhibit a substantial degree of gene repression. PMID:27107422

  9. Dual mode of cancer cell destruction for pancreatic cancer therapy using Hsp90 inhibitor loaded polymeric nano magnetic formulation.

    PubMed

    Rochani, Ankit K; Balasubramanian, Sivakumar; Ravindran Girija, Aswathy; Raveendran, Sreejith; Borah, Ankita; Nagaoka, Yutaka; Nakajima, Yoshikata; Maekawa, Toru; Kumar, D Sakthi

    2016-09-10

    Heat Shock Protein 90 (Hsp90) has been extensively explored as a potential drug target for cancer therapies. 17- N-allylamino- 17-demethoxygeldanamycin (17AAG) was the first Hsp90 inhibitor to enter clinical trials for cancer therapy. However, native drug is being shown to have considerable anticancer efficacy against pancreatic cancer when used in combination therapy regime. Further, magnetic hyperthermia has shown to have promising effects against pancreatic cancer in combination with known cyto-toxic drugs under both target and non-targeted scenarios. Hence, in order to enhance the efficacy of 17AAG against pancreatic cancer, we developed poly (lactic-co-glycolic acid) (PLGA) coated, 17AAG and Fe3O4 loaded magnetic nanoparticle formulations by varying the relative concentration of polymer. We found that polymer concentration affects the magnetic strength and physicochemical properties of formulation. We were also able to see that our aqueous dispensable formulations were able to provide anti-pancreatic cancer activity for MIA PaCa-2 cell line in dose and time dependent manner in comparison to mice fibroblast cell lines (L929). Moreover, the in-vitro magnetic hyperthermia against MIA PaCa-2 provided proof principle that our 2-in-1 particles may work against cancer cell lines effectively. PMID:27469073

  10. Hsp90 is involved in the regulation of cytosolic precursor protein abundance in tomato.

    PubMed

    Tillmann, Bodo; Röth, Sascha; Bublak, Daniela; Sommer, Manuel; Stelzer, Ernst H K; Scharf, Klaus-Dieter; Schleiff, Enrico

    2015-02-01

    Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein (Chip), appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response. PMID:25619681

  11. Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition

    PubMed Central

    Weigert, Oliver; Lane, Andrew A.; Bird, Liat; Kopp, Nadja; Chapuy, Bjoern; van Bodegom, Diederik; Toms, Angela V.; Marubayashi, Sachie; Christie, Amanda L.; McKeown, Michael; Paranal, Ronald M.; Bradner, James E.; Yoda, Akinori; Gaul, Christoph; Vangrevelinghe, Eric; Romanet, Vincent; Murakami, Masato; Tiedt, Ralph; Ebel, Nicolas; Evrot, Emeline; De Pover, Alain; Régnier, Catherine H.; Erdmann, Dirk; Hofmann, Francesco; Eck, Michael J.; Sallan, Stephen E.; Levine, Ross L.; Kung, Andrew L.; Baffert, Fabienne

    2012-01-01

    Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor–like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100–1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors. PMID:22271575

  12. TsDAF-21/Hsp90 is expressed in all examined stages of Trichinella spiralis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Trichinella is an important parasitic nematode of animals worldwide. Heat shock proteins are ubiquitous in nature and allow organisms to quickly respond to environmental stress. A portion of the Tsdaf-21 gene, a Caenorhabditis elegans daf-21 homologue encoding heat-shock protein 90 (Hsp90) was clone...

  13. Hsp90 in the continuum of breast ductal carcinogenesis: Evaluation in precursors, preinvasive and ductal carcinoma lesions

    PubMed Central

    2010-01-01

    Background Hsp90 (heat shock protein90) is a chaperone protein essential for preserving and regulating the function of various cellular proteins. Elevated Hsp90 expression seems to be a trait of breast cancer and may be an integral part of the coping mechanisms that cancer cells exhibit vis-à-vis stress. This manuscript tries to examine the immunohistochemical expression of Hsp90 all along the continuum of breast ductal lesions encompassing ductal hyperplasia without atypia (DHWithoutA), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Methods Tissue specimens were taken from 30 patients with DHWithoutA, 31 patients with ADH, 51 with DCIS and 51 with IDC. Immunohistochemical assessment of Hsp90 was performed both in the lesion and the adjacent normal breast ducts and lobules; the latter serving as control. Concerning Hsp90 assessment the percentage of positive cells and the intensity were separately analyzed. Subsequently, the Allred score was calculated. Post hoc analysis on the correlations between Hsp90 Allred score and possible predictors (grade, nodal status, tumor size, ER Allred score, PR Allred score, c-erbB-2 status and triple negative status) was conducted in IDC. Results Hsp90 exhibited mainly cytoplasmic immunoreactivity. Hsp90 Allred score exhibited an increasing trend along the continuum of breast ductal lesions (Spearman's rho = 0.169, p = 0.031). Compared to the adjacent normal ducts and lobules, no statistically significant differences were noted in DHwithoutA, ADH and DCIS. Hsp90 expression (intensity, positive cells, Allred score) was higher in IDC, compared to the adjacent normal tissue. Higher Hsp90 expression was observed in grade 2/3 IDCs (borderline association) and tumors of larger size. At the univariable analysis, higher Hsp90 expression was associated with higher ER Allred score, PR Allred score and c-erbB-2 positivity in IDC. Triple-negative IDCs exhibited significantly lower Hsp90

  14. A protective role of HSP90 chaperone in gamma-irradiated Arabidopsis thaliana seeds

    NASA Astrophysics Data System (ADS)

    Kozeko, Liudmyla; Talalaiev, Oleksandr; Neimash, Volodymyr; Povarchuk, Vasyl

    2015-07-01

    The heat shock protein 90 (HSP90) is required for the maturation and conformational regulation of many regulatory proteins affecting morphogenetic pathways and stress tolerance. The purpose of this work is to disclose a role of HSP90 in radioresistance of seeds. Arabidopsis thaliana (Ler) seeds were exposed to γ-ray irradiation with doses of 0.1-1 kGy using 60Co source to obtain a viable but polymorphic material. A comet assay of the seeds showed a dose-dependent increase in DNA damage. Phenotypic consequences of irradiation included growth stimulation at doses of 0.1-0.25 kGy and negative growth effects at doses from 0.5 kGy and beyond, along with increasing heterogeneity of seedling growth rate and phenotype. The frequencies of abnormal phenotypes were highly correlated with the degree of DNA damage in seeds. Treatment of seeds with geldanamycin (GDA), an inhibitor of HSP90, stimulated the seedling growth at all radiation doses and, at the same time, enhanced the growth rate and morphological diversity. It was also found that HSP70 induction by γ-rays was increased following GDA treatment (shown at 1 kGy). We suppose that the GDA-induced HSP70 can be involved in elimination of detrimental radiation effects that ultimately results in growth stimulation. On the other hand, the increase in phenotypic variation, when HSP90 function was impaired, confirms the supposition that the chaperone may control the concealment of cryptic genetic alterations and the developmental stability. In general, these results demonstrate that HSP90 may interface the stress response and phenotypic expression of genetic alterations induced by irradiation.

  15. Balance Between Folding and Degradation for Hsp90-Dependent Client Proteins: A Key Role for CHIP

    PubMed Central

    Kundrat, Lenka; Regan, Lynne

    2011-01-01

    Cells must regulate the synthesis and degradation of their proteins to maintain a balance that is appropriate for their specific growth conditions. Here we present the results of an investigation of the balance between protein folding and degradation for mammalian chaperone Hsp90-dependent client proteins. The central players are the molecular chaperones Hsp70 and Hsp90, the co-chaperone HOP, and ubiquitin ligase, CHIP. Hsp70 and Hsp90 bind to HOP thus forming a ternary folding complex whereas the binding of CHIP to the chaperones has previously been shown to lead to ubiquitination and ultimately to degradation of the client proteins as well as the chaperones. To understand the folding/degradation balance in more detail, we characterized the stoichiometries of the CHIP-Hsp70 and CHIP-Hsp90 complexes and measured the corresponding dissociation constants to be ~ 1 µM and ~ 4.5 µM respectively. We quantified the rate of ubiquitination of various substrates by CHIP in vitro. We further determined that the folding and degradation machineries cannot coexist in one complex. Lastly, we measured the in vivo concentrations of Hsp70, Hsp90, HOP, and CHIP under normal conditions and when client proteins are being degraded due to inhibition of the folding pathway. These in vivo measurements along with the in vitro data allowed us to calculate the approximate cellular concentrations of the folding and degradation complexes under both conditions and formulate a quantitative model for the balance between protein folding and degradation as well as an explanation for the shift to client protein degradation when the folding pathway is inhibited. PMID:20704274

  16. A protective role of HSP90 chaperone in gamma-irradiated Arabidopsis thaliana seeds.

    PubMed

    Kozeko, Liudmyla; Talalaiev, Oleksandr; Neimash, Volodymyr; Povarchuk, Vasyl

    2015-07-01

    The heat shock protein 90 (HSP90) is required for the maturation and conformational regulation of many regulatory proteins affecting morphogenetic pathways and stress tolerance. The purpose of this work is to disclose a role of HSP90 in radioresistance of seeds. Arabidopsis thaliana (Ler) seeds were exposed to γ-ray irradiation with doses of 0.1-1 kGy using (60)Co source to obtain a viable but polymorphic material. A comet assay of the seeds showed a dose-dependent increase in DNA damage. Phenotypic consequences of irradiation included growth stimulation at doses of 0.1-0.25 kGy and negative growth effects at doses from 0.5 kGy and beyond, along with increasing heterogeneity of seedling growth rate and phenotype. The frequencies of abnormal phenotypes were highly correlated with the degree of DNA damage in seeds. Treatment of seeds with geldanamycin (GDA), an inhibitor of HSP90, stimulated the seedling growth at all radiation doses and, at the same time, enhanced the growth rate and morphological diversity. It was also found that HSP70 induction by γ-rays was increased following GDA treatment (shown at 1 kGy). We suppose that the GDA-induced HSP70 can be involved in elimination of detrimental radiation effects that ultimately results in growth stimulation. On the other hand, the increase in phenotypic variation, when HSP90 function was impaired, confirms the supposition that the chaperone may control the concealment of cryptic genetic alterations and the developmental stability. In general, these results demonstrate that HSP90 may interface the stress response and phenotypic expression of genetic alterations induced by irradiation. PMID:26256628

  17. Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency.

    PubMed

    Ji, Yewei; Nie, Bin; Li, Ping; Xu, Xiaoyu; Zhou, Yuanguo

    2007-07-01

    The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β (Hsp90β) gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection. Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and BglII. Recombinant plasmids were transformed into Escherichia coli, DH5a, and the colonies were picked and grown in the Amp-agarose. The presence of positive clones was checked by the means of endodigestion and sequencing. Three cell strains, HepG2, Human umbilicus vein endothelium cells (HUVEC) and HeK293, were cultured. Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine. The transfection efficiency was measured by detection of enhanced green fluorescence protein (EGFP). The presence of positive recombinant clones were verified by double-digestion and sequencing. The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1, pSuper-Hsp90β2 and pSuper-Hsp90β3. After optimizing the ratio of plasmid to Lipofectamine, we achieved high transfection efficiency in HeK293 cells. Transfection efficiency was still low in the HepG2 cells. In conclusion, the si-RNA-synthesizing plasmids targeting Hsp90β were constructed and transfected into cells with different transfection efficiency.

  18. Down-regulation of cellular FLICE-inhibitory protein (Long Form) contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

    PubMed Central

    2012-01-01

    Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL) is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90) either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG) or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP)-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ) were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB), c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets in human lung cancer

  19. Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases.

    PubMed

    Blum, J J; Hayes, A

    1984-01-01

    The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the "light" 30S dynein ATPase fractions are more activated than the "heavy" 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase

  20. Targeting Hsp90 with small molecule inhibitors induces the over-expression of the anti-apoptotic molecule, survivin, in human A549, HONE-1 and HT-29 cancer cells

    PubMed Central

    2010-01-01

    Background Survivin is a dual functioning protein. It inhibits the apoptosis of cancer cells by inhibiting caspases, and also promotes cancer cell growth by stabilizing microtubules during mitosis. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that up-regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently enhance cancer anti-drug responses. The purpose of this study is to determine whether targeting Hsp90 can alter survivin expression differently in different cancer cell lines and to explore possible mechanisms that cause the alteration in survivin expression. Results Here, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by Western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced proteasomal activity while inhibition of 26S proteasome activity further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines. Conclusions We showed for the first time that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. Instead, targeting Hsp90 with small molecule inhibitors will induce the

  1. Two HSP90 genes in mandarin fish Siniperca chuatsi: identification, characterization and their specific expression profiles during embryogenesis and under stresses.

    PubMed

    Wang, Peng-Fei; Zeng, Shuang; Xu, Peng; Zhou, Lei; Li, Gui-Feng

    2016-08-01

    HSP90 plays important roles in multiple cellular stress responses. Here, two cytoplasmic HSP90 isoforms, ScHSP90α and ScHSP90β, were identified from Siniperca chuatsi. Their cDNA and gDNA structures, amino acid sequence features, and sequence identities and phylogenetic analysis with other species were described. Their expression profiles during embryonic development in different tissues and under stressful conditions were analyzed using real-time quantitative PCR. During embryogenesis, transcripts of both genes were detected at low levels during the early developmental stages and were up-regulated from appearance of myomere for ScHSP90a and closure of blastopore for ScHSP90β. ScHSP90α showed a tissue-specific variation with high expression in ovary and brain under non-stressed conditions, while ScHSP90β was ubiquitously highly expressed in different tissues. Acute heat shock resulted in a strong up-regulation of ScHSP90α in heart, liver, and head kidney, while it only weakly induced ScHSP90β in these tissues. ScHSP90α was also markedly induced in liver in a time-dependent manner under hypoxia, while the expression of ScHSP90β was not affected by hypoxia. Additionally, Aeromonas hydrophila infection markedly augmented ScHSP90α in head kidney and spleen and mildly up-regulated ScHSP90β in spleen, while suppressing ScHSP90β in head kidney. These results suggest that ScHSP90α and ScHSP90β are differently involved in embryogenesis and under different environmental conditions including high temperature, hypoxia, and bacterial infection. This study will benefit to further clarify the roles of fish HSP90 isoforms in embryogenesis and under stressful conditions and contribute to further study on enhancing stress tolerance and disease resistance of mandarin fish. PMID:26820141

  2. Two HSP90 genes in mandarin fish Siniperca chuatsi: identification, characterization and their specific expression profiles during embryogenesis and under stresses.

    PubMed

    Wang, Peng-Fei; Zeng, Shuang; Xu, Peng; Zhou, Lei; Li, Gui-Feng

    2016-08-01

    HSP90 plays important roles in multiple cellular stress responses. Here, two cytoplasmic HSP90 isoforms, ScHSP90α and ScHSP90β, were identified from Siniperca chuatsi. Their cDNA and gDNA structures, amino acid sequence features, and sequence identities and phylogenetic analysis with other species were described. Their expression profiles during embryonic development in different tissues and under stressful conditions were analyzed using real-time quantitative PCR. During embryogenesis, transcripts of both genes were detected at low levels during the early developmental stages and were up-regulated from appearance of myomere for ScHSP90a and closure of blastopore for ScHSP90β. ScHSP90α showed a tissue-specific variation with high expression in ovary and brain under non-stressed conditions, while ScHSP90β was ubiquitously highly expressed in different tissues. Acute heat shock resulted in a strong up-regulation of ScHSP90α in heart, liver, and head kidney, while it only weakly induced ScHSP90β in these tissues. ScHSP90α was also markedly induced in liver in a time-dependent manner under hypoxia, while the expression of ScHSP90β was not affected by hypoxia. Additionally, Aeromonas hydrophila infection markedly augmented ScHSP90α in head kidney and spleen and mildly up-regulated ScHSP90β in spleen, while suppressing ScHSP90β in head kidney. These results suggest that ScHSP90α and ScHSP90β are differently involved in embryogenesis and under different environmental conditions including high temperature, hypoxia, and bacterial infection. This study will benefit to further clarify the roles of fish HSP90 isoforms in embryogenesis and under stressful conditions and contribute to further study on enhancing stress tolerance and disease resistance of mandarin fish.

  3. A chemical-biological study reveals C9-type iridoids as novel heat shock protein 90 (Hsp90) inhibitors.

    PubMed

    Dal Piaz, Fabrizio; Vassallo, Antonio; Temraz, Abeer; Cotugno, Roberta; Belisario, Maria A; Bifulco, Giuseppe; Chini, Maria G; Pisano, Claudio; De Tommasi, Nunziatina; Braca, Alessandra

    2013-02-28

    The potential of heat shock protein 90 (Hsp90) as a therapeutic target for numerous diseases has made the identification and optimization of novel Hsp90 inhibitors an emerging therapeutic strategy. A surface plasmon resonance (SPR) approach was adopted to screen some iridoids for their Hsp90 α binding capability. Twenty-four iridoid derivatives, including 13 new natural compounds, were isolated from the leaves of Tabebuia argentea and petioles of Catalpa bignonioides. Their structures were elucidated by NMR, electrospray ionization mass spectrometry, and chemical methods. By means of a panel of chemical and biological approaches, four iridoids were demonstrated to bind Hsp90 α. In particular, the dimeric iridoid argenteoside A was shown to efficiently inhibit the chaperone in biochemical and cellular assays. Our results disclose C9-type iridoids as a novel class of Hsp90 inhibitors.

  4. Induction of heat shock protein HSPA6 (HSP70B') upon HSP90 inhibition in cancer cell lines.

    PubMed

    Kuballa, Petric; Baumann, Anna-Lena; Mayer, Klaus; Bär, Ute; Burtscher, Helmut; Brinkmann, Ulrich

    2015-06-01

    Genome-wide transcript profiling to elucidate responses to HSP90 inhibition revealed strong induction of HSPA6 in MCF-7 cells treated with 17-AAG. Time- and dose dependent induction of HSPA6 (confirmed by qPCR and Western Blots) occurred also upon treatment with Radicicol, another HSP90 inhibitor. HSPA6 was not detectable in untreated cells or cells treated with toxins that do not inhibit HSP90, or upon applying oxidative stress. Thus, HSPA6 induction is not a general response to cytotoxic insults. Modulation of HSPA6 levels by siRNA-mediated inhibition or recombinant expression did not influence 17-AAG mediated cell death. HSPA6 induction as a consequence of HSP90 inhibition occurs in various (but not all) cell lines and may be a more specific marker for HSP90 inhibition than induction of other HSP70 proteins.

  5. Hyperthyroidism increases the uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase.

    PubMed Central

    Arruda, Ana Paula; Da-Silva, Wagner S; Carvalho, Denise P; De Meis, Leopoldo

    2003-01-01

    The sarcoplasmic reticulum Ca2+-ATPase is able to modulate the distribution of energy released during ATP hydrolysis, so that a portion of energy is used for Ca2+ transport (coupled ATPase activity) and a portion is converted into heat (uncoupled ATPase activity). In this report it is shown that T4 administration to rabbits promotes an increase in the rates of both the uncoupled ATPase activity and heat production in sarcoplasmic reticulum vesicles, and that the degree of activation varies depending on the muscle type used. In white muscles hyperthyroidism promotes a 0.8-fold increase of the uncoupled ATPase activity and in red muscle a 4-fold increase. The yield of vesicles from hyperthyroid muscles is 3-4-fold larger than that obtained from normal muscles; thus the rate of heat production by the Ca2+-ATPase expressed in terms of g of muscle in hyperthyroidism is increased by a factor of 3.6 in white muscles and 12.0 in red muscles. The data presented suggest that the Ca2+-ATPase uncoupled activity may represent one of the heat sources that contributes to the enhanced thermogenesis noted in hyperthyroidism. PMID:12887329

  6. Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells

    PubMed Central

    Mimura, Naoya; Minami, Jiro; Ohguchi, Hiroto; Yoshida, Yasuhiro; Sagawa, Morihiko; Gorgun, Gullu; Cirstea, Diana; Cottini, Francesca; Jakubikova, Jana; Tai, Yu-Tzu; Chauhan, Dharminder; Richardson, Paul G.; Munshi, Nikhil; Ando, Kiyoshi; Utsugi, Teruhiro; Hideshima, Teru; Anderson, Kenneth C.

    2015-01-01

    Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM. PMID:26630652

  7. Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells.

