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

Molecular Chaperone Hsp90 Is a Therapeutic Target for Noroviruses

PubMed Central

Urena, Luis; Gonzalez-Hernandez, Mariam B.; Choi, Jayoung; de Rougemont, Alexis; Rocha-Pereira, Joana; Neyts, Johan; Hwang, Seungmin; Wobus, Christiane E.

2015-01-01

ABSTRACT Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5′ and 3′ extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections. IMPORTANCE HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients. PMID:25855731

Targeting Heat Shock Protein 90 for the Treatment of Malignant Pheochromocytoma

PubMed Central

Giubellino, Alessio; Sourbier, Carole; Lee, Min-Jung; Scroggins, Brad; Bullova, Petra; Landau, Michael; Ying, Weiwen; Neckers, Len

2013-01-01

Metastatic pheochromocytoma represents one of the major clinical challenges in the field of neuroendocrine oncology. Recent molecular characterization of pheochromocytoma suggests new treatment options with targeted therapies. In this study we investigated the 90 kDa heat shock protein (Hsp90) as a potential therapeutic target for advanced pheochromocytoma. Both the first generation, natural product Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), and the second-generation synthetic Hsp90 inhibitor STA-9090 (ganetespib) demonstrated potent inhibition of proliferation and migration of pheochromocytoma cell lines and induced degradation of key Hsp90 clients. Furthermore, ganetespib induced dose-dependent cytotoxicity in primary pheochromocytoma cells. Using metastatic models of pheochromocytoma, we demonstrate the efficacy of 17-AAG and ganetespib in reducing metastatic burden and increasing survival. Levels of Hsp70 in plasma from the xenograft studies served as a proximal biomarker of drug treatment. Our study suggests that targeting Hsp90 may benefit patients with advanced pheochromocytoma. PMID:23457505

Sorafenib prevents liver fibrosis in a non-alcoholic steatohepatitis (NASH) rodent model

PubMed Central

Stefano, J.T.; Pereira, I.V.A.; Torres, M.M.; Bida, P.M.; Coelho, A.M.M.; Xerfan, M.P.; Cogliati, B.; Barbeiro, D.F.; Mazo, D.F.C.; Kubrusly, M.S.; D'Albuquerque, L.A.C.; Souza, H.P.; Carrilho, F.J.; Oliveira, C.P.

2015-01-01

Liver fibrosis occurring as an outcome of non-alcoholic steatohepatitis (NASH) can precede the development of cirrhosis. We investigated the effects of sorafenib in preventing liver fibrosis in a rodent model of NASH. Adult Sprague-Dawley rats were fed a choline-deficient high-fat diet and exposed to diethylnitrosamine for 6 weeks. The NASH group (n=10) received vehicle and the sorafenib group (n=10) received 2.5 mg·kg-1·day-1 by gavage. A control group (n=4) received only standard diet and vehicle. Following treatment, animals were sacrificed and liver tissue was collected for histologic examination, mRNA isolation, and analysis of mitochondrial function. Genes related to fibrosis (MMP9, TIMP1, TIMP2), oxidative stress (HSP60, HSP90, GST), and mitochondrial biogenesis (PGC1α) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Liver mitochondrial oxidation activity was measured by a polarographic method, and cytokines by enzyme-linked immunosorbent assay (ELISA). Sorafenib treatment restored mitochondrial function and reduced collagen deposition by nearly 63% compared to the NASH group. Sorafenib upregulated PGC1α and MMP9 and reduced TIMP1 and TIMP2 mRNA and IL-6 and IL-10 protein expression. There were no differences in HSP60, HSP90 and GST expression. Sorafenib modulated PGC1α expression, improved mitochondrial respiration and prevented collagen deposition. It may, therefore, be useful in the treatment of liver fibrosis in NASH. PMID:25714891

Inhibition of Mitochondrial Matrix Chaperones and Antiapoptotic Bcl-2 Family Proteins Empower Antitumor Therapeutic Responses.