    PubMed

    Suzuki, Rikio; Kikuchi, Shohei; Harada, Takeshi; Mimura, Naoya; Minami, Jiro; Ohguchi, Hiroto; Yoshida, Yasuhiro; Sagawa, Morihiko; Gorgun, Gullu; Cirstea, Diana; Cottini, Francesca; Jakubikova, Jana; Tai, Yu-Tzu; Chauhan, Dharminder; Richardson, Paul G; Munshi, Nikhil; Ando, Kiyoshi; Utsugi, Teruhiro; Hideshima, Teru; Anderson, Kenneth C

    2015-01-01

    Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

  8. 2-Aroylquinoline-5,8-diones as potent anticancer agents displaying tubulin and heat shock protein 90 (HSP90) inhibition.

    PubMed

    Nepali, Kunal; Kumar, Sunil; Huang, Hsiang-Ling; Kuo, Fei-Chiao; Lee, Cheng-Hsin; Kuo, Ching-Chuan; Yeh, Teng-Kuang; Li, Yu-Hsuan; Chang, Jang-Yang; Liou, Jing-Ping; Lee, Hsueh-Yun

    2016-01-14

    This study reports the synthesis of a series of 2-aroylquinoline-5,8-diones (11-23) on the basis of scaffold hopping. The presence of a methoxy group at C6 assists the highly regioselective incorporation with various amines, and simplifies the structural identification process. Among the synthetic compounds, 6-dimethylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (12) and 7-pyrrolidin-1-yl-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (23) exhibit remarkable anti-proliferative activity against the cancer cell lines tested with mean IC50 values of 0.14 and 0.27 μM, respectively. Compound 23 showed moderate inhibitory activity against tubulin polymerization with an IC50 value of 5.9 μM. In a western blot analysis, 23 caused induction of HSP70 and degradation of Akt, revealing that it possesses HSP90 inhibitory activity.

  9. Antileukemic Scalarane Sesterterpenoids and Meroditerpenoid from Carteriospongia (Phyllospongia) sp., Induce Apoptosis via Dual Inhibitory Effects on Topoisomerase II and Hsp90

    PubMed Central

    Lai, Kuei-Hung; Liu, Yi-Chang; Su, Jui-Hsin; El-Shazly, Mohamed; Wu, Chih-Fung; Du, Ying-Chi; Hsu, Yu-Ming; Yang, Juan-Cheng; Weng, Ming-Kai; Chou, Chia-Hua; Chen, Guan-Yu; Chen, Yu-Cheng; Lu, Mei-Chin

    2016-01-01

    Two new scalarane sesterterpenoids, 12β-(3′β-hydroxybutanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (1) and 12β-(3′β-hydroxypentanoyloxy)-20,24-dimethyl-24-oxo-scalara-16-en-25-al (2), along with one known tetraprenyltoluquinol-related metabolite (3), were isolated from the sponge Carteriospongia sp. In leukemia Molt 4 cells, 1 at 0.0625 μg/mL (125 nM) triggered mitochondrial membrane potential (MMP) disruption and apoptosis showing more potent effect than 2 and 3. The isolates inhibited topoisomerase IIα expression. The apoptotic-inducing effect of 3 was supported by the in vivo experiment through suppressing the volume of xenograft tumor growth (47.58%) compared with the control. Compound 1 apoptotic mechanism of action in Molt 4 cells was further elucidated through inducing ROS generation, calcium release and ER stress. Using the molecular docking analysis, 1 exhibited more binding affinity to N-terminal ATP-binding pocket of Hsp90 protein than 17-AAG, a standard Hsp90 inhibitor. The expression of Hsp90 client proteins, Akt, p70S6k, NFκB, Raf-1, p-GSK3β, and XIAP, MDM 2 and Rb2, and CDK4 and Cyclin D3, HIF 1 and HSF1 were suppressed by the use of 1. However, the expression of Hsp70, acetylated tubulin, and activated caspase 3 were induced after 1 treatment. Our results suggested that the proapoptotic effect of the isolates is mediated through the inhibition of Hsp90 and topoisomerase activities. PMID:27796344

  10. Downhill running and exercise in hot environments increase leukocyte Hsp72 (HSPA1A) and Hsp90α (HSPC1) gene transcripts.

    PubMed

    Tuttle, James A; Castle, Paul C; Metcalfe, Alan J; Midgley, Adrian W; Taylor, Lee; Lewis, Mark P

    2015-04-15

    Stressors within humans and other species activate Hsp72 and Hsp90α mRNA transcription, although it is unclear which environmental temperature or treadmill gradient induces the largest increase. To determine the optimal stressor for priming the Hsp system, physically active but not heat-acclimated participants (19.8 ± 1.9 and 20.9 ± 3.6 yr) exercised at lactate threshold in either temperate (20°C, 50% relative humidity; RH) or hot (30°C, 50% RH) environmental conditions. Within each condition, participants completed a flat running (temperate flat or hot flat) and a downhill running (temperate downhill or hot downhill) experimental trial in a randomized counterbalanced order separated by at least 7 days. Venous blood samples were taken immediately before (basal), immediately after exercise, and 3 and 24 h postexercise. RNA was extracted from leukocytes and RT-quantitative PCR conducted to determine Hsp72 and Hsp90α mRNA relative expression. Leukocyte Hsp72 mRNA was increased immediately after exercise following downhill running (1.9 ± 0.9-fold) compared with flat running (1.3 ± 0.4-fold; P = 0.001) and in hot (1.9 ± 0.6-fold) compared with temperate conditions (1.1 ± 0.5-fold; P = 0.003). Leukocyte Hsp90α mRNA increased immediately after exercise following downhill running (1.4 ± 0.8-fold) compared with flat running (0.9 ± 0.6-fold; P = 0.002) and in hot (1.6 ± 1.0-fold) compared with temperate conditions (0.9 ± 0.6-fold; P = 0.003). Downhill running and exercise in hot conditions induced the largest stimuli for leukocyte Hsp72 and Hsp90α mRNA increases.

  11. Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy, induces complete responses in triple-negative breast cancer models.

    PubMed

    Caldas-Lopes, Eloisi; Cerchietti, Leandro; Ahn, James H; Clement, Cristina C; Robles, Ana I; Rodina, Anna; Moulick, Kamalika; Taldone, Tony; Gozman, Alexander; Guo, Yunke; Wu, Nian; de Stanchina, Elisa; White, Julie; Gross, Steven S; Ma, Yuliang; Varticovski, Lyuba; Melnick, Ari; Chiosis, Gabriela

    2009-05-19

    Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Because of the absence of identified targets and targeted therapies, and due to a heterogeneous molecular presentation, treatment guidelines for patients with TNBC include only conventional chemotherapy. Such treatment, while effective for some, leaves others with high rates of early relapse and is not curative for any patient with metastatic disease. Here, we demonstrate that these tumors are sensitive to the heat shock protein 90 (Hsp90) inhibitor PU-H71. Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with this agent. Notably, TNBC tumors respond to retreatment with PU-H71 for several cycles extending for over 5 months without evidence of resistance or toxicity. Through a proteomics approach, we show that multiple oncoproteins involved in tumor proliferation, survival, and invasive potential are in complex with PU-H71-bound Hsp90 in TNBC. PU-H71 induces efficient and sustained downregulation and inactivation, both in vitro and in vivo, of these proteins. Among them, we identify downregulation of components of the Ras/Raf/MAPK pathway and G(2)-M phase to contribute to its anti-proliferative effect, degradation of activated Akt and Bcl-xL to induce apoptosis, and inhibition of activated NF-kappaB, Akt, ERK2, Tyk2, and PKC to reduce TNBC invasive potential. The results identify Hsp90 as a critical and multimodal target in this most difficult to treat breast cancer subtype and support the use of the Hsp90 inhibitor PU-H71 for clinical trials involving patients with TNBC.

  12. The mechanism of acetylcholine receptor in binding MuSK in myasthenia gravis and the role of HSP90 molecular chaperone.

    PubMed

    Chen, Rongbo; Chen, Siqia; Liao, Juan; Chen, Xiaopu; Xu, Xiaoling

    2016-01-01

    As an autoimmune disease, myasthenia gravis is caused by the dysfunction of neural transmission. Acetylcholine is known to exert its function after entering into synaptic cleft through binding onto postsynaptic membrane. The role of acetylcholine in binding MuSK in myasthenia gravis, however, remains unknown. A total of 38 myasthenia gravis patients and 27 healthy controls were included in this study for the detection of the expression of MuSK using immunofluorescent method. Expression of both MuSK and interleukin-6 (IL-6) were measured by Western blot, followed by the correlation analysis between heat shock protein 90 (HSP90) and IL-6 which were measured by enzyme-linked immunosorbent assay (ELISA). In myasthenia gravis patients, MuSK was co-localized with acetylcholine at the postsynaptic membrane. Such accumulation of MuSK, however, did not occur in normal people. Meanwhile we also observed elevated expression of IL-6 in myasthenia gravis patients (p<0.05). ELISA assay showed higher expression of HSP90 in patients. Further signaling pathway screening revealed the activation of IL-6-mediated pathways including STAT3 and SPH2. In conclusion, MuSK was co-localized with acetylcholine in myasthenia gravis patients, with elevated expression. HSP90 in disease people can activate IL-6 mediated signaling pathways.

  13. The HSP90 inhibitor geldanamycin perturbs endosomal structure and drives recycling ErbB2 and transferrin to modified MVBs/lysosomal compartments

    PubMed Central

    Cortese, Katia; Howes, Mark T.; Lundmark, Richard; Tagliatti, Erica; Bagnato, Paola; Petrelli, Annalisa; Bono, Maria; McMahon, Harvey T.; Parton, Robert G.; Tacchetti, Carlo

    2013-01-01

    The ErbB2 receptor is a clinically validated cancer target whose internalization and trafficking mechanisms remain poorly understood. HSP90 inhibitors, such as geldanamycin (GA), have been developed to target the receptor to degradation or to modulate downstream signaling. Despite intense investigations, the entry route and postendocytic sorting of ErbB2 upon GA stimulation have remained controversial. We report that ErbB2 levels inversely impact cell clathrin-mediated endocytosis (CME) capacity. Indeed, the high levels of the receptor are responsible for its own low internalization rate. GA treatment does not directly modulate ErbB2 CME rate but it affects ErbB2 recycling fate, routing the receptor to modified multivesicular endosomes (MVBs) and lysosomal compartments, by perturbing early/recycling endosome structure and sorting capacity. This activity occurs irrespective of the cargo interaction with HSP90, as both ErbB2 and the constitutively recycled, HSP90-independent, transferrin receptor are found within modified endosomes, and within aberrant, elongated recycling tubules, leading to modified MVBs/lysosomes. We propose that GA, as part of its anticancer activity, perturbs early/recycling endosome sorting, routing recycling cargoes toward mixed endosomal compartments. PMID:23154999

  14. Discovery of an L-alanine ester prodrug of the Hsp90 inhibitor, MPC-3100.

    PubMed

    Kim, Se-Ho; Tangallapally, Rajendra; Kim, In Chul; Trovato, Richard; Parker, Daniel; Patton, J Scott; Reeves, Leslie; Bradford, Chad; Wettstein, Daniel; Baichwal, Vijay; Papac, Damon; Bajji, Ashok; Carlson, Robert; Yager, Kraig M

    2015-11-15

    Various types of Hsp90 inhibitors have been and continue to undergo clinical investigation. One development candidate is the purine-based, synthetic Hsp90 inhibitor 1 (MPC-3100), which successfully completed a phase I clinical study. However, further clinical development of 1 was hindered by poor solubility and consequent formulation issues and promoted development of a more water soluble prodrug. Towards this end, numerous pro-moieties were explored in vitro and in vivo. These studies resulted in identification of L-alanine ester mesylate, 2i (MPC-0767), which exhibited improved aqueous solubility, adequate chemical stability, and rapid bioconversion without the need for solubilizing excipients. Based on improved physical characteristics and favorable PK and PD profiles, 2i mesylate was selected for further development. A convergent, scalable, chromatography-free synthesis for 2i mesylate was developed to support further clinical evaluation.

  15. Synthesis and evaluation of new Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold.

    PubMed

    Taddei, Maurizio; Ferrini, Serena; Giannotti, Luca; Corsi, Massimo; Manetti, Fabrizio; Giannini, Giuseppe; Vesci, Loredana; Milazzo, Ferdinando M; Alloatti, Domenico; Guglielmi, Mario B; Castorina, Massimo; Cervoni, Maria L; Barbarino, Marcella; Foderà, Rosanna; Carollo, Valeria; Pisano, Claudio; Armaroli, Silvia; Cabri, Walter

    2014-03-27

    Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of an azido moiety installed on dihydroxycumene scaffold with differently substituted aryl propiolates gave a new family of 1,4,5-trisubstituted triazole carboxylic acid derivatives that showed high affinity toward Hsp90 associated with cell proliferation inhibition, both in nanomolar range. The 1,5 arrangement of the resorcinol, the aryl moieties, and the presence of an alkyl (secondary) amide in position 4 of the triazole ring were essential to get high activity. Docking simulations suggested that the triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstituted triazole carboxamides induced dramatic depletion of the examined client proteins and a very strong increase in the expression levels of the chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18, SST0287CL1) was selected for further investigation as the most promising drug candidate. PMID:24588105

  16. HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo

    PubMed Central

    Safavi, Setareh; Järnum, Sofia; Vannas, Christoffer; Udhane, Sameer; Jonasson, Emma; Tomic, Tajana Tesan; Grundevik, Pernilla; Fagman, Henrik; Hansson, Magnus; Kalender, Zeynep; Jauhiainen, Alexandra; Dolatabadi, Soheila; Stratford, Eva Wessel; Myklebost, Ola; Eriksson, Mikael; Stenman, Göran; Stock, Regine Schneider; Ståhlberg, Anders; Åman, Pierre

    2016-01-01

    Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS. PMID:26595521

  17. Plasma Hsp90 Level as a Marker of Early Acute Lymphoblastic Leukemia Engraftment and Progression in Mice.

    PubMed

    Milani, Mateus; Laranjeira, Angelo Brunelli Albertoni; de Vasconcellos, Jaíra Ferreira; Brandalise, Silvia Regina; Nowill, Alexandre Eduardo; Yunes, José Andrés

    2015-01-01

    Current monitoring of acute lymphoblastic leukemia (ALL) in living mice is based on FACS analysis of blood hCD45+ cells. In this work, we evaluated the use of human IGFBP2, B2M or Hsp90 as soluble markers of leukemia. ELISA for B2M and IGFBP2 resulted in high background levels in healthy animals, precluding its use. Conversely, plasma levels of Hsp90 showed low background and linear correlation to FACS results. In another experiment, we compared Hsp90 levels with percentage of hCD45+ cells in blood, bone marrow, liver and spleen of animals weekly sacrificed. Hsp90 levels proved to be a superior method for the earlier detection of ALL engraftment and correlated linearly to ALL burden and progression in all compartments, even at minimal residual disease levels. Importantly, the Hsp90/hCD45+ ratio was not altered when animals were treated with dexamethasone or a PI3K inhibitor, indicating that chemotherapy does not directly interfere with leukemia production of Hsp90. In conclusion, plasma Hsp90 was validated as a soluble biomarker of ALL, useful for earlier detection of leukemia engraftment, monitoring leukemia kinetics at residual disease levels, and pre-clinical or mouse avatar evaluations of anti-leukemic drugs. PMID:26068922

  18. Plasma Hsp90 Level as a Marker of Early Acute Lymphoblastic Leukemia Engraftment and Progression in Mice.

    PubMed

    Milani, Mateus; Laranjeira, Angelo Brunelli Albertoni; de Vasconcellos, Jaíra Ferreira; Brandalise, Silvia Regina; Nowill, Alexandre Eduardo; Yunes, José Andrés

    2015-01-01

    Current monitoring of acute lymphoblastic leukemia (ALL) in living mice is based on FACS analysis of blood hCD45+ cells. In this work, we evaluated the use of human IGFBP2, B2M or Hsp90 as soluble markers of leukemia. ELISA for B2M and IGFBP2 resulted in high background levels in healthy animals, precluding its use. Conversely, plasma levels of Hsp90 showed low background and linear correlation to FACS results. In another experiment, we compared Hsp90 levels with percentage of hCD45+ cells in blood, bone marrow, liver and spleen of animals weekly sacrificed. Hsp90 levels proved to be a superior method for the earlier detection of ALL engraftment and correlated linearly to ALL burden and progression in all compartments, even at minimal residual disease levels. Importantly, the Hsp90/hCD45+ ratio was not altered when animals were treated with dexamethasone or a PI3K inhibitor, indicating that chemotherapy does not directly interfere with leukemia production of Hsp90. In conclusion, plasma Hsp90 was validated as a soluble biomarker of ALL, useful for earlier detection of leukemia engraftment, monitoring leukemia kinetics at residual disease levels, and pre-clinical or mouse avatar evaluations of anti-leukemic drugs.

  19. Differential Gene Expression of Heat Shock Protein 90 (Hsp90) of Candida albicans obtained from Malaysian and Iranian Patients

    PubMed Central

    Khalili, Vajihe; Shokri, Hojjatollah; Md Akim, Abdah; Khosravi, Ali Reza

    2016-01-01

    Background Candida albicans (C. albicans) has several virulence factors, in particular heat shock protein 90 (Hsp90), which is expressed by Hsp90 gene. The purposes of this study were to assess the expression of Hsp90 gene in clinical and control isolates of C. albicans obtained from different geographical regions (Malaysia and Iran), different temperatures (25°C, 37°C and 42°C) and mice with candidiasis. Methods C. albicans isolates were cultured onto sabouraud dextrose agar (SDA). The assessment of the expression of Hsp90 gene was performed using real time-polymerase chain reaction (RT-PCR). Results The results showed a significant increase in the expression of C. albicans Hsp90 gene under high thermal shock (42°C) when compared to other temperatures tested (P-value = 0.001). The mean differences in the expression of Hsp90 gene at 37°C were 0.20 (95% confidence interval (CI) 0.13–0.29) between Malaysian and Iranian controls (P-value = 0.040) and 0.47 (95% CI 0.27–0.60) between Malaysian and Iranian patients (P-value = 0.040). Conclusion The results demonstrated that the expression of C. albicans Hsp90 gene varied between Malaysian and Iranian subjects, representing the efficacy of geographical and thermal conditions on virulence gene expression. PMID:27418871

  20. Progesterone receptor chaperone complex-based highthroughput screening assay: identification of capsaicin as inhibitor of Hsp90 machine

    PubMed Central

    Patwardhan, Chaitanya A.; Alfa, Eyad; Lu, Su; Chadli, Ahmed

    2016-01-01

    Hsp90 and its co-chaperones are known to be important for cancer cell survival. The N-terminal inhibitors of Hsp90 that are in ongoing clinical trials as anti-tumor agents have unfortunately shown disappointing efficacies in the clinic. Thus, novel inhibitors of the Hsp90 machine with different mechanism of action are urgently needed. We report here the development of a novel high-throughput drug-screening (HTS) assay platform to identify small molecule inhibitors of Hsp90 and its co-chaperones. This assay quantitatively measures the ability of Hsp90 and its co-chaperones to refold/protect the progesterone receptor (PR), a physiological client of Hsp90, in 96-well plate format. We screened the NIH clinical collection drug library and identified capsaicin as a hit molecule. Capsaicin is an FDA-approved drug for topical use in pain management. Cell survival assays showed that capsaicin selectively kills cancer cells and destabilizes several Hsp90 client proteins. Thus, our data may explain the seemingly pleotropic effect of capsaicin. PMID:25184514

  1. Sequence features and phylogenetic analysis of the stress protein Hsp90α in chinook salmon (Oncorhynchus tshawytscha), a poikilothermic vertebrate

    USGS Publications Warehouse

    Palmisano, Aldo N.; Winton, James R.; Dickhoff, Walton W.

    1999-01-01

    We cloned and sequenced a chinook salmon Hsp90 cDNA; sequence analysis shows it to be Hsp90??. Phylogenetic analysis supports the hypothesis that ?? and ?? paralogs of Hsp90 arose as a result of a gene duplication event and that they diverged early in the evolution of vertebrates, before tetrapods separated from the teleost lineage. Among several differences distinguishing poikilothermic Hsp90?? sequences from their bird and mammal orthologs, the teleost versions specifically lack a characteristic QTQDQP phosphorylation site near the N-terminus. We used the cDNA to develop an RNA (Northern) blot to quantify cellular Hsp90 mRNA levels. Chinook salmon embryonic (CHSE-214) cells responded to heat shock with a rapid rise in Hsp90 mRNA through 4 h, followed by a gradual decline over the next 20 h. Hsp90 mRNA level may be useful as a stress indicator, especially in a laboratory setting or in response to acute heat stress.

  2. Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (Penaeus monodon).

    PubMed

    Jiang, Shigui; Qiu, Lihua; Zhou, Falin; Huang, Jianhua; Guo, Yihui; Yang, Keng

    2009-01-01

    The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5' untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3' UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37 degrees C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.

  3. Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells

    SciTech Connect

    Tung, Chun-Liang; Jian, Yi-Jun; Syu, Jhan-Jhang; Wang, Tai-Jing; Chang, Po-Yuan; Chen, Chien-Yu; Jian, Yun-Ting; Lin, Yun-Wei

    2015-05-15

    Gefitinib (Iressa{sup R}, ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells. - Highlights: • Gefitinib treatment decreased XRCC1 mRNA and protein expression in NSCLC cells. • Knocking down XRCC1 expression enhanced the cytotoxic effect of gefitinib. • Gefitinib combined with an Hsp90 inhibitor resulted in synergistically cytotoxicity.

  4. Synthesis of Reblastatin, Autolytimycin, Non-Benzoquinone Analogs: Potent Inhibitors of Heat Shock Protein 90 (Hsp90)

    PubMed Central

    Wrona, Iwona E.; Gozman, Alexander; Taldone, Tony; Chiosis, Gabriela; Panek, James S.

    2010-01-01

    A full account of an asymmetric synthesis of reblastatin (1), the first total synthesis of autolytimycin (2) and related structural compounds is described. The syntheses expand the utility of a highly regio-and diastereoselective hydrometalation aldehyde addition sequence to assemble the fully functionalized ansa chain of the natural products. Also documented is an intramolecular copper-mediated amidation reaction to close the 19-membered macrolactams. The amidation reaction was also employed for the generation of structural derivatives (6–9) of phenolic ansamycins. Ansamycin natural products and selected structural analogs were evaluated in a competitive binding assay to breast cancer cell lysate and a cytotoxicity assay. Both reblastatin (1) and autolytimycin (2) were shown to bind the Hsp90 protein with enhanced binding activity (~25 nM) than 17-allylamino-17-demethoxygeldanamycin (17-AAG, 4), a geldanamycin (3) derivative currently under evaluation for treatment of cancer (~100 nM). PMID:20392070

  5. Aurora kinase A activates the vacuolar H+-ATPase (V-ATPase) in kidney carcinoma cells.

    PubMed

    Al-Bataineh, Mohammad M; Alzamora, Rodrigo; Ohmi, Kazuhiro; Ho, Pei-Yin; Marciszyn, Allison L; Gong, Fan; Li, Hui; Hallows, Kenneth R; Pastor-Soler, Núria M

    2016-06-01

    Extracellular proton-secreting transport systems that contribute to extracellular pH include the vacuolar H(+)-ATPase (V-ATPase). This pump, which mediates ATP-driven transport of H(+) across membranes, is involved in metastasis. We previously showed (Alzamora R, Thali RF, Gong F, Smolak C, Li H, Baty CJ, Bertrand CA, Auchli Y, Brunisholz RA, Neumann D, Hallows KR, Pastor-Soler NM. J Biol Chem 285: 24676-24685, 2010) that V-ATPase A subunit phosphorylation at Ser-175 is important for PKA-induced V-ATPase activity at the membrane of kidney intercalated cells. However, Ser-175 is also located within a larger phosphorylation consensus sequence for Aurora kinases, which are known to phosphorylate proteins that contribute to the pathogenesis of metastatic carcinomas. We thus hypothesized that Aurora kinase A (AURKA), overexpressed in aggressive carcinomas, regulates the V-ATPase in human kidney carcinoma cells (Caki-2) via Ser-175 phosphorylation. We found that AURKA is abnormally expressed in Caki-2 cells, where it binds the V-ATPase A subunit in an AURKA phosphorylation-dependent manner. Treatment with the AURKA activator anacardic acid increased V-ATPase expression and activity at the plasma membrane of Caki-2 cells. In addition, AURKA phosphorylates the V-ATPase A subunit at Ser-175 in vitro and in Caki-2 cells. Immunolabeling revealed that anacardic acid induced marked membrane accumulation of the V-ATPase A subunit in transfected Caki-2 cells. However, anacardic acid failed to induce membrane accumulation of a phosphorylation-deficient Ser-175-to-Ala (S175A) A subunit mutant. Finally, S175A-expressing cells had decreased migration in a wound-healing assay compared with cells expressing wild-type or a phospho-mimetic Ser-175-to-Asp (S175D) mutant A subunit. We conclude that AURKA activates the V-ATPase in kidney carcinoma cells via phosphorylation of Ser-175 in the V-ATPase A subunit. This regulation contributes to kidney carcinoma V-ATPase-mediated extracellular

  6. Combined inhibition of Hsp90 and heme oxygenase-1 induces apoptosis and endoplasmic reticulum stress in melanoma.

    PubMed

    Barbagallo, Ignazio; Parenti, Rosalba; Zappalà, Agata; Vanella, Luca; Tibullo, Daniele; Pepe, Francesco; Onni, Toniangelo; Li Volti, Giovanni

    2015-10-01

    Heat shock proteins are ubiquitous molecular chaperones involved in post-translational folding, stability, activation and maturation of many proteins that are essential mediators of signal transduction and cell cycle progression. Heat shock protein 90 (Hsp90) has recently emerged as an attractive therapeutic target in cancer treatment since it may act as a key regulator of various oncogene products and cell-signaling molecules. Heme oxygenase-1 (HO-1; also known as Hsp32) is an inducible enzyme participating in heme degradation and involved in oxidative stress resistance. Recent studies indicate that HO-1 activation may play a role in tumor development and progression. In the present study we investigated the chemotherapic effects of combining an Hsp90 inhibitor (NMS E973) and an HO-1 inhibitor (SnMP) on A375 melanoma cells. NMS E973 treatment was able to reduce cell viability and induce endoplasmic reticulum (ER) stress (i.e. Ire1α, ERO1, PDI, BIP and CHOP). Interestingly, no significant effect was observed in reactive oxygen species (ROS) formation. Finally, NMS E973 treatment resulted in a significant HO-1 overexpression, which in turn serves as a possible chemoresistance molecular mechanism. Interestingly, the combination of NMS E973 and SnMP produced an increase of ROS and reduced cell viability compared to NMS E973 treatment alone. The inhibitors combination exhibited higher ER stress, apoptosis as evidenced by bifunctional apoptosis regulator (BFAR) mRNA expression and lower phosphorylation of Akt when compared to NMS E973 alone. In conclusion, these data suggest that HO-1 inhibition potentiates NMS E973 toxicity and may be exploited as a strategy for melanoma treatment.

  7. Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors.

    PubMed

    Casale, Elena; Amboldi, Nadia; Brasca, Maria Gabriella; Caronni, Dannica; Colombo, Nicoletta; Dalvit, Claudio; Felder, Eduard R; Fogliatto, Gianpaolo; Galvani, Arturo; Isacchi, Antonella; Polucci, Paolo; Riceputi, Laura; Sola, Francesco; Visco, Carlo; Zuccotto, Fabio; Casuscelli, Francesco

    2014-08-01

    In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.

  8. The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage

    PubMed Central

    Bhide, Shreerang A.; Eccles, Suzanne A.; Workman, Paul; Nutting, Christopher M.; Huddart, Robert A.; Harrington, Kevin J.

    2012-01-01

    Background Heat shock protein 90 (HSP90) is a molecular chaperone responsible for the conformational maintenance of a number of client proteins that play key roles in cell cycle arrest, DNA damage repair and apoptosis following radiation. HSP90 inhibitors exhibit antitumor activity by modulating the stabilisation and activation of HSP90 client proteins. We sought to evaluate NVP-AUY922, the most potent HSP90 inhibitor yet reported, in preclinical radiosensitization studies. Principal Findings NVP-AUY922 potently radiosensitized cells in vitro at low nanomolar concentrations with a concurrent depletion of radioresistance-linked client proteins. Radiosensitization by NVP-AUY922 was verified for the first time in vivo in a human head and neck squamous cell carcinoma xenograft model in athymic mice, as measured by delayed tumor growth and increased surrogate end-point survival (p = <0.0001). NVP-AUY922 was shown to ubiquitously inhibit resolution of dsDNA damage repair correlating to delayed Rad51 foci formation in all cell lines tested. Additionally, NVP-AUY922 induced a stalled mitotic phenotype, in a cell line-dependent manner, in HeLa and HN5 cell lines irrespective of radiation exposure. Cell cycle analysis indicated that NVP-AUY922 induced aberrant mitotic entry in all cell lines tested in the presence of radiation-induced DNA damage due to ubiquitous CHK1 depletion, but resultant downstream cell cycle effects were cell line dependent. Conclusions These results identify NVP-AUY922 as the most potent HSP90-mediated radiosensitizer yet reported in vitro, and for the first time validate it in a clinically relevant in vivo model. Mechanistic analysis at clinically achievable concentrations demonstrated that radiosensitization is mediated by the combinatorial inhibition of cell growth and survival pathways, ubiquitous delay in Rad51-mediated homologous recombination and CHK1-mediated G2/M arrest, but that the contribution of cell cycle perturbation to

  9. Hsp90 inhibitors, part 1: definition of 3-D QSAutogrid/R models as a tool for virtual screening.

    PubMed

    Ballante, Flavio; Caroli, Antonia; Wickersham, Richard B; Ragno, Rino

    2014-03-24

    The multichaperone heat shock protein (Hsp) 90 complex mediates the maturation and stability of a variety of oncogenic signaling proteins. For this reason, Hsp90 has emerged as a promising target for anticancer drug development. Herein, we describe a complete computational procedure for building several 3-D QSAR models used as a ligand-based (LB) component of a comprehensive ligand-based (LB) and structure-based (SB) virtual screening (VS) protocol to identify novel molecular scaffolds of Hsp90 inhibitors. By the application of the 3-D QSAutogrid/R method, eight SB PLS 3-D QSAR models were generated, leading to a final multiprobe (MP) 3-D QSAR pharmacophoric model capable of recognizing the most significant chemical features for Hsp90 inhibition. Both the monoprobe and multiprobe models were optimized, cross-validated, and tested against an external test set. The obtained statistical results confirmed the models as robust and predictive to be used in a subsequent VS.

  10. C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

    PubMed

    Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

    2009-10-12

    Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

  11. Expression patterns and structural modelling of Hsp70 and Hsp90 in a fish-borne zoonotic nematode Anisakis pegreffii.

    PubMed

    Chen, Hui-Yu; Cheng, Yi-Sheng; Shih, Hsiu-Hui

    2015-09-15

    Heat shock proteins (HSPs) are essential molecular chaperones that are highly conserved across organisms. They have a pivotal function in responding to thermal stress and are responsible for many cellular functions. Here, we aimed to elucidate the possible roles of Hsp70 and Hsp90 in the life cycle of the parasitic nematode Anisakis, particularly third- and fourth-stage larvae, from cold-blooded fish to warm-blooded marine mammals or accidentally to human hosts. We examined the expression profiles of Hsp70 and Hsp90 in different developmental stages of Anisakis pegreffii. The open reading frame of Hsp70 of A. pegreffii was 1950 bp, and deduced amino acid sequence showed high homology with those of other nematodes. Heatmap analysis revealed sequence identity of Hsp70 and Hsp90 in 13 important parasitic species, human and yeast. On heatmap and phylogenetic analysis, ApHsp70 and ApHsp90 shared the highest amino acid sequence identity with other nematodes and formed a monophyletic clade. The three-dimensional (3D) structure prediction of the newly characterized ApHsp70 and known ApHsp90 gene showed highly conserved motifs between A. pegreffii and other species. Quantitative real-time PCR and western blot analysis revealed higher mRNA and protein expression for ApHsp70 and ApHsp90 in fourth- than third-stage larvae, with higher mRNA and protein expression for ApHsp70 than ApHsp90. ApHsp70 and ApHsp90 may play important roles in Anisakis in response to thermal stress and might be important molecules in the development of A. pegreffii, which has implications for its control. PMID:26215928

  12. Polymorphisms in the HSP90AA1 5' flanking region are associated with scrapie incubation period in sheep.

    PubMed

    Marcos-Carcavilla, Ane; Moreno, Carole; Serrano, Magdalena; Laurent, Pascal; Cribiu, Edmond P; Andréoletti, Olivier; Ruesche, Julien; Weisbecker, Jean-Louis; Calvo, Jorge H; Moazami-Goudarzi, Katayoun

    2010-07-01

    Susceptibility to scrapie is mainly controlled by point mutations at the PRNP locus. However, additional quantitative trait loci (QTL) have been identified across the genome including a region in OAR18. The gene which encodes the inducible form of the cytoplasmic Hsp90 chaperone (HSP90AA1) maps within this region and seems to be associated with the resistance/susceptibility to scrapie in sheep. Here, we have analyzed several polymorphisms which were previously described in the ovine HSP90AA1 5' flanking region and in intron 10 in two naturally scrapie infected Romanov sheep populations. First, we have studied 58 ARQ/VRQ animals pertaining to the sire family where the QTL influencing scrapie incubation period in OAR18 was detected. We have found a significant association between polymorphisms localized at -660 and -528 in the HSP90AA1 5' flanking region and the scrapie incubation period. These two polymorphisms have also been studied in a second sample constituted by 62 VRQ/VRQ sheep showing an extreme incubation period. Results are concordant with the first dataset. Finally, we have studied the HSP90AA1 expression in scrapie and control animals (N = 41) with different HSP90AA1 genotypes by real time PCR on blood samples. The HSP90AA1 expression rate was equivalent in CC(-600)AA(-528) and CG(-600)AG(-528) scrapie resistant animals (ARR/ARR) and was higher in their CC(-600)AA(-528) than in their CG(-600)AG(-528) scrapie susceptible counterparts (VRQ/VRQ). Our results support the hypothesis that the ovine HSP90AA1 gene acts as a modulator of scrapie susceptibility, contributing to the observed differences in the incubation period of scrapie infected animals with the same PRNP genotype.

  13. Hsp90 (heat shock protein 90) inhibitor occupancy is a direct determinant of client protein degradation and tumor growth arrest in vivo.

    PubMed

    Tillotson, Bonnie; Slocum, Kelly; Coco, John; Whitebread, Nigel; Thomas, Brian; West, Kip A; MacDougall, John; Ge, Jie; Ali, Janid A; Palombella, Vito J; Normant, Emmanuel; Adams, Julian; Fritz, Christian C

    2010-12-17

    Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.

  14. PU-H71: An improvement on nature's solutions to oncogenic Hsp90 addiction.

    PubMed

    Trendowski, Matthew

    2015-09-01

    Despite recent advances in precision medicine, many molecular-based antineoplastic agents do not potentiate sustainable long term remissions, warranting the investigation of novel therapeutic strategies. Heat shock protein 90 (Hsp90) is a molecular chaperone that not only has oncogenic properties, but also has distinct expression profiles in malignant and normal cells, providing a rational strategy to attain preferential damage. Prior attempts to target Hsp90 with natural product-based compounds have been hampered by their associated off target toxicities, suggesting that novel, fully synthetic inhibitors may be required to achieve the specificity necessary for therapeutic efficacy. Therefore, this review highlights the antineoplastic potential of PU-H71 (8-[(6-iodo-1,3-benzodioxol-5-yl)sulfanyl]-9-[3-(propan-2-ylamino)propyl]purin-6-amine), a novel purine based analog that has shown efficacy in many preclinical models of malignancy, and is now under clinical examination. In addition, the review suggests potential concomitant therapeutic approaches that may be particularly beneficial to molecular-based, as well as traditional cytotoxic cancer chemotherapy.

  15. A role of Hsp90 chaperones in stabilization of plant growth and morphogenesis in gamma-irradiated Arabidopsis thaliana seeds

    NASA Astrophysics Data System (ADS)

    Kozeko, Liudmyla; Talalaiev, Oleksandr; Povarchuk, Vasyl; Neimash, Volodymyr

    An insight into the molecular basis of survival and renewal of the irradiated seeds is important for the reliable functioning of plant systems during long space and planetary missions. We have investigated a role of HSP90 chaperones in protection of Arabidopsis thaliana seeds against radiation damage. HSP90 are important in the maturation and conformational regulation of a diverse set of proteins that regulate key steps in different biological processes. It has been supposed that HSP90s can prevent a part of cryptic genetic changes from displaying in a phenotype [Queitsch et al., 2002]. So reduction of HSP90 functioning may destabilize development and uncover some cryptic genetic variations that can increase phenotypic variability. Moreover, binding of Hsf, HSP90s may negatively regulate HSP expression - an important component of the stress reaction. To study this, dry A. thaliana (Ler) seeds were exposed to gamma-radiation (100-1000 Gy) and treated with geldanamycin (GDA) - an inhibitor of HSP90. The affect of cosmic radiation was simulated by gamma rays irradiation from the radioactive isotope cobalt-60 (15 rads at 23ºC). A significant increase in variability in growth rates and phenotypes as well as appearance of strong morphological abnormalities were detected in the seedlings grown from the irradiated seeds. GDA treatment resulted in 1.5-2-fold increase in a number of altered phenotypes. In addition, the antibiotic essentially stimulated the seedling growth after irradiation, but this effect was time-lagged at 500-1000 Gy. It was also shown that GDA treatment induced HSP synthesis. These results indicate diverse cellular functions of HSP90: autoregulation of stress reaction, restoration after geno- and cytotoxic effects, concealment of genetic changes and stabilization of plant development.

  16. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    NASA Technical Reports Server (NTRS)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  17. Down-regulation of ERK1/2 and AKT-mediated X-ray repair cross-complement group 1 protein (XRCC1) expression by Hsp90 inhibition enhances the gefitinib-induced cytotoxicity in human lung cancer cells.

    PubMed

    Tung, Chun-Liang; Jian, Yi-Jun; Syu, Jhan-Jhang; Wang, Tai-Jing; Chang, Po-Yuan; Chen, Chien-Yu; Jian, Yun-Ting; Lin, Yun-Wei

    2015-05-15

    Gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreased XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells. PMID:25662161

  18. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    PubMed

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  19. The Hsp90 Co-chaperones Sti1, Aha1, and P23 Regulate Adaptive Responses to Antifungal Azoles

    PubMed Central

    Gu, Xiaokui; Xue, Wei; Yin, Yajing; Liu, Hongwei; Li, Shaojie; Sun, Xianyun

    2016-01-01

    Heat Shock Protein 90 (Hsp90) is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes) to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast), Aha1, and P23 (Sba1 in yeast) were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography–mass spectrometry (LC-MS) analysis showed that the toxic sterols eburicol and 14α-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents. PMID:27761133

  20. High-resolution structural analysis shows how Tah1 tethers Hsp90 to the R2TP complex.

    PubMed

    Back, Régis; Dominguez, Cyril; Rothé, Benjamin; Bobo, Claude; Beaufils, Chrystel; Moréra, Solange; Meyer, Philippe; Charpentier, Bruno; Branlant, Christiane; Allain, Frédéric H-T; Manival, Xavier

    2013-10-01

    The ubiquitous Hsp90 chaperone participates in snoRNP and RNA polymerase assembly through interaction with the R2TP complex. This complex includes the proteins Tah1, Pih1, Rvb1, and Rvb2. Tah1 bridges Hsp90 to R2TP. Its minimal TPR domain includes two TPR motifs and a capping helix. We established the high-resolution solution structures of Tah1 free and in complex with the Hsp90 C-terminal peptide. The TPR fold is similar in the free and bound forms and we show experimentally that in addition to its solvating/stabilizing role, the capping helix is essential for the recognition of the Hsp90 (704)EMEEVD(709) motif. In addition to Lys79 and Arg83 from the carboxylate clamp, this helix bears Tyr82 forming a π/S-CH3 interaction with Hsp90 M(705) from the peptide 310 helix. The Tah1 C-terminal region is unfolded, and we demonstrate that it is essential for the recruitment of the Pih1 C-terminal domain and folds upon binding. PMID:24012479

  1. Identification and characterization of novel ER-based hsp90 gene in the red flour beetle, Tribolium castaneum.

    PubMed

    Zhang, Yi; Gu, Shasha; Li, Chengjun; Sang, Ming; Wu, Wei; Yun, Xiaopei; Hu, Xingxing; Li, Bin

    2014-09-01

    Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone found in all species except for Archaea, which is required not only for stress tolerance but also for normal development. Recently, it was reported that HSP83, one member of the cytosolic HSP90 family, contributes to oogenesis and responds to heat resistance in Tribolium castaneum. Here, a novel ER-based HSP90 gene, Tchsp90, has been identified in T. castaneum. Phylogenetic analysis showed that hsp90s and hsp83s evolved separately from a common ancestor but that hsp90s originated earlier. Quantitative real-time polymerase chain reaction illustrated that Tchsp90 is expressed in all developmental stages and is highly expressed at early pupa and late adult stages. Tchsp90 was upregulated in response to heat stress but not to cold stress. Laval RNAi revealed that Tchsp90 is important for larval/pupal development. Meanwhile, parental RNAi indicated that it completely inhibited female fecundity and partially inhibited male fertility once Tchsp90 was knocked down and that it will further shorten the lifespan of T. castaneum. These results suggest that Tchsp90 is essential for development, lifespan, and reproduction in T. castaneum in addition to its response to heat stress.