PubMed

Karpel-Massler, Georg; Ishida, Chiaki Tsuge; Bianchetti, Elena; Shu, Chang; Perez-Lorenzo, Rolando; Horst, Basil; Banu, Matei; Roth, Kevin A; Bruce, Jeffrey N; Canoll, Peter; Altieri, Dario C; Siegelin, Markus D

2017-07-01

Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof of concept that targeting the mitochondrial Hsp90 chaperone network (mtHsp90) and inhibition of Bcl-2, Bcl-xL, and Mcl-1 is sufficient to elicit synthetic lethality in tumors recalcitrant to therapy. Our analyses focused on BH3 mimetics that are broad acting (ABT263 and obatoclax) or selective (ABT199, WEHI-539, and A1210477), along with the established mitochondrial matrix chaperone inhibitor gamitrinib-TPP. Drug combinations were tested in various therapy-resistant tumors in vitro and in vivo in murine model systems of melanoma, triple-negative breast cancer, and patient-derived orthotopic xenografts (PDX) of human glioblastoma. We found that combining BH3 mimetics and gamitrinib-TPP blunted cellular proliferation in a synergistic manner by massive activation of intrinsic apoptosis. In like manner, suppressing either Bcl-2, Bcl-xL, or Mcl-1 recapitulated the effects of BH3 mimetics and enhanced the effects of gamitrinib-TPP. Mechanistic investigations revealed that gamitrinib-TPP activated a PERK-dependent integrated stress response, which activated the proapoptotic BH3 protein Noxa and its downstream targets Usp9X and Mcl-1. Notably, in the PDX glioblastoma and BRAFi-resistant melanoma models, this drug combination safely and significantly extended host survival. Our results show how combining mitochondrial chaperone and Bcl-2 family inhibitors can synergize to safely degrade the growth of tumors recalcitrant to other treatments. Cancer Res; 77(13); 3513-26. ©2017 AACR . ©2017 American Association for Cancer Research.

Impaired Mitochondrial Dynamics Underlie Axonal Defects in Hereditary Spastic Paraplegias.

PubMed

Denton, Kyle; Mou, Yongchao; Xu, Chong-Chong; Shah, Dhruvi; Chang, Jaerak; Blackstone, Craig; Li, Xue-Jun

2018-05-02

Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.

Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms.

PubMed

Chen, Bin; Zhong, Daibin; Monteiro, Antónia

2006-06-17

HSP90 proteins are essential molecular chaperones involved in signal transduction, cell cycle control, stress management, and folding, degradation, and transport of proteins. HSP90 proteins have been found in a variety of organisms suggesting that they are ancient and conserved. In this study we investigate the nuclear genomes of 32 species across all kingdoms of organisms, and all sequences available in GenBank, and address the diversity, evolution, gene structure, conservation and nomenclature of the HSP90 family of genes across all organisms. Twelve new genes and a new type HSP90C2 were identified. The chromosomal location, exon splicing, and prediction of whether they are functional copies were documented, as well as the amino acid length and molecular mass of their polypeptides. The conserved regions across all protein sequences, and signature sequences in each subfamily were determined, and a standardized nomenclature system for this gene family is presented. The proeukaryote HSP90 homologue, HTPG, exists in most Bacteria species but not in Archaea, and it evolved into three lineages (Groups A, B and C) via two gene duplication events. None of the organellar-localized HSP90s were derived from endosymbionts of early eukaryotes. Mitochondrial TRAP and endoplasmic reticulum HSP90B separately originated from the ancestors of HTPG Group A in Firmicutes-like organisms very early in the formation of the eukaryotic cell. TRAP is monophyletic and present in all Animalia and some Protista species, while HSP90B is paraphyletic and present in all eukaryotes with the exception of some Fungi species, which appear to have lost it. Both HSP90C (chloroplast HSP90C1 and location-undetermined SP90C2) and cytosolic HSP90A are monophyletic, and originated from HSP90B by independent gene duplications. HSP90C exists only in Plantae, and was duplicated into HSP90C1 and HSP90C2 isoforms in higher plants. HSP90A occurs across all eukaryotes, and duplicated into HSP90AA and HSP90AB in vertebrates. Diplomonadida was identified as the most basal organism in the eukaryote lineage. The present study presents the first comparative genomic study and evolutionary analysis of the HSP90 family of genes across all kingdoms of organisms. HSP90 family members underwent multiple duplications and also subsequent losses during their evolution. This study established an overall framework of information for the family of genes, which may facilitate and stimulate the study of this gene family across all organisms.

Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms

PubMed Central

Chen, Bin; Zhong, Daibin; Monteiro, Antónia

2006-01-01

Background HSP90 proteins are essential molecular chaperones involved in signal transduction, cell cycle control, stress management, and folding, degradation, and transport of proteins. HSP90 proteins have been found in a variety of organisms suggesting that they are ancient and conserved. In this study we investigate the nuclear genomes of 32 species across all kingdoms of organisms, and all sequences available in GenBank, and address the diversity, evolution, gene structure, conservation and nomenclature of the HSP90 family of genes across all organisms. Results Twelve new genes and a new type HSP90C2 were identified. The chromosomal location, exon splicing, and prediction of whether they are functional copies were documented, as well as the amino acid length and molecular mass of their polypeptides. The conserved regions across all protein sequences, and signature sequences in each subfamily were determined, and a standardized nomenclature system for this gene family is presented. The proeukaryote HSP90 homologue, HTPG, exists in most Bacteria species but not in Archaea, and it evolved into three lineages (Groups A, B and C) via two gene duplication events. None of the organellar-localized HSP90s were derived from endosymbionts of early eukaryotes. Mitochondrial TRAP and endoplasmic reticulum HSP90B separately originated from the ancestors of HTPG Group A in Firmicutes-like organisms very early in the formation of the eukaryotic cell. TRAP is monophyletic and present in all Animalia and some Protista species, while HSP90B is paraphyletic and present in all eukaryotes with the exception of some Fungi species, which appear to have lost it. Both HSP90C (chloroplast HSP90C1 and location-undetermined SP90C2) and cytosolic HSP90A are monophyletic, and originated from HSP90B by independent gene duplications. HSP90C exists only in Plantae, and was duplicated into HSP90C1 and HSP90C2 isoforms in higher plants. HSP90A occurs across all eukaryotes, and duplicated into HSP90AA and HSP90AB in vertebrates. Diplomonadida was identified as the most basal organism in the eukaryote lineage. Conclusion The present study presents the first comparative genomic study and evolutionary analysis of the HSP90 family of genes across all kingdoms of organisms. HSP90 family members underwent multiple duplications and also subsequent losses during their evolution. This study established an overall framework of information for the family of genes, which may facilitate and stimulate the study of this gene family across all organisms. PMID:16780600

HSP72 is a mitochondrial stress sensor critical for Parkin action, oxidative metabolism, and insulin sensitivity in skeletal muscle.

PubMed

Drew, Brian G; Ribas, Vicente; Le, Jamie A; Henstridge, Darren C; Phun, Jennifer; Zhou, Zhenqi; Soleymani, Teo; Daraei, Pedram; Sitz, Daniel; Vergnes, Laurent; Wanagat, Jonathan; Reue, Karen; Febbraio, Mark A; Hevener, Andrea L

2014-05-01

Increased heat shock protein (HSP) 72 expression in skeletal muscle prevents obesity and glucose intolerance in mice, although the underlying mechanisms of this observation are largely unresolved. Herein we show that HSP72 is a critical regulator of stress-induced mitochondrial triage signaling since Parkin, an E3 ubiquitin ligase known to regulate mitophagy, was unable to ubiquitinate and control its own protein expression or that of its central target mitofusin (Mfn) in the absence of HSP72. In wild-type cells, we show that HSP72 rapidly translocates to depolarized mitochondria prior to Parkin recruitment and immunoprecipitates with both Parkin and Mfn2 only after specific mitochondrial insult. In HSP72 knockout mice, impaired Parkin action was associated with retention of enlarged, dysmorphic mitochondria and paralleled by reduced muscle respiratory capacity, lipid accumulation, and muscle insulin resistance. Reduced oxygen consumption and impaired insulin action were recapitulated in Parkin-null myotubes, confirming a role for the HSP72-Parkin axis in the regulation of muscle insulin sensitivity. These data suggest that strategies to maintain HSP72 may provide therapeutic benefit to enhance mitochondrial quality and insulin action to ameliorate complications associated with metabolic diseases, including type 2 diabetes.

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

PubMed Central

Presley, Tennille; Vedam, Kaushik; Liu, Xiaoping; Zweier, Jay L.

2009-01-01

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO2. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO2, but it was unaffected at low pO2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42°C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O2 binding at CcO at complex IV. PMID:19412660

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

Inhibition of stress-inducible HSP70 impairs mitochondrial proteostasis and function.

PubMed

Leu, Julia I-Ju; Barnoud, Thibaut; Zhang, Gao; Tian, Tian; Wei, Zhi; Herlyn, Meenhard; Murphy, Maureen E; George, Donna L