  2. Tel2 structure and function in the Hsp90-dependent maturation of mTOR and ATR complexes

    SciTech Connect

    Takai, Hiroyuki; Xie, Yihu; de Lange, Titia; Pavletich, Nikola P.

    2010-09-20

    We reported previously that the stability of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs) depends on their interaction with Tel2, the ortholog of yeast Tel2 and Caenorhabditis elegans Clk-2. Here we provide evidence that Tel2 acts with Hsp90 in the maturation of PIKK complexes. Quantitative immunoblotting showed that the abundance of Tel2 is low compared with the PIKKs, and Tel2 preferentially bound newly synthesized ATM, ATR, mTOR, and DNA-PKcs. Tel2 complexes contained, in addition to Tti1-Tti2, the Hsp90 chaperone, and inhibition of Hsp90 interfered with the interaction of Tel2 with the PIKKs. Analysis of in vivo labeled nascent protein complexes showed that Tel2 and Hsp90 mediate the formation of the mTOR TORC1 and TORC2 complexes and the association of ATR with ATRIP. The structure of yeast Tel2, reported here, shows that Tel2 consists of HEAT-like helical repeats that assemble into two separate {alpha}-solenoids. Through mutagenesis, we identify a surface patch of conserved residues involved in binding to the Tti1-Tti2 complex in vitro. In vivo, mutation of this conserved patch affects cell growth, levels of PIKKs, and ATM/ATR-mediated checkpoint signaling, highlighting the importance of Tti1-Tti2 binding to the function of Tel2. Taken together, our data suggest that the Tel2-Tti1-Tti2 complex is a PIKK-specific cochaperone for Hsp90.

  3. The Hsp90 inhibitor SNX-7081 is synergistic with fludarabine nucleoside via DNA damage and repair mechanisms in human, p53-negative chronic lymphocytic leukemia

    PubMed Central

    Alomari, Munther; Mirzaei, Mehdi; Best, O. Giles; Pascovici, Dana; Mactier, Swetlana; Mulligan, Stephen P.; Haynes, Paul A.; Christopherson, Richard I.

    2015-01-01

    Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker ϕH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered. PMID:26556860

  4. A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

    PubMed Central

    Young, Christopher NJ; Sinadinos, Anthony; Lefebvre, Alexis; Chan, Philippe; Arkle, Stephen; Vaudry, David; Gorecki, Dariusz C

    2015-01-01

    P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmdmdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca2+ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy. PMID:25700737

  5. Targeting of multiple oncogenic signaling pathways by Hsp90 inhibitor alone or in combination with berberine for treatment of colorectal cancer.

    PubMed

    Su, Yen-Hao; Tang, Wan-Chun; Cheng, Ya-Wen; Sia, Peik; Huang, Chi-Chen; Lee, Yi-Chao; Jiang, Hsin-Yi; Wu, Ming-Heng; Lai, I-Lu; Lee, Jun-Wei; Lee, Kuen-Haur

    2015-10-01

    There is a wide range of drugs and combinations under investigation and/or approved over the last decade to treat colorectal cancer (CRC), but the 5-year survival rate remains poor at stages II-IV. Therefore, new, more-efficient drugs still need to be developed that will hopefully be included in first-line therapy or overcome resistance when it appears, as part of second- or third-line treatments in the near future. In this study, we revealed that heat shock protein 90 (Hsp90) inhibitors have high therapeutic potential in CRC according to combinative analysis of NCBI's Gene Expression Omnibus (GEO) repository and chemical genomic database of Connectivity Map (CMap). We found that second generation Hsp90 inhibitor, NVP-AUY922, significantly downregulated the activities of a broad spectrum of kinases involved in regulating cell growth arrest and death of NVP-AUY922-sensitive CRC cells. To overcome NVP-AUY922-induced upregulation of survivin expression which causes drug insensitivity, we found that combining berberine (BBR), a herbal medicine with potency in inhibiting survivin expression, with NVP-AUY922 resulted in synergistic antiproliferative effects for NVP-AUY922-sensitive and -insensitive CRC cells. Furthermore, we demonstrated that treatment of NVP-AUY922-insensitive CRC cells with the combination of NVP-AUY922 and BBR caused cell growth arrest through inhibiting CDK4 expression and induction of microRNA-296-5p (miR-296-5p)-mediated suppression of Pin1-β-catenin-cyclin D1 signaling pathway. Finally, we found that the expression level of Hsp90 in tumor tissues of CRC was positively correlated with CDK4 and Pin1 expression levels. Taken together, these results indicate that combination of NVP-AUY922 and BBR therapy can inhibit multiple oncogenic signaling pathways of CRC. PMID:25982393

  6. Methylglyoxal, a glycolysis side-product, induces Hsp90 glycation and YAP-mediated tumor growth and metastasis

    PubMed Central

    Nokin, Marie-Julie; Durieux, Florence; Peixoto, Paul; Chiavarina, Barbara; Peulen, Olivier; Blomme, Arnaud; Turtoi, Andrei; Costanza, Brunella; Smargiasso, Nicolas; Baiwir, Dominique; Scheijen, Jean L; Schalkwijk, Casper G; Leenders, Justine; De Tullio, Pascal; Bianchi, Elettra; Thiry, Marc; Uchida, Koji; Spiegel, David A; Cochrane, James R; Hutton, Craig A; De Pauw, Edwin; Delvenne, Philippe; Belpomme, Dominique; Castronovo, Vincent; Bellahcène, Akeila

    2016-01-01

    Metabolic reprogramming toward aerobic glycolysis unavoidably induces methylglyoxal (MG) formation in cancer cells. MG mediates the glycation of proteins to form advanced glycation end products (AGEs). We have recently demonstrated that MG-induced AGEs are a common feature of breast cancer. Little is known regarding the impact of MG-mediated carbonyl stress on tumor progression. Breast tumors with MG stress presented with high nuclear YAP, a key transcriptional co-activator regulating tumor growth and invasion. Elevated MG levels resulted in sustained YAP nuclear localization/activity that could be reverted using Carnosine, a scavenger for MG. MG treatment affected Hsp90 chaperone activity and decreased its binding to LATS1, a key kinase of the Hippo pathway. Cancer cells with high MG stress showed enhanced growth and metastatic potential in vivo. These findings reinforce the cumulative evidence pointing to hyperglycemia as a risk factor for cancer incidence and bring renewed interest in MG scavengers for cancer treatment. DOI: http://dx.doi.org/10.7554/eLife.19375.001 PMID:27759563

  7. NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG.

    PubMed

    Kasai, Shuya; Arakawa, Nobuyuki; Okubo, Ayaka; Shigeeda, Wataru; Yasuhira, Shinji; Masuda, Tomoyuki; Akasaka, Toshihide; Shibazaki, Masahiko; Maesawa, Chihaya

    2016-01-01

    The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression. PMID:27045471

  8. Expression of Hsp90α and cyclin B1 were related to prognosis of esophageal squamous cell carcinoma and keratin pearl formation.

    PubMed

    Huang, Tingyuan; Chen, Size; Han, Hongyu; Li, Huadan; Huang, Zhizhou; Zhang, Jianming; Yin, Qiangbin; Wang, Xiaojie; Ma, Xiaojiao; Dai, Peijuan; Duan, Danping; Zou, Fei; Chen, Xuemei

    2014-01-01

    Hsp90α (heat shock protein 90α), one of the important molecular chaperones in cancer cell signal transduction, has been a new candidate target for cancer therapy. Cyclin B1, the client protein of Hsp90α, plays a key role as a mitotic cyclin in the G2-M phase transition during the cell cycle progression. However, the relationship between the level of HSP90α and cyclin B1, the location of Hsp90α and cyclin B1 in prognosis of esophageal squamous cell carcinoma (ESCC) has not been examined. Here, we demonstrate that the diagnostic significance of Hsp90α and cyclin B1 by immunohistochemistry and the association of Hsp90α and cyclin B1 expression in ESCC. In the specimens from 105 ESCC patients (81 stained with Hsp90α antibody by Immunohistochemistry, 65 with cyclin B1 antibody, and among them, 41 paired specimens were stained with Hsp90α and cyclin B1 respectively, and then checked for the correlation of the level and location of Hsp90α and cylcin B1. The positivity rate of Hsp90α and cyclin B1 expression were 96.3% (78 of 81) and 84.6% (55 of 65) respectively. Both of them, the expression levels are associated with the clinical pathological stage (Hsp90α, p=0.027; cyclin B1, p=0.007). No association was found between Hsp90α or cyclin B1 and gender, age, tumor location. As to TMN stage, there is no association with the level of Hsp90α, However, cyclin B1 expression is significantly related to tumor status (p=0.002). Interestingly, Hsp90α expression was negatively correlated to cyclin B1 expression (Gamma=-0.692, p=0.007) in the keratin pearls though there is a positive correlation in the other areas of tumor (Gamma=0.503, p=0.015), which suggest Hsp90α might play diverse roles in the cyclin B1 expression and cyclin B1 related cell cycle regulation in the different area of tumor. These findings demonstrated that the expression of Hsp90α, cyclin B1 protein is associated with tumor malignancy and prognosis for patients with human esophageal squamous cell

  9. Hsp90 Inhibitors, Part 2: Combining Ligand-Based and Structure-Based Approaches for Virtual Screening Application

    PubMed Central

    2015-01-01

    Hsp90 continues to be an important target for pharmaceutical discovery. In this project, virtual screening (VS) for novel Hsp90 inhibitors was performed using a combination of Autodock and Surflex-Sim (LB) scoring functions with the predictive ability of 3-D QSAR models, previously generated with the 3-D QSAutogrid/R procedure. Extensive validation of both structure-based (SB) and ligand-based (LB), through realignments and cross-alignments, allowed the definition of LB and SB alignment rules. The mixed LB/SB protocol was applied to virtually screen potential Hsp90 inhibitors from the NCI Diversity Set composed of 1785 compounds. A selected ensemble of 80 compounds were biologically tested. Among these molecules, preliminary data yielded four derivatives exhibiting IC50 values ranging between 18 and 63 μM as hits for a subsequent medicinal chemistry optimization procedure. PMID:24555544

  10. Assessment and Reconstruction of Novel HSP90 Genes: Duplications, Gains and Losses in Fungal and Animal Lineages

    PubMed Central

    Pantzartzi, Chrysoula N.; Drosopoulou, Elena; Scouras, Zacharias G.

    2013-01-01

    Hsp90s, members of the Heat Shock Protein class, protect the structure and function of proteins and play a significant task in cellular homeostasis and signal transduction. In order to determine the number of hsp90 gene copies and encoded proteins in fungal and animal lineages and through that key duplication events that this family has undergone, we collected and evaluated Hsp90 protein sequences and corresponding Expressed Sequence Tags and analyzed available genomes from various taxa. We provide evidence for duplication events affecting either single species or wider taxonomic groups. With regard to Fungi, duplicated genes have been detected in several lineages. In invertebrates, we demonstrate key duplication events in certain clades of Arthropoda and Mollusca, and a possible gene loss event in a hymenopteran family. Finally, we infer that the duplication event responsible for the two (a and b) isoforms in vertebrates occurred probably shortly after the split of Hyperoartia and Gnathostomata. PMID:24066039

  11. Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

    PubMed

    Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

    2010-07-30

    Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway.

  12. The differential regulation of human ACT1 isoforms by Hsp90 in IL-17 signaling1

    PubMed Central

    Wu, Ling; Wang, Chenhui; Boisson, Bertrand; Misra, Saurav; Rayman, Patricia; Finke, James H.; Puel, Anne; Casanova, Jean-Laurent; Li, Xiaoxia

    2014-01-01

    IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of autoimmune diseases including psoriasis. ACT1 is an essential adaptor molecule in the IL-17 signaling pathway. A missense single nucleotide polymorphism (rs33980500; SNP-D10N) that resulted in the substitution of an asparagine for an aspartic acid at position 10 of ACT1 (ACT1-D10N) is associated with psoriasis susceptibility. Due to alternative splicing in humans, SNP-D10N encodes two mutated ACT1 proteins, ACT1-D10N and ACT1-D19N. Though both ACT1 isoforms are Hsp90 ‘client’ proteins, the nine additional amino acids in ACT1-D19N provide an additional Hsp90 binding site that is absent in ACT1-D10N. Therefore, while ACT1-D10N is a dead protein that is unable to transduce IL-17 signals for gene expression, ACT1-D19N is fully responsive to IL-17. Intriguingly, the two ACT1 isoforms are differentially expressed in ACT1D10N/D10N fibroblasts and T cells. Fibroblasts express both isoforms equally, enabling ACT1-D19N to compensate for the loss of ACT1-D10N function. ACT1D10N/D10N T cells, however, express predominantly ACT1-D10N. Lacking this compensatory mechanism, ACT1D10N/D10N T cells behave like ACT1-deficient T cells, exhibiting a dysregulated and hyperactive Th17 phenotype with overproduction of IL-22 and IL-17. The hyperactive Th17 response combined with fully responsive fibroblasts likely synergized to contribute to psoriasis susceptibility in SNP-D10N patients. PMID:25024377

  13. The differential regulation of human ACT1 isoforms by Hsp90 in IL-17 signaling.

    PubMed

    Wu, Ling; Wang, Chenhui; Boisson, Bertrand; Misra, Saurav; Rayman, Patricia; Finke, James H; Puel, Anne; Casanova, Jean-Laurent; Li, Xiaoxia

    2014-08-15

    IL-17 is a proinflammatory cytokine implicated in the pathogenesis of autoimmune diseases including psoriasis. ACT1 is an essential adaptor molecule in the IL-17 signaling pathway. A missense single nucleotide polymorphism (rs33980500; SNP-D10N) that resulted in the substitution of an asparagine for an aspartic acid at position 10 of ACT1 (ACT1-D10N) is associated with psoriasis susceptibility. Due to alternative splicing in humans, SNP-D10N encodes two mutated ACT1 proteins, ACT1-D10N and ACT1-D19N. Although both ACT1 isoforms are Hsp90 client proteins, the nine additional amino acids in ACT1-D19N provide an additional Hsp90 binding site that is absent in ACT1-D10N. Therefore, whereas ACT1-D10N is a dead protein that is unable to transduce IL-17 signals for gene expression, ACT1-D19N is fully responsive to IL-17. Intriguingly, the two ACT1 isoforms are differentially expressed in ACT1(D10N/D10N) fibroblasts and T cells. Fibroblasts express both isoforms equally, enabling ACT1-D19N to compensate for the loss of ACT1-D10N function. ACT1(D10N/D10N) T cells, however, express predominantly ACT1-D10N. Lacking this compensatory mechanism, ACT1(D10N/D10N) T cells behave like ACT1-deficient T cells, exhibiting a dysregulated and hyperactive Th17 phenotype with overproduction of IL-22 and IL-17. The hyperactive Th17 response combined with fully responsive fibroblasts likely synergized to contribute to psoriasis susceptibility in SNP-D10N patients. PMID:25024377

  14. Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90).

    PubMed

    Brasca, Maria Gabriella; Mantegani, Sergio; Amboldi, Nadia; Bindi, Simona; Caronni, Dannica; Casale, Elena; Ceccarelli, Walter; Colombo, Nicoletta; De Ponti, Anna; Donati, Daniele; Ermoli, Antonella; Fachin, Gabriele; Felder, Eduard R; Ferguson, Ronald D; Fiorelli, Claudio; Guanci, Marco; Isacchi, Antonella; Pesenti, Enrico; Polucci, Paolo; Riceputi, Laura; Sola, Francesco; Visco, Carlo; Zuccotto, Fabio; Fogliatto, Gianpaolo

    2013-11-15

    Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.

  15. Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90).

    PubMed

    Brasca, Maria Gabriella; Mantegani, Sergio; Amboldi, Nadia; Bindi, Simona; Caronni, Dannica; Casale, Elena; Ceccarelli, Walter; Colombo, Nicoletta; De Ponti, Anna; Donati, Daniele; Ermoli, Antonella; Fachin, Gabriele; Felder, Eduard R; Ferguson, Ronald D; Fiorelli, Claudio; Guanci, Marco; Isacchi, Antonella; Pesenti, Enrico; Polucci, Paolo; Riceputi, Laura; Sola, Francesco; Visco, Carlo; Zuccotto, Fabio; Fogliatto, Gianpaolo

    2013-11-15

    Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile. PMID:24100158

  16. Polypeptide from Chlamys farreri inhibits UVB-induced apoptosis of HaCaT cells via iNOS/NO and HSP90

    NASA Astrophysics Data System (ADS)

    Zhang, Zhengyang; Liu, Xiaojin; Liu, Tuo; Yan, Lin; Wang, Yuejun; Wang, Chunbo

    2009-09-01

    Polypeptide from Chlamys farreri (PCF) is a novel marine bioactive product that was isolated from the gonochoric Chinese scallop Chlamys farreri, and was found to be an effective antioxidant in our recent studies. In this study, we investigated the effect of PCF on ultraviolet B (UVB)-induced apoptosis of HaCaT cells and the intracellular signaling pathways involved. Pretreatment with the inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea sulfate inhibited UVB-induced apoptosis, indicating that iNOS and NO play important roles in apoptosis. On the other hand, the inhibition of UVB-induced apoptosis in the immortalized keratinocyte (HaCaT) cells by PCF was estimated using a DNA ladder. PCF treatment inhibited UVB-induced iNOS activation, as determined by RT-PCR, NO production, as determined by ESR, and up-regulated heat shock protein (HSP) 90 activation, as determined by Western blotting. Our results indicate that iNOS and NO are involved in UVB-induced apoptosis of HaCaT cells and the protective effect of PCF against UVB irradiation is exerted by suppressing the expression of iNOS, followed by inhibition of NO release and enhanced activation of HSP90.

  17. Concomitant Inhibition of HSP90, Its Mitochondrial Localized Homologue TRAP1 and HSP27 by Green Tea in Pancreatic Cancer HPAF-II Cells

    PubMed Central

    Zhang, Lifeng; Pang, Eric; Ogorzalek Loo, Rachel R.; Rao, Jianyu; Go, Vay-Liang W.; Loo, Joseph A.; Lu, Qing-Yi

    2012-01-01

    Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer. PMID:22116673

  18. Different Poses for Ligand and Chaperone in Inhibitor Bound Hsp90 and GRP94: Implications for Paralog-specific Drug Design

    PubMed Central

    Immormino, Robert M.; Metzger, Louis E.; Reardon, Patrick N.; Dollins, D. Eric; Blagg, Brian S.J.; Gewirth, Daniel T.

    2009-01-01

    Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian hsp90 paralogs, which are cytoplasmic Hsp90α and β, ER GRP94, and mitochondrial Trap-1. Given that each of the hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each hsp90 paralog. Here we present side by side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-hsp90 inhibitors Geldanamycin and Radamide. These structures reveal paralog specific differences in the Hsp90 and GRP94 conformations in response to Geldanamycin binding. We also report significant variation in the pose and disparate binding affinities for the Geldanamycin-Radicicol chimera Radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors. PMID:19361515

  19. Consecutive interactions with HSP90 and eEF1A underlie a functional maturation and storage pathway of AID in the cytoplasm.

    PubMed

    Methot, Stephen P; Litzler, Ludivine C; Trajtenberg, Felipe; Zahn, Astrid; Robert, Francis; Pelletier, Jerry; Buschiazzo, Alejandro; Magor, Brad G; Di Noia, Javier M

    2015-04-01

    Activation-induced deaminase (AID) initiates mutagenic pathways to diversify the antibody genes during immune responses. The access of AID to the nucleus is limited by CRM1-mediated nuclear export and by an uncharacterized mechanism of cytoplasmic retention. Here, we define a conformational motif in AID that dictates its cytoplasmic retention and demonstrate that the translation elongation factor eukaryotic elongation factor 1 α (eEF1A) is necessary for AID cytoplasmic sequestering. The mechanism is independent of protein synthesis but dependent on a tRNA-free form of eEF1A. Inhibiting eEF1A prevents the interaction with AID, which accumulates in the nucleus and increases class switch recombination as well as chromosomal translocation byproducts. Most AID is associated to unspecified cytoplasmic complexes. We find that the interactions of AID with eEF1A and heat-shock protein 90 kD (HSP90) are inversely correlated. Despite both interactions stabilizing AID, the nature of the AID fractions associated with HSP90 or eEF1A are different, defining two complexes that sequentially produce and store functional AID in the cytoplasm. In addition, nuclear export and cytoplasmic retention cooperate to exclude AID from the nucleus but might not be functionally equivalent. Our results elucidate the molecular basis of AID cytoplasmic retention, define its functional relevance and distinguish it from other mechanisms regulating AID.

  20. Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperone-dependent regulation of ribonucleotide reductase

    PubMed Central

    Truman, Andrew W.; Kristjansdottir, Kolbrun; Wolfgeher, Donald; Ricco, Natalia; Mayampurath, Anoop; Volchenboum, Samuel L.; Clotet, Josep; Kron, Stephen J.

    2015-01-01

    The highly conserved molecular chaperones Hsp90 and Hsp70 are indispensible for folding and maturation of a significant fraction of the proteome, including many proteins involved in signal transduction and stress response. To examine the dynamics of chaperone-client interactions after DNA damage, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to characterize interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. Of 256 proteins identified and quantified via 16O/18O labeling and LC-MS/MS, 142 are novel Hsp70/90 interactors. Nearly all interactions remained unchanged or decreased after DNA damage, but 5 proteins increased interactions with Ssa1 and/or Hsp82, including the ribonucleotide reductase (RNR) subunit Rnr4. Inhibiting Hsp70 or 90 chaperone activity destabilized Rnr4 in yeast and its vertebrate homolog hRMM2 in breast cancer cells. In turn, pre-treatment of cancer cells with chaperone inhibitors sensitized cells to the RNR inhibitor gemcitabine, suggesting a novel chemotherapy strategy. All MS data have been deposited in the ProteomeXchange with identifier PXD001284. PMID:25452130

  1. Measuring In Vitro ATPase Activity for Enzymatic Characterization.

    PubMed

    Rule, Chelsea S; Patrick, Marcella; Sandkvist, Maria

    2016-01-01

    Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary. PMID:27584824

  2. Electrostatic interactions of Hsp-organizing protein tetratricopeptide domains with Hsp70 and Hsp90: computational analysis and protein engineering.

    PubMed

    Kajander, Tommi; Sachs, Jonathan N; Goldman, Adrian; Regan, Lynne

    2009-09-11

    The Hsp-organizing protein (HOP) binds to the C termini of the chaperones Hsp70 and Hsp90, thus bringing them together so that substrate proteins can be passed from Hsp70 to Hsp90. Because Hsp90 is essential for the correct folding and maturation of many oncogenic proteins, it has become a significant target for anti-cancer drug design. HOP binds to Hsp70 and Hsp90 via two independent tetratricopeptide (TPR) domains, TPR1 and TPR2A, respectively. We have analyzed ligand binding using Poisson-Boltzmann continuum electrostatic calculations, free energy perturbation, molecular dynamics simulations, and site-directed mutagenesis to delineate the contribution of different interactions to the affinity and specificity of the TPR-peptide interactions. We found that continuum electrostatic calculations could be used to guide protein design by removing unfavorable interactions to increase binding affinity, with an 80-fold increase in affinity for TPR2A. Contributions at buried charged residues, however, were better predicted by free energy perturbation calculations. We suggest using a combination of the two approaches for increasing the accuracy of results, with free energy perturbation calculations used only at selected buried residues of the ligand binding pocket. Finally we present the crystal structure of TPR2A in complex with its non-cognate Hsp70 ligand, which provides insight on the origins of specificity in TPR domain-peptide recognition. PMID:19586912

  3. Synthesis of 19-substituted geldanamycins with altered conformations and their binding to heat shock protein Hsp90

    NASA Astrophysics Data System (ADS)

    Kitson, Russell R. A.; Chang, Chuan-Hsin; Xiong, Rui; Williams, Huw E. L.; Davis, Adrienne L.; Lewis, William; Dehn, Donna L.; Siegel, David; Roe, S. Mark; Prodromou, Chrisostomos; Ross, David; Moody, Christopher J.

    2013-04-01

    The benzoquinone ansamycin geldanamycin and its derivatives are inhibitors of heat shock protein Hsp90, an emerging target for novel therapeutic agents both in cancer and in neurodegeneration. However, the toxicity of these compounds to normal cells has been ascribed to reaction with thiol nucleophiles at the quinone 19-position. We reasoned that blocking this position would ameliorate toxicity, and that it might also enforce a favourable conformational switch of the trans-amide group into the cis-form required for protein binding. Here, we report an efficient synthesis of such 19-substituted compounds and realization of our hypotheses. Protein crystallography established that the new compounds bind to Hsp90 with, as expected, a cis-amide conformation. Studies on Hsp90 inhibition in cells demonstrated the molecular signature of Hsp90 inhibitors: decreases in client proteins with compensatory increases in other heat shock proteins in both human breast cancer and dopaminergic neural cells, demonstrating their potential for use in the therapy of cancer or neurodegenerative diseases.

  4. 17-DMAG, an HSP90 Inhibitor, Ameliorates Multiple Organ Dysfunction Syndrome via Induction of HSP70 in Endotoxemic Rats

    PubMed Central

    Wang, Yi-Li; Shen, Hsin-Hsueh; Cheng, Pao-Yun; Chu, Yen-Ju; Hwang, Hwong-Ru; Lam, Kwok-Keung; Lee, Yen-Mei

    2016-01-01

    Sepsis is a systemic inflammatory disorder, accompanied with elevated oxidative stress, leading to multiple organ dysfunction syndrome (MODS), and disseminated intravascular coagulation. 17-Dimethylaminoethylamino- 17-demethoxygeldanamycin (17-DMAG), a heat shock protein (HSP) 90 inhibitor, has been reported to possess anti-inflammatory effects. In this study, the beneficial effects of 17-DMAG on lipopolysaccharide (LPS) induced MODS and DIC was evaluated in anesthetized rats. 17-DMAG (5 mg/kg, i.p.) was significantly increased survival rate, and prevented hypotension in LPS (30 mg/kg i.v. infused for 4 h) induced endotoxemia. The elevated levels of alanine aminotransferase (ALT), creatine phosphokinase (CPK), lactate dehydrogenase, creatinine, nitric oxide (NO) metabolites, IL-6, and TNF-α in LPS-exposed rat plasma were significantly reduced by 17-DMAG. Moreover, 17-DMAG suppressed LPS-induced superoxide anion production and caspase 3 activation in heart tissues. LPS induced the prolongation of prothrombin time, and a pronounced decrease in platelet count, which were improved by 17-DMAG. 17-DMAG markedly induced HSP70 and heme oxygenase (HO)-1, and suppressed inducible nitric oxide synthase (iNOS) and phosphorylated NF-κB p65 protein expression in organs 6 h after LPS initiation. Pretreatment with high dose of quercetin (300 mg/kg, i.p.), as an HSP70 inhibitor, reversed the beneficial effects of 17-DMAG on survival rate, plasma levels of ALT, CPK, creatinine, IL-6, and NO metabolites, iNOS induction, and caspase-3 activation in LPS-treated rats. In conclusion, 17-DMAG possesses the anti-inflammatory and antioxidant effects that were proved through LPS-induced acute inflammation, which is associated with induction of HSP70 and HO-1, leading to prevent MODS in sepsis. PMID:27224288

  5. Molecular chaperons and co-chaperons, Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Ito, Makoto; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2015-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.

  6. A structural pathway for activation of the kinesin motor ATPase

    PubMed Central

    Yun, Mikyung; Zhang, Xiaohua; Park, Cheon-Gil; Park, Hee-Won; Endow, Sharyn A.

    2001-01-01

    Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPase. The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules. PMID:11387196

  7. Synthesis of 5,6-dihydro-4H-benzo[d]isoxazol-7-one and 5,6-dihydro-4H-isoxazolo[5,4-c]pyridin-7-one derivatives as potential Hsp90 inhibitors.

    PubMed

    Musso, Loana; Cincinelli, Raffaella; Giannini, Giuseppe; Manetti, Fabrizio; Dallavalle, Sabrina

    2015-11-01

    A novel class of 5,6-dihydro-4H-benzo[d]isoxazol-7-ones and 5,6-dihydro-4H-isoxazolo[5,4-c]pyridin-7-ones was designed, synthesized, and assayed to investigate the affinity toward Hsp90 protein. The synthetic route was based on a 1,3-dipolar cycloaddition of nitriloxides, generated in situ from suitable benzaldoximes, with 2-bromocyclohex-2-enones or 3-bromo-5,6-dihydro-1H-pyridin-2-ones. Whereas all the compounds bearing a benzamide group on the bicyclic scaffold were devoid of activity, the derivatives carrying a resorcinol-like fragment showed a remarkable inhibitory effect on Hsp90. Docking calculations were performed to investigate the orientation of the new compounds within the binding site of the enzyme.

  8. Synthesis of 5,6-dihydro-4H-benzo[d]isoxazol-7-one and 5,6-dihydro-4H-isoxazolo[5,4-c]pyridin-7-one derivatives as potential Hsp90 inhibitors.

    PubMed

    Musso, Loana; Cincinelli, Raffaella; Giannini, Giuseppe; Manetti, Fabrizio; Dallavalle, Sabrina

    2015-11-01

    A novel class of 5,6-dihydro-4H-benzo[d]isoxazol-7-ones and 5,6-dihydro-4H-isoxazolo[5,4-c]pyridin-7-ones was designed, synthesized, and assayed to investigate the affinity toward Hsp90 protein. The synthetic route was based on a 1,3-dipolar cycloaddition of nitriloxides, generated in situ from suitable benzaldoximes, with 2-bromocyclohex-2-enones or 3-bromo-5,6-dihydro-1H-pyridin-2-ones. Whereas all the compounds bearing a benzamide group on the bicyclic scaffold were devoid of activity, the derivatives carrying a resorcinol-like fragment showed a remarkable inhibitory effect on Hsp90. Docking calculations were performed to investigate the orientation of the new compounds within the binding site of the enzyme. PMID:25855505

  9. Influence of activating hormones on human platelet membrane Ca/sup 2 +/-ATPase activity

    SciTech Connect

    Resink, T.J.; Dimitrov, D.; Stucki, S.; Buehler, F.R.

    1986-07-16

    Intact platelets were pretreated with hormones and thereafter membranes were prepared and Ca/sup 2 +/-ATPase activity determined. Thrombin decreased the V/sub max/ of Ca/sup 2 +/-ATPase after pretreatment of intact platelets. Platelet activating factor, vasopressin and ADP also decreased Ca/sup 2 +/-ATPase activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) or A23187 or ionomycin alone had no effect, while the simultaneous pretreatment with TPA and Ca/sup 2 +/-ionophore decreased Ca/sup 2 +/-ATPase activity. cAMP elevating agents prostaglandin E/sub 1/ (PGE/sub 1/) and forskolin had no influence per se on Ca/sup 2 +/-ATPase, but antagonized the inhibitory effect of thrombin. The data suggest a close connection between phosphoinositide metabolism and the Ca/sup 2 +/-ATPase system.

  10. [Low dose benzo(a)pyrene up-regulated the transcription of HSP70 and HSP90 in Eisenia fetida].

    PubMed

    Zheng, Sen-Lin; Sun, Tie-Heng; Xiao, Hong; Qiu, Xiao-Yan; Song, Yu-Fang

    2008-02-01

    To search for the molecular biomarkers of sub-lethal polycyclic aromatic hydrocarbons (PAHs)-contamination of soil, the subtractive cDNA libraries of earthworm Eisenia fetida exposed to benzo(a)pyrene (BaP) in artificial soil were constructed by suppression subtractive hybridization. After sequencing and analyzing with basic local alignment search tool (BLAST), two clones matching heat shock protein 70 k Da (HSP70) and one clone matching heat shock protein 90 k Da (HSP90) were isolated from the up-regulated library, and subsequently, the up-regulation of HSP70 and HSP90 was verified by real-time PCR in E. fetida exposed to 0.1 mg x kg(-1) and 1.0 mg x kg(-1) BaP. It was indicated that these two newly identified HSPs in E. fetida were the potential molecular biomarkers for soil contamination monitoring.

  11. Tumor-specific HSP90 inhibition as a therapeutic approach in JAK-mutant acute lymphoblastic leukemias.

    PubMed

    Kucine, Nicole; Marubayashi, Sachie; Bhagwat, Neha; Papalexi, Efthymia; Koppikar, Priya; Sanchez Martin, Marta; Dong, Lauren; Tallman, Marty S; Paietta, Elisabeth; Wang, Kai; He, Jie; Lipson, Doron; Stephens, Phil; Miller, Vince; Rowe, Jacob M; Teruya-Feldstein, Julie; Mullighan, Charles G; Ferrando, Adolfo A; Krivtsov, Andrei; Armstrong, Scott; Leung, Laura; Ochiana, Stefan O; Chiosis, Gabriela; Levine, Ross L; Kleppe, Maria

    2015-11-26

    The development of the dual Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib for the treatment of myeloproliferative neoplasms (MPNs) has led to studies of ruxolitinib in other clinical contexts, including JAK-mutated acute lymphoblastic leukemia (ALL). However, the limited ability of JAK inhibition to induce molecular or clinicopathological responses in MPNs suggests a need for development of better therapies for JAK kinase-dependent malignancies. Here, we demonstrate that heat shock protein 90 (HSP90) inhibition using a purine-scaffold HSP90 inhibitor in early clinical development is an effective therapeutic approach in JAK-dependent ALL and can overcome persistence to JAK-inhibitor therapy in ALL cells. PMID:26443624

  12. Benefit of HSP90α intervention on ischemia-reperfusion injury of venous blood-congested flaps

    PubMed Central

    HU, XIAO-YING; CHEN, ZHEN-YU; ZHANG, BIN; LENG, XIANG-FENG; FAN, XIAO-JIAN; LIU, TAO

    2016-01-01

    In order to decrease the incidence of flap necrosis after reconstructive surgeries, new approaches are required. In the present study, a model of venous congested flaps in rats was established to test the heat shock protein (HSP) 90α, ‘F-5’, protein as an intervention therapy to alleviate ischemia-reperfusion injury. A recombinant plasmid pET15b-F-5 carrying the HSP90α gene was constructed and the induced protein was purified from bacterial cell cultures. The rats in the study were divided into three different intervention groups: group A rats were treated with normal saline prior to flap establishment, group B rats were treated with HSP90α, ‘F-5’, protein prior to flap establishment, and group C rats were treated with the same ‘F-5’ protein after the surgical procedure. Additionally, the reperfusion time-points, ischemia for 6 or 8 h (5 rats each), were established in each group. After set periods of time, the flaps were observed for skin appearance, blood flow, survival rate and histological changes including neovascularization and re-epithelialization. The results showed that the flaps in the rats pre-treated with ‘F-5’ protein performed better than the flaps of rats in the other two groups: the blood flow was higher, flap survival rate was increased, inflammatory cell infiltration was decreased and angiogenesis increased, and new skin structure was better completed by the end of the experiment. The parameters examind were improved for all the groups when the ischemia time was 6 h instead of 8 h. In conclusion, HSP90α intervention prior to flap establishment was shown to be beneficial in the model of ischemia-reperfusion injury in venous-congested flaps. PMID:27347036

  13. A novel Hsp90 inhibitor AT13387 induces senescence in EBV-positive nasopharyngeal carcinoma cells and suppresses tumor formation

    PubMed Central

    2013-01-01

    Background Nasopharyngeal carcinoma (NPC) is an epithelial malignancy strongly associated with Epstein-Barr virus (EBV). AT13387 is a novel heat shock protein 90 (Hsp90) inhibitor, which inhibits the chaperone function of Hsp90 and reduces expression of Hsp90-dependent client oncoproteins. This study aimed to evaluate both the in vitro and in vivo antitumor effects of AT13387 in the EBV-positive NPC cell line C666-1. Results Our results showed that AT13387 inhibited C666-1 cell growth and induced cellular senescence with the downregulation of multiple Hsp90 client oncoproteins EGFR, AKT, CDK4, and restored the protein expression of negative cell cycle regulator p27. We also studied the ability of AT13387 to restore p27 expression by downregulation of AKT and the p27 ubiquitin mediator, Skp2, using AKT inhibitor and Skp2 siRNA. In the functional study, AT13387 inhibited cell migration with downregulation of a cell migration regulator, HDAC6, and increased the acetylation and stabilization of α-tubulin. We also examined the effect of AT13387 on putative cancer stem cells (CSC) by 3-D tumor sphere formation assay. AT13387 effectively reduced both the number and size of C666-1 tumor spheres with decreased expression of NPC CSC-like markers CD44 and SOX2. In the in vivo study, AT13387 significantly suppressed tumor formation in C666-1 NPC xenografts. Conclusion AT13387 suppressed cell growth, cell migration, tumor sphere formation and induced cellular senescence on EBV-positive NPC cell line C666-1. Also, the antitumor effect of AT13387 was demonstrated in an in vivo model. This study provided experimental evidence for the preclinical value of using AT13387 as an effective antitumor agent in treatment of NPC. PMID:24156782

  14. Cadmium inhibits motility, activities of plasma membrane Ca(2+)-ATPase and axonemal dynein-ATPase of human spermatozoa.

    PubMed

    Da Costa, R; Botana, D; Piñero, S; Proverbio, F; Marín, R

    2016-05-01

    Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd(2+) on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca(2+)-ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd(2+) concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd(2+) showed a significant inhibition of progressive motility, reaching values even lower at higher Cd(2+) concentrations. In addition, it was found that the activity of the plasma membrane Ca(2+)-ATPase reached maximal inhibition at 50 nm free Cd(2+), with a K50% inhibition of 18.3 nm free Cd(2+). The dynein-ATPase activity was maximally inhibited by 25 nm free Cd(2+) in the assay medium, with a K50% inhibition of 11.3 nm Cd(2+). Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd(2+). PMID:26259968

  15. HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation

    PubMed Central

    Zepeda, Andrea B.; Figueroa, Carolina A.; Abdalla, Dulcineia S.P.; Maranhão, Andrea Q.; Ulloa, Patricio H.; Pessoa, Adalberto; Farías, Jorge G.

    2014-01-01

    Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol −10 °C, 4X = 3% methanol −30 °C, and 5X = 1% methanol −10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris. PMID:25242931

  16. Jasmonate signalling in Arabidopsis involves SGT1b–HSP70–HSP90 chaperone complexes

    PubMed Central

    Zhang, Xue-Cheng; Millet, Yves A.; Cheng, Zhenyu; Bush, Jenifer; Ausubel, Frederick M.