2017-07-11

Protein quality control is an important component of survival for all cells. The use of proteasome inhibitors for cancer therapy derives from the fact that tumor cells generally exhibit greater levels of proteotoxic stress than do normal cells, and thus cancer cells tend to be more sensitive to proteasome inhibition. However, this approach has been limited in some cases by toxicity to normal cells. Recently, the concept of inhibiting proteostasis in organelles for cancer therapy has been advanced, in part because it is predicted to have reduced toxicity for normal cells. Here we demonstrate that a fraction of the major stress-induced chaperone HSP70 (also called HSPA1A or HSP72, but hereafter HSP70) is abundantly present in mitochondria of tumor cells, but is expressed at quite low or undetectable levels in mitochondria of most normal tissues and non-tumor cell lines. We show that treatment of tumor cells with HSP70 inhibitors causes a marked change in mitochondrial protein quality control, loss of mitochondrial membrane potential, reduced oxygen consumption rate, and loss of ATP production. We identify several nuclear-encoded mitochondrial proteins, including polyadenylate binding protein-1 (PABPC1), which exhibit decreased abundance in mitochondria following treatment with HSP70 inhibitors. We also show that targeting HSP70 function leads to reduced levels of several mitochondrial-encoded RNA species that encode components of the electron transport chain. Our data indicate that small molecule inhibitors of HSP70 represent a new class of organelle proteostasis inhibitors that impair mitochondrial function in cancer cells, and therefore constitute novel therapeutics.

Hsp90 Is a Novel Target Molecule of CDDO-Me in Inhibiting Proliferation of Ovarian Cancer Cells.

PubMed

Qin, Dong-Jun; Tang, Cai-Xia; Yang, Li; Lei, Hu; Wei, Wei; Wang, Ying-Ying; Ma, Chun-Min; Gao, Feng-Hou; Xu, Han-Zhang; Wu, Ying-Li

2015-01-01

Synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO-Me) has been shown as a promising agent against ovarian cancer. However, the underlying mechanism is not well understood. Here, we demonstrate that CDDO-Me directly interacts with Hsp90 in cells by cellular thermal shift assay. CDDO-Me treatment leads to upregulation of Hsp70 and degradation of Hsp90 clients (ErbB2 and Akt), indicating the inhibition of Hsp90 by CDDO-Me in cells. Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells. Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition. This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90. This study identifies Hsp90 as a novel target protein of CDDO-Me, and provides a novel insight into the mechanism of action of CDDO-Me in ovarian cancer cells.

DMPS reverts morphologic and mitochondrial damage in OK cells exposed to toxic concentrations of HgCl2.

PubMed

Carranza-Rosales, Pilar; Guzmán-Delgado, Nancy E; Cruz-Vega, Delia E; Balderas-Rentería, Isaías; Gandolfi, A Jay

2007-05-01

Mercuric chloride (HgCl(2)) is a highly toxic compound, which can cause nephrotoxic damage. In the present study effects of HgCl(2) on mitochondria integrity and energy metabolism, as well as antidotal effects of 2,3-dimercaptopropane-1-sulfonate (DMPS) were investigated in the opossum kidney derived cell line (OK). OK cell monolayers were incubated during 0, 1, 3, 6, and 9 h in serum-free culture medium containing 15 microM HgCl(2), either in the absence or in the presence of 60 microM DMPS in a 1:4 ratio. Intracellular ATP content, MTT reduction, and HSP70/HSP90 induction were studied; confocal, transmission electron microscopy, and light microscopy studies were also performed. For confocal analysis, a mitochondrial selective probe (MitoTracker Red CMXH2Ros) was used. Antioxidant activity of DMPS was also studied by the scavenging of the free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) technique. A decrease of ATP content, an impaired ability to reduce tetrazolium, and dramatic changes on cellular and mitochondrial morphology, and energetic levels were found after either 6 or 9 h of HgCl(2) exposure. Increased expression of HSP90 and HSP70 were also seen. When OK cells were co-incubated with HgCl(2) and DMPS, cellular morphology, viability, intracellular ATP, and mitochondrial membrane potential were partially restored; a protective effect on mitochondrial morphology was also seen. DMPS also showed potent antioxidant activity in vitro. Mitochondrial protection could be the cellular mechanism mediated by DMPS in OK cells exposed to a toxic concentration of HgCl(2).

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.

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

PubMed Central

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

2014-01-01

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

Protein chaperones: a composition of matter review (2008 – 2013)

PubMed Central

Taldone, Tony; Patel, Hardik J; Bolaender, Alexander; Patel, Maulik R; Chiosis, Gabriela