    2016-01-01

    Plant hormones play pivotal roles in growth, development and stress responses. Although it is essential to our understanding of hormone signalling, how plants maintain a steady state level of hormone receptors is poorly understood. We show that mutation of the Arabidopsis thaliana co-chaperone SGT1b impairs responses to the plant hormones jasmonate, auxin and gibberellic acid, but not brassinolide and abscisic acid, and that SGT1b and its homologue SGT1a are involved in maintaining the steady state levels of the F-box proteins COI1 and TIR1, receptors for jasmonate and auxin, respectively. The association of SGT1b with COI1 is direct and is independent of the Arabidopsis SKP1 protein, ASK1. We further show that COI1 is a client protein of SGT1b–HSP70–HSP90 chaperone complexes and that the complexes function in hormone signalling by stabilizing the COI1 protein. This study extends the SGT1b–HSP90 client protein list and broadens the functional scope of SGT1b–HSP70–HSP90 chaperone complexes. PMID:27054042

  17. Molecular Characteristic, Protein Distribution and Potential Regulation of HSP90AA1 in the Anadromous Fish Coilia nasus

    PubMed Central

    Fang, Di-An; Duan, Jin-Rong; Zhou, Yan-Feng; Zhang, Min-Ying; Xu, Dong-Po; Liu, Kai; Xu, Pao

    2016-01-01

    Heat shock proteins play essential roles in basic cellular events. Spawning migration is a complex process, with significant structural and biochemical changes taking place in the adult gonad. To date, the molecular mechanisms underlying migration reproductive biology remain undetermined. In this regard, a full length HSP90AA1 comprising 2608 nucleotides from the anadromous fish Coilia nasus was characterized, encoding 742 amino acid (aa) residues with potential phosphorylation sites. HSP90AA1 mRNA transcripts were detected in all organs, especially in the gonad. Furthermore, the greatest transcript levels were found during the developmental phase, while the lowest levels were found during the resting phase. In addition, the strongest immunolabeling positive signal was found in the primary spermatocyte and oocyte, with lower positive staining in secondary germ cells, and a weak or absent level in the mature sperm and oocyte. Interestingly, HSP90AA1 was mainly located in the cytoplasm of germ cells. These results are important for understanding the molecular mechanism of anadromous migration reproductive biology. In combination with data from other fish species, the result of this present study may facilitate further investigations on the spawning migration mechanism. PMID:26828521

  18. HSF-1, HIF-1 and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation.

    PubMed

    Zepeda, Andrea B; Figueroa, Carolina A; Abdalla, Dulcineia S P; Maranhão, Andrea Q; Ulloa, Patricio H; Pessoa, Adalberto; Farías, Jorge G

    2014-01-01

    Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol -10 °C, 4X = 3% methanol -30 °C, and 5X = 1% methanol -10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris. PMID:25242931

  19. Folding of Toll-like receptors by the HSP90 paralogue gp96 requires a substrate-specific cochaperone

    PubMed Central

    Liu, Bei; Yang, Yi; Qiu, Zhijuan; Staron, Matthew; Hong, Feng; Li, Yi; Wu, Shuang; Li, Yunfeng; Hao, Bing; Bona, Robert; Han, David; Li, Zihai

    2010-01-01

    Cytosolic HSP90 requires multiple cochaperones in folding client proteins. However, the function of gp96 (HSP90b1, grp94), an HSP90 paralogue in the endoplasmic reticulum (ER), is believed to be independent of cochaperones. Here, we demonstrate that gp96 chaperones multiple Toll-like receptors (TLRs), but not TLR3, in a manner that is dependent on another ER luminal protein, CNPY3. gp96 directly interacts with CNPY3, and the complex dissociates in the presence of adenosine triphosphate (ATP). Genetic disruption of gp96–CNPY3 interaction completely abolishes their TLR chaperone function. Moreover, we demonstrate that TLR9 forms a multimolecular complex with gp96 and CNPY3, and the binding of TLR9 to either molecule requires the presence of the other. We suggest that CNPY3 interacts with the ATP-sensitive conformation of gp96 to promote substrate loading. Our study has thus established CNPY3 as a TLR-specific cochaperone for gp96. PMID:20865800

  20. Design and synthesis of N-(5-chloro-2,4-dihydroxybenzoyl)-(R)-1,2,3,4-tetrahydroisoquinoline-3-carboxamides as novel Hsp90 inhibitors.

    PubMed

    Liang, Chuanpeng; Hao, Huilin; Wu, Xingkang; Li, Zhenyu; Zhu, Jing; Lu, Chunhua; Shen, Yuemao

    2016-10-01

    Heat shock protein 90 (Hsp90) is an attractive chemotherapeutic target for antitumor drug development. Herein, we reported the design and synthesis of two series of novel N-(5-chloro-2,4-dihydroxybenzoyl)-1,2,3,4-tetrahydroisoquinoline-3- carboxamides as Hsp90 inhibitors using (S)-Tic (1,2,3,4-tetrahydroisoquinoline-3- carboxylic acid) (A1-13) and (R)-Tic (B1-13) as scaffold, respectively. Cellular thermal shift assay (CETSA) screening showed that compounds B1-13 with (R)-Tic scaffold exhibited potent ability to stabilize Hsp90α. Compound B7 showed not only the most potent ability to induce thermal stabilization of Hsp90α but also the strongest cytotoxicity. The IC50 values of B7 were 0.98 μM and 1.74 μM against the proliferation of human breast cancer MDA-MB-231 and human cervical cancer HeLa cell lines, respectively. Moreover, CETSA melt and ITDRFCETSA (isothermal dose-response fingerprint) curves confirmed that B7 bound to Hsp90α in 293T cells. Western blotting results indicated that B7 induced the degradation of Hsp90 clients CDK4, Her2, Cdc-2 and C-raf. In addition, docking and Molecular dynamics (MD) refinement of the B7-Hsp90 complex showed that the binding model of B7 to Hsp90 was similar with that of 8-benzyladenines. The overall properties warrant compound B7 a promising lead for the development of Hsp90 inhibitor antitumor drugs. PMID:27266997

  1. Pre-clinical efficacy of PU-H71, a novel HSP90 inhibitor, alone and in combination with bortezomib in Ewing sarcoma.

    PubMed

    Ambati, Srikanth R; Lopes, Eloisi Caldas; Kosugi, Kohji; Mony, Ullas; Zehir, Ahmet; Shah, Smit K; Taldone, Tony; Moreira, Andre L; Meyers, Paul A; Chiosis, Gabriela; Moore, Malcolm A S

    2014-03-01

    Ewing sarcoma is characterized by multiple deregulated pathways that mediate cell survival and proliferation. Heat shock protein 90 (HSP90) is a critical component of the multi-chaperone complexes that regulate the disposition and activity of a large number of proteins involved in cell-signaling systems. We tested the efficacy of PU-H71, a novel HSP90 inhibitor in Ewing sarcoma cell lines, primary samples, benign mesenchymal stromal cells and hematopoietic stem cells. We performed cell cycle analysis, clonogenic assay, immunoblot analysis and reverse phase protein array in Ewing cell lines and in vivo experiments in NSG and nude mice using the A673 cell line. We noted a significant therapeutic window in the activity of PU-H71 against Ewing cell lines and benign cells. PU-H71 treatment resulted in G2/M phase arrest. Exposure to PU-H71 resulted in depletion of critical proteins including AKT, pERK, RAF-1, c-MYC, c-KIT, IGF1R, hTERT and EWS-FLI1 in Ewing cell lines. Our results indicated that Ewing sarcoma tumor growth and the metastatic burden were significantly reduced in the mice injected with PU-H71 compared to the control mice. We also investigated the effects of bortezomib, a proteasome inhibitor, alone and in combination with PU-H71 in Ewing sarcoma. Combination index (CI)-Fa plots and normalized isobolograms indicated synergism between PU-H71 and bortezomib. Ewing sarcoma xenografts were significantly inhibited when mice were treated with the combination compared to vehicle or either drug alone. This provides a strong rationale for clinical evaluation of PU-H71 alone and in combination with bortezomib in Ewing sarcoma.

  2. Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge.

    PubMed

    Fu, Dingkun; Chen, Jinhui; Zhang, Yang; Yu, Ziniu

    2011-07-01

    Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3', 5'-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important

  3. Nitric oxide suppresses LPS-induced inflammation in a mouse asthma model by attenuating the interaction of IKK and Hsp90

    PubMed Central

    Lee, Ming-Yung; Sun, Kuang-Hui; Chiang, Chien-Ping; Huang, Ching-Feng; Sun, Guang-Huan; Tsou, Yu-Chi; Liu, Huan-Yun

    2015-01-01

    A feature of allergic airway disease is the observed increase of nitric oxide (NO) in exhaled breath. Gram-negative bacterial infections have also been linked with asthma exacerbations. However, the role of NO in asthma exacerbations with gram-negative bacterial infections is still unclear. In this study, we examined the role of NO in lipopolysaccharide (LPS)-induced inflammation in an ovalbumin (OVA)-challenged mouse asthma model. To determine whether NO affected the LPS-induced response, a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) or a selective inhibitor of NO synthase (1400W) was injected intraperitoneally into the mice before the LPS stimulation. Decreased levels of proinflammatory cytokines were demonstrated in the bronchoalveolar lavage fluid from mice treated with SNAP, whereas increased levels of cytokines were found in the 1400W-treated mice. To further explore the molecular mechanism of NO-mediated inhibition of proinflammatory responses in macrophages, RAW 264.7 cells were treated with 1400W or SNAP before LPS stimulation. LPS-induced inflammation in the cells was attenuated by the presence of NO. The LPS-induced IκB kinase (IKK) activation and the expression of IKK were reduced by NO through attenuation of the interaction between Hsp90 and IKK in the cells. The IKK decrease in the lung immunohistopathology was verified in SNAP-treated asthma mice, whereas IKK increased in the 1400W-treated group. We report for the first time that NO attenuates the interaction between Hsp90 and IKK, decreasing the stability of IKK and causing the down-regulation of the proinflammatory response. Furthermore, the results suggest that NO may repress LPS-stimulated innate immunity to promote pulmonary bacterial infection in asthma patients. PMID:25519430

  4. Tissue-specific induction of Hsp90 mRNA and plasma cortisol response in chinook salmon following heat shock, seawater challenge, and handling challenge

    USGS Publications Warehouse

    Palmisano, Aldo N.; Winton, J.R.; Dickhoff, Walton W.

    2000-01-01

    In studying the whole-body response of chinook salmon (Oncorhynchus tshawytscha) to various stressors, we found that 5-hour exposure to elevated temperature (mean 21.6??C; + 10.6??C over ambient) induced a marked increase in Hsp90 messenger RNA accumulation in heart, brain, gill, muscle, liver, kidney, and tail fin tissues. The most vital tissues (heart, brain, gill, and muscle) showed the greatest Hsp90-mRNA response, with heart tissue increasing approximately 35-fold, Heat shock induced no increase in plasma cortisol. In contrast, a standard handling challenge induced high plasma cortisol levels, but no elevation in Hsp90 mRNA in any tissue, clearly separating the physiological and cellular stress responses. We saw no increase either in tissue Hsp90 mRNA levels or in plasma cortisol concentrations after exposing the fish to seawater overnight.

  5. Brain Na+, K+-ATPase Activity In Aging and Disease

    PubMed Central

    de Lores Arnaiz, Georgina Rodríguez; Ordieres, María Graciela López

    2014-01-01

    Na+/K+ pump or sodium- and potassium-activated adenosine 5’-triphosphatase (Na+, K+-ATPase), its enzymatic version, is a crucial protein responsible for the electrochemical gradient across the cell membranes. It is an ion transporter, which in addition to exchange cations, is the ligand for cardenolides. This enzyme regulates the entry of K+ with the exit of Na+ from cells, being the responsible for Na+/K+ equilibrium maintenance through neuronal membranes. This transport system couples the hydrolysis of one molecule of ATP to exchange three sodium ions for two potassium ions, thus maintaining the normal gradient of these cations in animal cells. Oxidative metabolism is very active in brain, where large amounts of chemical energy as ATP molecules are consumed, mostly required for the maintenance of the ionic gradients that underlie resting and action potentials which are involved in nerve impulse propagation, neurotransmitter release and cation homeostasis. Protein phosphorylation is a key process in biological regulation. At nervous system level, protein phosphorylation is the major molecular mechanism through which the function of neural proteins is modulted in response to extracellular signals, including the response to neurotransmitter stimuli. It is the major mechanism of neural plasticity, including memory processing. The phosphorylation of Na+, K+-ATPase catalytic subunit inhibits enzyme activity whereas the inhibition of protein kinase C restores the enzyme activity. The dephosphorylation of neuronal Na+, K+-ATPase is mediated by calcineurin, a serine / threonine phosphatase. The latter enzyme is involved in a wide range of cellular responses to Ca2+ mobilizing signals, in the regulation of neuronal excitability by controlling the activity of ion channels, in the release of neurotransmitters and hormones, as well as in synaptic plasticity and gene transcription. In the present article evidence showing Na+, K+-ATPase involvement in signaling pathways

  6. The effect of basketball training on the players' erythrocyte membrane acetylcholinesterase, (Na+,K+)-ATPase and Mg2+-ATPase activities.

    PubMed

    Parthimos, T; Tsopanakis, C; Angelogianni, P; Schulpis, K H; Parthimos, N; Tsakiris, S

    2007-08-01

    The aim of this study was to investigate whether the activities of erythrocyte membrane acetylcholinesterase (AChE), (Na (+),K (+))-ATPase and Mg (2+)-ATPase are modulated by a basketball training. Blood was obtained from 10 basketball players pre- and postexercise. Total antioxidant status (TAS), lactate and pyruvate concentrations were determined with kits, while the enzyme activities were determined spectrophotometrically. Post-training blood lactate and pyruvate concentrations as well as AChE (2.90 +/- 0.05 vs. 3.98 +/- 0.09 Delta OD/min . mg protein, p < 0.01) and Na (+),K (+)-ATPase (0.58 +/- 0.04 vs. 1.27 +/- 0.12 micromol Pi/h . mg protein, p < 0.001) activities were remarkably increased, whereas TAS was significantly decreased. Mg (2+)-ATPase activity remained unaltered at the end of the training. In conclusion, the stimulation of AChE and Na (+),K (+)-ATPase by the training may be due to the rise of blood catecholamine oxidation contributing to TAS decrease and/or the increase of serotonin levels. This stress condition may modulate cholinergic and catecholaminergic/serotoninergic functions in players.

  7. Binding of the pathogen receptor HSP90AA1 to avibirnavirus VP2 induces autophagy by inactivating the AKT-MTOR pathway.

    PubMed

    Hu, Boli; Zhang, Yina; Jia, Lu; Wu, Huansheng; Fan, Chengfei; Sun, Yanting; Ye, Chengjin; Liao, Min; Zhou, Jiyong

    2015-01-01

    Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.

  8. Decreased thioredoxin-1 and increased HSP90 expression in skeletal muscle in subjects with type 2 diabetes or impaired glucose tolerance.

    PubMed

    Venojärvi, M; Korkmaz, A; Aunola, S; Hällsten, K; Virtanen, K; Marniemi, J; Halonen, J-P; Hänninen, O; Nuutila, P; Atalay, M

    2014-01-01

    In diabetes, the endogenous defence systems are overwhelmed, causing various types of stress in tissues. In this study, newly diagnosed or diet-treated type 2 diabetics (T2D) (n = 10) were compared with subjects with impaired glucose tolerance (IGT) (n = 8). In both groups, at resting conditions, blood samples were drawn for assessing metabolic indices and skeletal muscle samples (m. vastus lateralis) were taken for the measurements of cellular defence markers: thioredoxin-1 (TRX-1) and stress proteins HSP72, HSP90. The protein level of TRX-1 was 36.1% lower (P = 0.031) and HSP90 was 380% higher (P < 0.001) in the T2D than in the IGT subjects, with no significant changes in HSP72. However, after the adjustment of both analyses with HOMA-IR only HSP90 difference remained significant. In conclusion, level of TRX-1 in skeletal muscle tissue was lower while that of HSP90 was higher in T2D than in IGT subjects. This may impair antioxidant defence and lead to disruptions of protein homoeostasis and redox regulation of cellular defences. Because HSP90 may be involved in sustaining functional insulin signalling pathway in type 2 diabetic muscles and higher HSP90 levels can be a consequence of type 2 diabetes, our results are potentially important for the diabetes research. PMID:24689038

  9. The neuroregenerative mechanism mediated by the Hsp90-binding immunophilin FKBP52 resembles the early steps of neuronal differentiation

    PubMed Central

    Quintá, HR; Galigniana, MD

    2012-01-01

    BACKGROUND AND PURPOSE The immunosuppressive macrolide FK506 (tacrolimus) shows neuroregenerative action by a mechanism that appears to involve the Hsp90-binding immunophilin FKBP52. This study analyses some aspects of the early steps of neuronal differentiation and neuroregeneration. EXPERIMENTAL APPROACH Undifferentiated murine neuroblastoma cells and hippocampal neurones isolated from embryonic day-17 rat embryos were induced to differentiate with FK506. Subcellular relocalization of FKBP52, Hsp90 and its co-chaperone p23 was analysed by indirect immunofluorescence confocal microscopy and by Western blots of axonal fractions isolated from cells grown on a porous transwell cell culture chamber. Neuroregeneration was evaluated using a scratch-wound assay. KEY RESULTS In undifferentiated cells, FKBP52, Hsp90 and p23 are located in the cell nucleus, forming an annular structure that disassembles when the differentiation process is triggered by FK506. This was observed in the N2a cell line and in hippocampal neurones. More importantly, the annular structure of chaperones is reassembled after damaging the neurones, whereas FK506 prompts their rapid regeneration, a process linked to the subcellular redistribution of the heterocomplex. CONCLUSIONS AND IMPLICATIONS There is a direct relationship between the disassembly of the chaperone complex and the progression of neuronal differentiation upon stimulation with the immunophilin ligand FK506. Both neuronal differentiation and neuroregeneration appear to be mechanistically linked, so the elucidation of one mechanism may lead to unravel the properties of the other. This study also implies that the discovery of FK506 derivatives, devoid of immunosuppressive action, would be therapeutically significant for neurotrophic use. PMID:22091865

  10. An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters.

    PubMed Central

    Conaway, R C; Conaway, J W

    1989-01-01

    A transcription factor required for synthesis of accurately initiated run-off transcripts by RNA polymerase II has been purified and shown to have an associated DNA-dependent ATPase (dATPase) activity that is strongly stimulated by the TATA region of promoters. This transcription factor, designated delta, was purified more than 3000-fold from extracts of crude rat liver nuclei and has a native molecular mass of approximately 230 kDa. DNA-dependent ATPase (dATPase) and transcription activities copurify when delta is analyzed by hydrophobic interaction and ion-exchange HPLC, arguing that transcription factor delta possesses an ATPase (dATPase) activity. ATPase (dATPase) is specific for adenine nucleotides; ATP and dATP, but not CTP, UTP, or GTP, are hydrolyzed. ATPase (dATPase) is stimulated by both double-stranded and single-stranded DNAs, including pUC18, ssM13, and poly(dT); however, DNA fragments containing the TATA region of either the adenovirus 2 major late or mouse interleukin 3 promoters stimulate ATPase as much as 10-fold more effectively than DNA fragments containing nonpromoter sequences. These data suggest the intriguing possibility that delta plays a critical role in the ATP (dATP)-dependent activation of run-off transcription through a direct interaction with the TATA region of promoters. Images PMID:2552440

  11. Dietary selenium increases the antioxidant levels and ATPase activity in the arteries and veins of poultry.

    PubMed

    Cao, Changyu; Zhao, Xia; Fan, Ruifeng; Zhao, Jinxin; Luan, Yilin; Zhang, Ziwei; Xu, Shiwen

    2016-07-01

    Selenium (Se) deficiency is associated with the pathogenesis of vascular diseases. It has been shown that oxidative levels and ATPase activity were involved in Se deficiency diseases in humans and mammals; however, the mechanism by how Se influences the oxidative levels and ATPase activity in the poultry vasculature is unclear. We assessed the effects of dietary Se deficiency on the oxidative stress parameters (superoxide dismutase, catalase, and hydroxyl radical) and ATPase (Na(+)K(+)-ATPase, Ca(++)-ATPase, Mg(++)-ATPase, and Ca(++)Mg(++)-ATPase) activity in broiler poultry. A total of 40 broilers (1-day old) were randomly divided into a Se-deficient group (L group, fed a Se-deficient diet containing 0.08 mg/kg Se) and a control group (C group, fed a diet containing sodium selenite at 0.20 mg/kg Se). Then, arteries and veins were collected following euthanasia when typical symptoms of Se deficiency appeared. Antioxidant indexes and ATPase activity were evaluated using standard assays in arteries and veins. The results indicated that superoxide dismutase activity in the artery according to dietary Se deficiency was significantly lower (p < 0.05) compared with the C group. The catalase activity in the veins and hydroxyl radical inhibition in the arteries and veins by dietary Se deficiency were significantly higher (p < 0.05) compared with the C group. The Se-deficient group showed a significantly lower (p < 0.05) tendency in Na(+)K(+)-ATPase activity, Ca(++)-ATPase activity, and Ca(++)Mg(++)-ATPase activity. There were strong correlations between antioxidant indexes and Ca(++)-ATPase activity. Thus, these results indicate that antioxidant indexes and ATPases may have special roles in broiler artery and vein injuries under Se deficiency. PMID:26637493

  12. Oscillations in glycolysis in Saccharomyces cerevisiae: the role of autocatalysis and intracellular ATPase activity.

    PubMed

    Kloster, Antonina; Olsen, Lars Folke

    2012-05-01

    We have investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations. The amplitude dependence on cell density shows the same behavior as that observed in cells in a CSTR. Furthermore, the amplitude decreases with increasing inhibition of the three ATPases (i) F(0)F(1) ATPase, (ii) plasma membrane ATPase (Pma1p) and (iii) vacuolar ATPase (V-ATPase). The amplitude of the oscillations also decreases by stimulating the ATPase activity, e.g. by FCCP or Amphotericin B. Thus, ATPase activity strongly affects the glycolytic oscillations. We discuss these data in relation to a simple autocatalytic model of glycolysis which can reproduce the experimental data and explain the role of membrane-bound ATPases . In addition we also studied a recent detailed model of glycolysis and found that, although this model faithfully reproduces the oscillations of glycolytic intermediates observed experimentally, it is not able to explain the role of ATPase activity on the oscillations.

  13. Overexpression of G6PD and HSP90 Beta in Mice with Benzene Exposure Revealed by Serum Peptidome Analysis

    PubMed Central

    Zhang, Juan; Tan, Kehong; Meng, Xing; Yang, Wenwen; Wei, Haiyan; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-01-01

    The small peptides representation of the original proteins are a valuable source of information that can be used as biomarkers involved in toxicity mechanism for chemical exposure. The aim of this study is to investigate serum peptide biomarkers of benzene exposure. C57BL/6 mice were enrolled into control group and benzene groups of 150 and 300 mg/kg/d Serum peptides were identified by mass spectrometry using an assisted laser desorption ionization/time of flight mass spectrometry (MS). Differential peptide spectra were obtained by tandem mass spectrometry and analyzed by searching the International Protein Index using the Sequest program. Forty-one peptide peaks were found in the range of 1000–10,000 Da molecular weight. Among them, seven peaks showed significantly different expression between exposure groups and control group. Two peptide peaks (1231.2 and 1241.8), which showed a two-fold increase in expression, were sequenced and confirmed as glucose 6-phosphate dehydrogenase (G6PD) and heat shock protein 90 Beta (HSP90 Beta), respectively. Furthermore, the expression of the two proteins in liver cells showed the same trend as in serum. In conclusion, G6PD and HSP90 beta might be the candidate serum biomarkers of benzene exposure. It also provided possible clues for the molecular mechanism of benzene-induced oxidative stress. PMID:26378550

  14. Overexpression of G6PD and HSP90 Beta in Mice with Benzene Exposure Revealed by Serum Peptidome Analysis.

    PubMed

    Zhang, Juan; Tan, Kehong; Meng, Xing; Yang, Wenwen; Wei, Haiyan; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-09-10

    The small peptides representation of the original proteins are a valuable source of information that can be used as biomarkers involved in toxicity mechanism for chemical exposure. The aim of this study is to investigate serum peptide biomarkers of benzene exposure. C57BL/6 mice were enrolled into control group and benzene groups of 150 and 300 mg/kg/d Serum peptides were identified by mass spectrometry using an assisted laser desorption ionization/time of flight mass spectrometry (MS). Differential peptide spectra were obtained by tandem mass spectrometry and analyzed by searching the International Protein Index using the Sequest program. Forty-one peptide peaks were found in the range of 1000-10,000 Da molecular weight. Among them, seven peaks showed significantly different expression between exposure groups and control group. Two peptide peaks (1231.2 and 1241.8), which showed a two-fold increase in expression, were sequenced and confirmed as glucose 6-phosphate dehydrogenase (G6PD) and heat shock protein 90 Beta (HSP90 Beta), respectively. Furthermore, the expression of the two proteins in liver cells showed the same trend as in serum. In conclusion, G6PD and HSP90 beta might be the candidate serum biomarkers of benzene exposure. It also provided possible clues for the molecular mechanism of benzene-induced oxidative stress.

  15. Corresponding Functional Dynamics across the Hsp90 Chaperone Family: Insights from a Multiscale Analysis of MD Simulations

    PubMed Central

    Morra, Giulia; Potestio, Raffaello; Micheletti, Cristian; Colombo, Giorgio

    2012-01-01

    Understanding how local protein modifications, such as binding small-molecule ligands, can trigger and regulate large-scale motions of large protein domains is a major open issue in molecular biology. We address various aspects of this problem by analyzing and comparing atomistic simulations of Hsp90 family representatives for which crystal structures of the full length protein are available: mammalian Grp94, yeast Hsp90 and E.coli HtpG. These chaperones are studied in complex with the natural ligands ATP, ADP and in the Apo state. Common key aspects of their functional dynamics are elucidated with a novel multi-scale comparison of their internal dynamics. Starting from the atomic resolution investigation of internal fluctuations and geometric strain patterns, a novel analysis of domain dynamics is developed. The results reveal that the ligand-dependent structural modulations mostly consist of relative rigid-like movements of a limited number of quasi-rigid domains, shared by the three proteins. Two common primary hinges for such movements are identified. The first hinge, whose functional role has been demonstrated by several experimental approaches, is located at the boundary between the N-terminal and Middle-domains. The second hinge is located at the end of a three-helix bundle in the Middle-domain and unfolds/unpacks going from the ATP- to the ADP-state. This latter site could represent a promising novel druggable allosteric site common to all chaperones. PMID:22457611

  16. Choice of Biological Source Material Supersedes Oxidative Stress in Its Influence on DJ-1 in Vivo Interactions with Hsp90

    PubMed Central

    Knobbe, Christiane B.; Revett, Timothy J.; Bai, Yu; Chow, Vinca; Jeon, Amy Hye Won; Böhm, Christopher; Ehsani, Sepehr; Kislinger, Thomas; Mount, Howard T.; Mak, Tak W.; St. George-Hyslop, Peter; Schmitt-Ulms, Gerold

    2016-01-01

    DJ-1 is a small but relatively abundant protein of unknown function that may undergo stress-dependent cellular translocation and has been implicated in both neurodegenerative diseases and cancer. As such, DJ-1 may be an excellent study object to elucidate the relative influence of the cellular context on its interactome and for exploring whether acute exposure to oxidative stressors alters its molecular environment. Using quantitative mass spectrometry, we conducted comparative DJ-1 interactome analyses from in vivo cross-linked brains or livers and from hydrogen peroxide-treated or naïve embryonic stem cells. The analysis identified a subset of glycolytic enzymes, heat shock proteins 70 and 90, and peroxiredoxins as interactors of DJ-1. Consistent with a role of DJ-1 in Hsp90 chaperone biology, we document destabilization of Hsp90 clients in DJ-1 knockout cells. We further demonstrate the existence of a C106 sulfinic acid modification within DJ-1 and thereby establish that this previously inferred modification also exists in vivo. Our data suggest that caution has to be exerted in interpreting interactome data obtained from a single biological source material and identify a role of DJ-1 as an oxidative stress sensor and partner of a molecular machinery notorious for its involvement in cell fate decisions. PMID:21819105

  17. Substrate independent ATPase activity may complicate high throughput screening.

    PubMed

    Tuntland, Micheal L; Fung, L W-M

    2016-10-01

    Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening. PMID:27430931

  18. A Homogeneous Polysaccharide from Fructus Schisandra chinensis (Turz.) Baill Induces Mitochondrial Apoptosis through the Hsp90/AKT Signalling Pathway in HepG2 Cells.

    PubMed

    Chen, Yonglin; Shi, Songshan; Wang, Huijun; Li, Ning; Su, Juan; Chou, Guixin; Wang, Shunchun

    2016-06-28

    According to the potential anti-hepatoma therapeutic effect of Schisandra chinensis polysaccharides presented in previous studies, a bioactive constituent, homogeneous Schisandra chinensis polysaccharide-0-1 (SCP-0-1), molecular weight (MW) circa 69.980 kDa, was isolated and purified. We assessed the efficacy of SCP-0-1 against human hepatocellular liver carcinoma (HepG2) cells to investigate the effects of its antitumour activity and molecular mechanisms. Anticancer activity was evaluated using microscopy, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst 33258 staining, acridine orange (AO) staining, flow cytometry (FCM), and cell-cycle analysis. SCP-0-1 inhibited the HepG2 cells' growth via inducing apoptosis and second gap/mitosis (G2/M) arrest dose-dependently, with a half maximal inhibitory concentration (IC50) value of 479.63 µg/mL. Western blotting of key proteins revealed the apoptotic and autophagic potential of SCP-0-1. Besides, SCP-0-1 upregulated Bcl-2 Associated X Protein (Bax) and downregulated B-cell leukemia/lymphoma 2 (Bcl-2) in the HepG2 cells. The expression of caspase-3, -8, and -9; poly (ADP-ribose) polymerase (PARP); cytochrome c (Cyt C); tumor protein 53 (p53); survivin; sequestosome 1 (p62); microtubule-associated protein 1 light chain-3B (LC3B); mitogen-activated protein kinase p38 (p38); extracellular regulated protein kinases (ERK); c-Jun N-terminal kinase (JNK); protein kinase B (AKT); and heat shock protein 90 (Hsp90) were evaluated using Western blotting. Our findings demonstrate a novel mechanism through which SCP-0-1 exerts its antiproliferative activity and induces mitochondrial apoptosis rather than autophagy. The induction of mitochondrial apoptosis was attributed to the inhibition of the Hsp90/AKT signalling pathway in an extracellular signal-regulated kinase-independent manner. The results also provide initial evidence on a molecular basis that SCP-0-1 can be used as an anti

  19. A Homogeneous Polysaccharide from Fructus Schisandra chinensis (Turz.) Baill Induces Mitochondrial Apoptosis through the Hsp90/AKT Signalling Pathway in HepG2 Cells

    PubMed Central

    Chen, Yonglin; Shi, Songshan; Wang, Huijun; Li, Ning; Su, Juan; Chou, Guixin; Wang, Shunchun

    2016-01-01

    According to the potential anti-hepatoma therapeutic effect of Schisandra chinensis polysaccharides presented in previous studies, a bioactive constituent, homogeneous Schisandra chinensis polysaccharide-0-1 (SCP-0-1), molecular weight (MW) circa 69.980 kDa, was isolated and purified. We assessed the efficacy of SCP-0-1 against human hepatocellular liver carcinoma (HepG2) cells to investigate the effects of its antitumour activity and molecular mechanisms. Anticancer activity was evaluated using microscopy, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst 33258 staining, acridine orange (AO) staining, flow cytometry (FCM), and cell-cycle analysis. SCP-0-1 inhibited the HepG2 cells’ growth via inducing apoptosis and second gap/mitosis (G2/M) arrest dose-dependently, with a half maximal inhibitory concentration (IC50) value of 479.63 µg/mL. Western blotting of key proteins revealed the apoptotic and autophagic potential of SCP-0-1. Besides, SCP-0-1 upregulated Bcl-2 Associated X Protein (Bax) and downregulated B-cell leukemia/lymphoma 2 (Bcl-2) in the HepG2 cells. The expression of caspase-3, -8, and -9; poly (ADP-ribose) polymerase (PARP); cytochrome c (Cyt C); tumor protein 53 (p53); survivin; sequestosome 1 (p62); microtubule-associated protein 1 light chain-3B (LC3B); mitogen-activated protein kinase p38 (p38); extracellular regulated protein kinases (ERK); c-Jun N-terminal kinase (JNK); protein kinase B (AKT); and heat shock protein 90 (Hsp90) were evaluated using Western blotting. Our findings demonstrate a novel mechanism through which SCP-0-1 exerts its antiproliferative activity and induces mitochondrial apoptosis rather than autophagy. The induction of mitochondrial apoptosis was attributed to the inhibition of the Hsp90/AKT signalling pathway in an extracellular signal-regulated kinase-independent manner. The results also provide initial evidence on a molecular basis that SCP-0-1 can be used as an anti

  20. The targeted inhibition of mitochondrial Hsp90 overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis

    SciTech Connect

    Yan, Chunlan; Oh, Joon Seok; Yoo, Seung Hee; Lee, Jee Suk; Yoon, Young Geol; Oh, Yoo Jin; Jang, Min Seok; Lee, Sang Yeob; Yang, Jun; Lee, Sang Hwa; Kim, Hye Young; Yoo, Young Hyun

    2013-01-01

    Previous studies have reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity, thus inducing the apoptotic pathway in the cells. Accordingly, G-TPP shows a potential as a promising drug for the treatment of cancer. A cell can die from different types of cell death such as apoptosis, necrosis, necroptosis, and autophagic cell death. In this study, we further investigated the mechanisms and modes of cell death in the G-TPP-treated Hep3B and U937 cell lines. We discovered that G-TPP kills the U937 cells through the apoptotic pathway and the overexpression of Bcl-2 significantly inhibits U937 cell death to G-TPP. We further discovered that G-TPP kills the Hep3B cells by activating necroptosis in combination with the partial activation of caspase-dependent apoptosis. Importantly, G-TPP overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. We also observed that G-TPP induces compensatory autophagy in the Hep3B cell line. We further found that whereas there is a Bcl-2-Beclin 1 interaction in response to G-TPP, silencing the beclin 1 gene failed to block LC3-II accumulation in the Hep3B cells, indicating that G-TPP triggers Beclin 1-independent protective autophagy in Hep3B cells. Taken together, these data reveal that G-TPP induces cell death through a combination of death pathways, including necroptosis and apoptosis, and overcomes the apoptosis resistance conferred by Bcl-2 in Hep3B cells via necroptosis. These findings are important for the therapeutic exploitation of necroptosis as an alternative cell death program to bypass the resistance to apoptosis. Highlights: ► G-TPP binds to mitochondrial Hsp90. ► G-TPP induces apoptosis in U937 human leukemia cancer cells. ► G-TPP induces combination of death pathways in Hep3B cell. ► G-TPP overcomes the resistance conferred by Bcl-2 in Hep3B cells via necroptosis. ► G-TPP triggers Beclin 1-independent

  1. Measuring the Pharmacodynamic Effects of a Novel Hsp90 Inhibitor on HER2/neu Expression in Mice Using 89Zr-DFO-Trastuzumab

    PubMed Central

    Holland, Jason P.; Caldas-Lopes, Eloisi; Divilov, Vadim; Longo, Valerie A.; Taldone, Tony; Zatorska, Danuta; Chiosis, Gabriela; Lewis, Jason S.

    2010-01-01

    Background The positron-emitting radionuclide 89Zr (t1/2 = 3.17 days) was used to prepare 89Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted. Methodology/Principal Findings Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [89Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in 89Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. 89Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3±2.1 MBq/mg (2.82±0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68±13.06%ID/g; 71.71±10.35%ID/g and 85.18±11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of 89Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75±4.43%ID/g and 41.42±3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64±12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels. Conclusions/Significance The results indicate that 89Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has

  2. [The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

    PubMed

    Snigireva, A V; Vrublevskaya, V V; Skarga, Y Y; Morenkov, O S

    2016-01-01

    Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90β, we showed that membrane-bound Hsp90α and Hsp90β play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90β to the plasma membrane.

  3. Geldanamycin and its anti-cancer activities.

    PubMed

    Fukuyo, Yayoi; Hunt, Clayton R; Horikoshi, Nobuo

    2010-04-01

    Geldanamycin is a benzoquinone ansamycin antibiotic that manifests anti-cancer activity through the inhibition of HSP90-chaperone function. The HSP90 molecular chaperone is expressed at high levels in a wide variety of human cancers including melanoma, leukemia, and cancers in colon, prostate, lung, and breast. In cancer cells dependent upon mutated and/or over-expressed oncogene proteins, HSP90 is thought to have a critical role in regulating the stability, folding, and activity of HSP90-associated proteins, so-called "client proteins". These client proteins include the growth-stimulating proteins and kinases that support malignant transformation. Recently, oncogenic activating BRAF mutants have been identified in variety of cancers where constitutive activation of the MEK/ERK MAPK signaling pathway is the key for tumorigenesis, and they have been shown to be client proteins for HSP90. Accordingly, HSP90 inhibition can suppress certain cancer-causing client proteins and therefore represents an important therapeutic target. The molecular mechanism underlying the anti-cancer effect of HSP90 inhibition is complicated. Geldanamycin and its derivatives have been shown to induce the depletion of mutationally-activated BRAF through several mechanisms. In this review, we will describe the HSP90-inhibitory mechanism, focusing on recent progress in understanding HSP90 chaperone structure-function relationships, the identification of new HSP90 client proteins and the development of HSP90 inhibitors for clinical applications.

  4. Targeting Proteostasis Through the Protein Quality Control Function of the Hsp90/Hsp70-based Chaperone Machinery for Treatment of Adult Onset Neurodegenerative Diseases

    PubMed Central

    Pratt, William B.; Gestwicki, Jason E.; Osawa, Yoichi; Lieberman, Andrew P.

    2015-01-01

    Currently available therapies for adult onset neurodegenerative diseases provide symptomatic relief, but are not disease modifying. We explore here a new neuroprotective approach based on drugs targeting chaperone-directed protein quality control. Critical target proteins that unfold and aggregate in these diseases, such as the polylglutamine androgen receptor (spinal and bulbar muscular atrophy), huntingtin (Huntington’s disease), α-synuclein (Parkinson’s disease) and tau (Alzheimer’s disease) are client proteins of Hsp90, and their turnover is regulated by the protein quality control function of the Hsp90/Hsp70-based chaperone machinery. In protein quality control Hsp90 and Hsp70 have opposing effects on client protein stability; Hsp90 stabilizes the clients and inhibits their ubiquitination, whereas Hsp70 promotes CHIP-dependent ubiquitination and proteasomal degradation. We discuss how drugs that modulate proteostasis by inhibiting Hsp90 function or by promoting Hsp70 function enhance the degradation of the critical aggregating proteins and ameliorate toxic symptoms in cell and animal disease models. PMID:25292434

  5. A SNP in the HSP90AA1 gene 5' flanking region is associated with the adaptation to differential thermal conditions in the ovine species.

    PubMed

    Marcos-Carcavilla, Ane; Mutikainen, Mari; González, Carmen; Calvo, Jorge H; Kantanen, Juha; Sanz, Albina; Marzanov, Nurbiy S; Pérez-Guzmán, María D; Serrano, Magdalena

    2010-01-01

    Molecular chaperones have long been understood to be preferentially transcribed in response to multiple perturbations of the cellular homeostasis. In this study, several polymorphisms in the gene encoding the inducible form of the cytoplasmic Hsp90 (HSP90AA1) were addressed in 24 sheep breeds reared in different climatic regions of Europe, Africa, and Asia. Significant differences in the genotype frequencies for a C/G single nucleotide polymorphism (SNP) located at position -660 in the HSP90AA1 5'flanking region were found between the different breeds. Regression analyses reflected significant correlations (from 0.41 to 0.62) between the alternative genotypes of this polymorphism and several climatic and geographic variables characteristic of the regions where these breeds are reared. Real-time analysis revealed that animals bearing the CC(-660) genotype presented higher expression levels than those presenting the CG(-660) or GG(-660) in summer, but not in spring. Mutation at -660 site seems to affect HSP90AA1 transcription rates which could have important effects on the adaptation to different environmental conditions in sheep. Thus, the variability found in the genotype frequencies for the SNP at -660 in the ovine HSP90AA1 locus could be the result of the different environmental pressures occurring in the regions where these breed are maintained.

  6. A Model in which Hsp90 Targets Protein Folding Clefts: rationale for a New Approach to Neuroprotective Treatment of Protein Folding Diseases

    PubMed Central

    Pratt, William B.; Morishima, Yoshihiro; Gestwicki, Jason E.; Lieberman, Andrew P.; Osawa, Yoichi

    2015-01-01

    In an EBM Minireview published in 2010, we proposed that the Hsp90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation (1). The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts (2). The model provides a framework for thinking about the development of neuroprotective therapies for protein folding diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD) and the polyglutamine expansion disorders, such as Huntington’s disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are ‘client’ proteins of the abundant and ubiquitous stress chaperone Hsp90 (3). These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD) and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this minireview we update our model in which Hsp90 acts on protein folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins. PMID:24990484

  7. Origin of concurrent ATPase activities in skinned cardiac trabeculae from rat.

    PubMed Central

    Ebus, J P; Stienen, G J

    1996-01-01

    1. To determine the rate of ATP turnover by the sarcoplasmic reticulum (SR) Ca2+ pump in cardiac muscle, and to assess the contributions of other ATPase activities to the overall ATP turnover rate, ATPase activity and isometric force production were studied in saponin-skinned trabeculae from rat. ATP hydrolysis was enzymatically coupled to the oxidation of NADH; the concentration of NADH was monitored photometrically. All measurements were performed at 20 +/- 1 degrees C and pH 7.0. Resting sarcomere length was adjusted to 2.1 microns. All solutions contained 5 mM caffeine to ensure continuous release of Ca2+ from the SR. 2. The Ca(2+)-independent ATPase activity, determined in relaxing solution (pCa 9), amounted to 130 +/- 13 microM s-1 (mean +/- S.E.M., n = 7) at the beginning of an experiment. During subsequent measurements in relaxing solution, a decrease in ATPase activity was observed, indicative of loss of membrane-bound ATPase activity. The steady-state Ca(2+)-independent (basal) ATPase activity was 83 +/- 5 microM s-1 (n = 66). 3. Treatment of saponin-skinned preparations with Triton X-100 abolished 50 microM s-1 (60%) of the basal ATPase activity. Addition of ouabain (1 mM) suppressed 14 +/- 5% of the basal activity, whereas 8 +/- 3% was suppressed by 20 microM cyclopiazonic acid (CPA). It is argued that 31 microM s-1 of the basal ATPase activity may be associated with MgATPase from the transverse tubular system. 4. The maximal Ca(2+)-activated ATPase activity, i.e. the total ATPase activity (determined in activating solution, pCa 4.3) corrected for basal ATPase activity, was found to be 409 +/- 15 microM s-1 (n = 66). Experiments with CPA indicated that at least 9 +/- 6% of the maximal Ca(2+)-activated ATPase activity originates from the sarcoplasmic Ca2+ pump. These experiments indicate that the rate of ATP consumption by the SR Ca2+ transporting ATPase amounts to at least 37 microM s-1. 5. Treatment of preparations with Triton X-100 abolished 15 +/- 3

  8. Rapid activation of gill Na+,K+-ATPase in the euryhaline teleost Fundulus heteroclitus

    USGS Publications Warehouse

    Mancera, J.M.; McCormick, S.D.

    2000-01-01

    The rapid activation of gill Na+,K+-ATPase was analyzed in the mummichog (Fundulus heteroclitus) and Atlantic salmon (Salmo salar) transferred from low salinity (0.1 ppt) to high salinity (25-35 ppt). In parr and presmolt, Salmo salar gill Na+,K+-ATPase activity started to increase 3 days after transfer. Exposure of Fundulus heteroclitus to 35 ppt seawater (SW) induced a rise in gill Na+,K+-ATPase activity 3 hr after transfer. After 12 hr, the values dropped to initial levels but showed a second significant increase 3 days after transfer. The absence of detergent in the enzyme assay resulted in lower values of gill Na+,K+-ATPase, and the rapid increase after transfer to SW was not observed. Na+,K+-ATPase activity of gill filaments in vitro for 3 hr increased proportionally to the osmolality of the culture medium (600 mosm/kg > 500 mosm/kg > 300 mosm/kg). Osmolality of 800 mosm/kg resulted in lower gill Na+,K+-ATPase activity relative to 600 mosm/kg. Increasing medium osmolality to 600 mosm/kg with mannitol also increased gill Na+,K+-ATPase. Cycloheximide inhibited the increase in gill Na+,K+-ATPase activity observed in hyperosmotic medium in a dose-dependent manner (10-4 M > 10-5 M > 10-6 M). Actinomycin D or bumetanide in the culture (doses of 10-4 M, 10-5 M, and 10-6 M) did not affect gill Na+,K+-ATPase. Injection of fish with actinomycin D prior to gill organ culture, however, prevented the increase in gill Na+,K+-ATPase activity in hyperosmotic media. The results show a very rapid and transitory increase in gill Na+,K+-ATPase activity in the first hours after the transfer of Fundulus heteroclitus to SW that is dependent on translational and transcriptional processes. (C) 2000 Wiley-Liss, Inc.

  9. Sperm sorting procedure induces a redistribution of Hsp70 but not Hsp60 and Hsp90 in boar spermatozoa.

    PubMed

    Spinaci, Marcella; Volpe, Sara; Bernardini, Chiara; de Ambrogi, Marco; Tamanini, Carlo; Seren, Eraldo; Galeati, Giovanna

    2006-01-01

    Heat shock proteins, besides their protective function against stresses, have been recently indicated as key factors for sperm fertilizing ability. Since sexing sperm by high-speed flow-cytometry subjects them to different physical, mechanical, and chemical stresses, the present study was designed to verify, by immunofluorescence and Western blot, whether the sorting procedure induces any modification in the amount and cellular distribution of heat shock proteins 60, 70, and 90 (Hsp60, Hsp70, Hsp90). Immunolocalization and Western blot quantification of both Hsp60 and Hsp90 did not reveal differences between unsorted and sorted semen. On the contrary, a redistribution of Hsp70 immunoreactivity from the equatorial subsegment toward the equator of sperm cells was recorded after sorting; this relocation suggests capacitation-like changes of sperm membrane. This modification seems to be caused mainly by incubation with Hoechst 33342, while both passage of sperm through flow cytometer and laser beam represent only minor stimuli. A further Hsp70 redistribution seems to be due to the final steps of sperm sorting, charging, and deflection of drops, and to the dilution during collection. On the other hand, staining procedure and mechanical stress seem to be the factors most injurious to sperm viability. Moreover, Hsp70 relocation was deeply influenced by the storage method. In fact, storing sexed spermatozoa, after centrifugation, in a small volume in presence of seminal plasma induced a reversion of Hsp70 redistribution, while storage in the diluted catch fluid of collection tubes caused Hsp70 relocation in most sorted spermatozoa. PMID:16870948

  10. Potential diagnostic significance of HSP90, ACS/TMS1, and L-plastin in the identification of melanoma.

    PubMed

    Strickler, Allen G; Vasquez, Juan G; Yates, Nathan; Ho, Jonhan

    2014-12-01

    Melanoma is one of the deadliest cancers, yet it remains a diagnostic and prognostic challenge. The lack of effective treatment modalities compounds this challenge. Characterizing the molecular mechanisms leading to the development of melanoma is the first step to understanding the pathophysiology of melanoma. Numerous molecular studies have helped us understand critical changes that occur in the transition from a benign nevus to melanoma. However, many of these processes remain undiscovered. The goal of the current project was to characterize the proteomes of benign nevi and malignant melanomas using proteomic methods, with confirmation by immunohistochemical analysis. Using tandem mass spectrometry, we identified proteins potentially involved in melanoma pathogenesis. Several of the identified proteins have known roles in oncogenesis, melanogenesis, or both. We selected Hsp90-β, apoptosis-associated speck-like protein containing a CARD (ASC/TMS1), and L-plastin from these to analyze nevi and melanoma samples by immunohistochemical analysis. Hsp90-β and ASC/TMS1 staining was higher in melanoma when compared with nevi, whereas L-plastin protein expression was not significantly different between cells of these tumor types; however, it was expressed in the inflammatory milieu of melanoma. ACS/TMS1 showed staining in normal and junctional melanocytes, as well as in superficial nevomelanocytes, but deeper dermal nevomelanocytes gradually lost expression. This study helps validate the use of proteomics to aid in characterizing protein differences between nevi and melanomas and also underscores the importance of correlating proteomic results with histomorphology to understand the context of the information. The proteins in the current study may hold potential in differentiating between melanoma and benign nevi in diagnostically challenging cases.

  11. Combining solvent thermodynamic profiles with functionality maps of the Hsp90 binding site to predict the displacement of water molecules.

    PubMed

    Haider, Kamran; Huggins, David J

    2013-10-28

    Intermolecular interactions in the aqueous phase must compete with the interactions between the two binding partners and their solvating water molecules. In biological systems, water molecules in protein binding sites cluster at well-defined hydration sites and can form strong hydrogen-bonding interactions with backbone and side-chain atoms. Displacement of such water molecules is only favorable when the ligand can form strong compensating hydrogen bonds. Conversely, water molecules in hydrophobic regions of protein binding sites make only weak interactions, and the requirements for favorable displacement are less stringent. The propensity of water molecules for displacement can be identified using inhomogeneous fluid solvation theory (IFST), a statistical mechanical method that decomposes the solvation free energy of a solute into the contributions from different spatial regions and identifies potential binding hotspots. In this study, we employed IFST to study the displacement of water molecules from the ATP binding site of Hsp90, using a test set of 103 ligands. The predicted contribution of a hydration site to the hydration free energy was found to correlate well with the observed displacement. Additionally, we investigated if this correlation could be improved by using the energetic scores of favorable probe groups binding at the location of hydration sites, derived from a multiple copy simultaneous search (MCSS) method. The probe binding scores were not highly predictive of the observed displacement and did not improve the predictivity when used in combination with IFST-based hydration free energies. The results show that IFST alone can be used to reliably predict the observed displacement of water molecules in Hsp90. However, MCSS can augment IFST calculations by suggesting which functional groups should be used to replace highly displaceable water molecules. Such an approach could be very useful in improving the hit-to-lead process for new drug targets.

  12. Inhibition of HSP90 by AT13387 delays the emergence of resistance to BRAF inhibitors and overcomes resistance to dual BRAF and MEK inhibition in melanoma models.

    PubMed

    Smyth, Tomoko; Paraiso, Kim H T; Hearn, Keisha; Rodriguez-Lopez, Ana M; Munck, Joanne M; Haarberg, H Eirik; Sondak, Vernon K; Thompson, Neil T; Azab, Mohammad; Lyons, John F; Smalley, Keiran S M; Wallis, Nicola G

    2014-12-01

    Emergence of clinical resistance to BRAF inhibitors, alone or in combination with MEK inhibitors, limits clinical responses in melanoma. Inhibiting HSP90 offers an approach to simultaneously interfere with multiple resistance mechanisms. Using the HSP90 inhibitor AT13387, which is currently in clinical trials, we investigated the potential of HSP90 inhibition to overcome or delay the emergence of resistance to these kinase inhibitors in melanoma models. In vitro, treating vemurafenib-sensitive cells (A375 or SK-MEL-28) with a combination of AT13387 and vemurafenib prevented colony growth under conditions in which vemurafenib treatment alone generated resistant colonies. In vivo, when AT13387 was combined with vemurafenib in a SK-MEL-28, vemurafenib-sensitive model, no regrowth of tumors was observed over 5 months, although 2 of 7 tumors in the vemurafenib monotherapy group relapsed in this time. Together, these data suggest that the combination of these agents can delay the emergence of resistance. Cell lines with acquired vemurafenib resistance, derived from these models (A375R and SK-MEL-28R) were also sensitive to HSP90 inhibitor treatment; key clients were depleted, apoptosis was induced, and growth in 3D culture was inhibited. Similar effects were observed in cell lines with acquired resistance to both BRAF and MEK inhibitors (SK-MEL-28RR, WM164RR, and 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combating resistance to BRAF and MEK inhibition in melanoma. Moreover, frontline combination of these agents with an HSP90 inhibitor could delay the emergence of resistance, providing a strong rationale for clinical investigation of such combinations in BRAF-mutated melanoma.

  13. Genome-wide dissection of AP2/ERF and HSP90 gene families in five legumes and expression profiles in chickpea and pigeonpea.

    PubMed

    Agarwal, Gaurav; Garg, Vanika; Kudapa, Himabindu; Doddamani, Dadakhalandar; Pazhamala, Lekha T; Khan, Aamir W; Thudi, Mahendar; Lee, Suk-Ha; Varshney, Rajeev K

    2016-07-01

    APETALA2/ethylene response factor (AP2/ERF) and heat-shock protein 90 (HSP90) are two significant classes of transcription factor and molecular chaperone proteins which are known to be implicated under abiotic and biotic stresses. Comprehensive survey identified a total of 147 AP2/ERF genes in chickpea, 176 in pigeonpea, 131 in Medicago, 179 in common bean and 140 in Lotus, whereas the number of HSP90 genes ranged from 5 to 7 in five legumes. Sequence alignment and phylogenetic analyses distinguished AP2, ERF, DREB, RAV and soloist proteins, while HSP90 proteins segregated on the basis of their cellular localization. Deeper insights into the gene structure allowed ERF proteins to be classified into AP2s based on DNA-binding domains, intron arrangements and phylogenetic grouping. RNA-seq and quantitative real-time PCR (qRT-PCR) analyses in heat-stressed chickpea as well as Fusarium wilt (FW)- and sterility mosaic disease (SMD)-stressed pigeonpea provided insights into the modus operandi of AP2/ERF and HSP90 genes. This study identified potential candidate genes in response to heat stress in chickpea while for FW and SMD stresses in pigeonpea. For instance, two DREB genes (Ca_02170 and Ca_16631) and three HSP90 genes (Ca_23016, Ca_09743 and Ca_25602) in chickpea can be targeted as potential candidate genes. Similarly, in pigeonpea, a HSP90 gene, C.cajan_27949, was highly responsive to SMD in the resistant genotype ICPL 20096, can be recommended for further functional validation. Also, two DREB genes, C.cajan_41905 and C.cajan_41951, were identified as leads for further investigation in response to FW stress in pigeonpea. PMID:26800652

  14. Studies on lipids and the activity of Na,K-ATPase in lens fibre cells.

    PubMed Central

    Dean, W L; Delamere, N A; Borchman, D; Moseley, A E; Ahuja, R P

    1996-01-01

    Na,K-ATPase was studied in the two cell types that make up the lens of the eye. Membrane material was isolated from lens fibre cells, which make up the bulk of the lens cell mass, and also from lens epithelial cells, which are present only as a monolayer on the anterior lens surface. Judged by immunoblotting, greater amounts of Na,K-ATPase alpha1 and beta1 polypeptides were found in fibre cell membrane material than in epithelial cell membrane material. However, the NA,K-ATPase activity in epithelial cell membrane material was 20 times that measured in fibre cell membrane material. In 86Rb uptake experiments with intact lenses, ouabain-inhibitable 86Rb uptake was observed for lens epithelium but not for lens fibres. These findings are consistent with a low Na,K-ATPase activity in lens fibre cells even though these cells express a considerable amount of Na,K-ATPase alpha1 and beta1 polypeptides. The lipid composition of lens fibre cell membranes causes them to be more ordered than epithelial cell membranes; this was confirmed by measurements of the infrared CH2 symmetric stretching band frequency. Because lipid composition can influence Na,K-ATPase activity, experiments were conducted to determine whether the activity of Na,K-ATPase alpha1 beta1 is inhibited by lens fibre lipid. However, no significant difference in Na,K-ATPase activity was detected when Na,K-ATPase alpha1 beta1 was purified from rabbit kidney and then reconstituted with lipid that had been isolated from either lens epithelium or lens fibre cells. These studies indicate that lens fibre cells contain both Na,K-ATPase alpha1 and beta1 polypeptides but have low Na,K-ATPase activity. However, the results do not support the notion that this is due to the lipid composition of lens fibre cell membranes. PMID:8615795

  15. [ATPase activity of the guinea pig central nervous system tissue with experimental allergic encephalomyelitis].

    PubMed

    Metal'nikova, N P; Terlets'ka, Ia T; Belik, Ia V; Chepurko, V N

    1977-01-01

    It is established that the activity of Na+, K+ = ATPase and Mg2+, Ca2+ = ATPase lowers significantly at the paralytic stage of the disease. At earlier stages of the disease preceding the appearance of peculiar neurological symptoms (the seventh and fourteenth days of the incubation period) a decrease of the Mg2+, Ca2+ = ATPase activity was observed in the brain and spinal cord, whereas the Na+, K+ = ATPase activity in the brain remained at the control level up to the appearance of the disease clearly developed symptoms. The Na+, K+ = ATPase activity of the brain on the fourteenth day of the incubation period corresponded to the level of the activity at the paralytic stage of the disease.

  16. Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90.

    PubMed

    Zhang, Yan; Dorey, Stephan; Swiderski, Michal; Jones, Jonathan D G

    2004-10-01

    The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR). The same phenotype is seen after expression of a full-length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P-loop motif of the NB domain abolish the HR. Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.

  17. Targeting β-tubulin:CCT-β complexes incurs Hsp90- and VCP-related protein degradation and induces ER stress-associated apoptosis by triggering capacitative Ca2+ entry, mitochondrial perturbation and caspase overactivation

    PubMed Central

    Lin, Y-F; Lee, Y-F; Liang, P-H

    2012-01-01

    We have previously demonstrated that interrupting the protein–protein interaction (PPI) of β-tubulin:chaperonin-containing TCP-1β (CCT-β) induces the selective killing of multidrug-resistant cancer cells due to CCT-β overexpression. However, the molecular mechanism has not yet been identified. In this study, we found that CCT-β interacts with a myriad of intracellular proteins involved in the cellular functions of the endoplasmic reticulum (ER), mitochondria, cytoskeleton, proteasome and apoptosome. Our data show that the targeted cells activate both the heat-shock protein 90 (Hsp90)-associated protein ubiquitination/degradation pathway to eliminate misfolded proteins in the cytoplasm and the valosin-containing protein (VCP)-centered ER-associated protein degradation pathway to reduce the excessive levels of unfolded polypeptides from the ER, thereby mitigating ER stress, at the onset of β-tubulin:CCT-β complex disruption. Once ER stress is expanded, ER stress-associated apoptotic signaling is enforced, as exhibited by cellular vacuolization and intracellular Ca2+ release. Furthermore, the elevated intracellular Ca2+ levels resulting from capacitative Ca2+ entry augments apoptotic signaling by provoking mitochondrial perturbation and caspase overactivation in the targeted cells. These findings not only provide a detailed picture of the apoptotic signaling cascades evoked by targeting the β-tubulin:CCT-β complex but also demonstrate a strategy to combat malignancies with chemoresistance to Hsp90- and VCP-related anticancer agents. PMID:23190606

  18. Effect of hindlimb unweighting on single soleus fiber maximal shortening velocity and ATPase activity

    NASA Technical Reports Server (NTRS)

    Mcdonald, K. S.; Fitts, R. H.

    1993-01-01

    The effect of hindlimb unweighting (HU) for 1 to 3 wks on the shortening velocity of a soleus fiber, its ATPase content, and the relative contents of the slow and fast myosin was investigated by measuring fiber force, V(0), ATPase activity, and myosin content in SDS protein profiles of a single rat soleus fiber suspended between a motor arm and a transducer. It was found that HU induces a progressive increase in fiber V(0) that is likely caused, at least in part, by an increase in the fiber's myofibrillar ATPase activity. The HU-induced increases in V(0) and ATPase were associated with the presence of a greater percentage of fast type IIa fibers. However, a large population of fibers after 1, 2, and 3 wks of HU showed increases in V(0) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers.

  19. Chromosomal assignment of six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) in four species of the genus Equus.

    PubMed

    Vidale, Pamela; Piras, Francesca M; Nergadze, Solomon G; Bertoni, Livia; Verini-Supplizi, Andrea; Adelson, David; Guérin, Gérard; Giulotto, Elena

    2011-01-01

    We mapped six genes (EIF4G3, HSP90, RBBP6, IL8, TERT, and TERC) on the chromosomes of Equus caballus, Equus asinus, Equus grevyi, and Equus burchelli by fluorescence in situ hybridization. Our results add six type I markers to the cytogenetic map of these species and provide new information on the comparative genomics of the genus Equus.

  20. The combination of Hsp90 inhibitor 17AAG and heavy-ion irradiation provides effective tumor control in human lung cancer cells

    PubMed Central

    Hirakawa, Hirokazu; Fujisawa, Hiroshi; Masaoka, Aya; Noguchi, Miho; Hirayama, Ryoichi; Takahashi, Momoko; Fujimori, Akira; Okayasu, Ryuichi

    2015-01-01

    Hsp90 inhibitors have become well-studied antitumor agents for their selective property against tumors versus normal cells. The combined treatment of Hsp90 inhibitor and conventional photon radiation also showed more effective tumor growth delay than radiation alone. However, little is known regarding the combined treatment of Hsp90 inhibitor and heavy-ion irradiation. In this study, SQ5 human lung tumor cells were used in vitro for clonogenic cell survival and in vivo for tumor growth delay measurement using a mouse xenograft model after 17-allylamino-17-demethoxygeldanamycin (17AAG) pretreatment and carbon ion irradiation. Repair of DNA double strand breaks (DSBs) was also assessed along with expressions of DSB repair-related proteins. Cell cycle analysis after the combined treatment was also performed. The combined treatment of 17AAG and carbon ions revealed a promising treatment option in both in vitro and in vivo studies. One likely cause of this effectiveness was shown to be the inhibition of homologous recombination repair by 17AAG. The more intensified G2 cell cycle delay was also associated with the combined treatment when compared with carbon ion treatment alone. Our findings indicate that the combination of Hsp90 inhibition and heavy-ion irradiation provides a new effective therapeutic alternative for treatment of solid tumors. PMID:25582113