2014-01-01

Introduction Heat shock proteins (Hsps) are proteins with important functions in regulating disease phenotypes. Historically, Hsp90 has first received recognition as a target in cancer, with consequent efforts extending its potential role to other diseases. Hsp70 has also attracted interest as a therapeutic target for its role as a co-chaperone to Hsp90 as well as its own anti-apoptotic roles. Areas covered Herein, patents from 2008 to 2013 are reviewed to identify those that disclose composition of matter claimed to inhibit Hsp90 or Hsp70. Expert opinion For Hsp90, there has been considerable creativity in the discovery of novel pharmacophores that fall outside the three initially discovered scaffolds (i.e., ansamycins, resorcinols and purines). Nonetheless, much of the patent literature appears to build on previously reported structure activity relationship through slight modifications of Hsp90 inhibitor space by finding weaknesses in existing patents. The major goal of future development of Hsp90 inhibitors is not necessarily identifying better molecules but rather understanding how to rationally use these agents in the clinic. The development of Hsp70 inhibitors has lagged behind. It will require a more concerted effort from the drug discovery community in order to begin to realize the potential of this target. PMID:24742089

Hexavalent chromium targets mitochondrial respiratory chain complex I to induce reactive oxygen species-dependent caspase-3 activation in L-02 hepatocytes.

PubMed

Xiao, Fang; Li, Yanhong; Dai, Lu; Deng, Yuanyuan; Zou, Yue; Li, Peng; Yang, Yuan; Zhong, Caigao

2012-09-01

Hexavalent chromium [Cr(VI)], which is used for various industrial applications, such as leather tanning and chroming, can cause a number of human diseases including inflammation and cancer. Cr(VI) exposure leads to severe damage to the liver, but the mechanisms involved in Cr(VI)-mediated toxicity in the liver are unclear. The present study provides evidence that Cr(VI) enhances reactive oxygen species (ROS) accumulation by inhibiting the mitochondrial respiratory chain complex (MRCC) I. Cr(VI) did not affect the expression levels of antioxidative proteins such as superoxide dismutase (SOD), catalase and thioredoxin (Trx), indicating that the antioxidative system was not involved in Cr(VI)-induced ROS accumulation. We found that ROS mediated caspase-3 activation partially depends on the downregulation of the heat shock protein (HSP) 70 and 90. In order to confirm our hypothesis that ROS plays a key role in Cr(VI)-mediated cytotoxicity, we used N-acetylcysteine (NAC) to inhibit the accumulation of ROS. NAC successfully blocked the inhibition of HSP70 and HSP90 as well as the activation of caspase-3, suggesting that ROS is essential in Cr(VI)-induced caspase-3 activation. By applying different MRCC substrates as electron donors, we also confirmed that Cr(VI) could accept the electrons leaked from MRCC I and the reduction occurs at MRCC I. In conclusion, the present study demonstrates that Cr(VI) induces ROS-dependent caspase-3 activation by inhibiting MRCC I activity, and MRCC I has been identified as a new target and a new mechanism for the apoptosis-inducing activity displayed by Cr(VI).

Targeting mitochondrial STAT3 with the novel phospho-valproic acid (MDC-1112) inhibits pancreatic cancer growth in mice.

PubMed

Mackenzie, Gerardo G; Huang, Liqun; Alston, Ninche; Ouyang, Nengtai; Vrankova, Kvetoslava; Mattheolabakis, George; Constantinides, Panayiotis P; Rigas, Basil

2013-01-01

New agents are needed to treat pancreatic cancer, one of the most lethal human malignancies. We synthesized phospho-valproic acid, a novel valproic acid derivative, (P-V; MDC-1112) and evaluated its efficacy in the control of pancreatic cancer. P-V inhibited the growth of human pancreatic cancer xenografts in mice by 60%-97%, and 100% when combined with cimetidine. The dominant molecular target of P-V was STAT3. P-V inhibited the phosphorylation of JAK2 and Src, and the Hsp90-STAT3 association, suppressing the activating phosphorylation of STAT3, which in turn reduced the expression of STAT3-dependent proteins Bcl-xL, Mcl-1 and survivin. P-V also reduced STAT3 levels in the mitochondria by preventing its translocation from the cytosol, and enhanced the mitochondrial levels of reactive oxygen species, which triggered apoptosis. Inhibition of mitochondrial STAT3 by P-V was required for its anticancer effect; mitochondrial STAT3 overexpression rescued animals from the tumor growth inhibition by P-V. Our results indicate that P-V is a promising candidate drug against pancreatic cancer and establish mitochondrial STAT3 as its key molecular target.

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10.

PubMed Central

Dubaquié, Y; Looser, R; Fünfschilling, U; Jenö, P; Rospert, S

1998-01-01

The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo. PMID:9774331

Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9.

PubMed

Rozenberg, Perri; Ziporen, Lea; Gancz, Dana; Saar-Ray, Moran; Fishelson, Zvi

2018-02-02

Cancer cells are commonly more resistant to cell death activated by the membranolytic protein complex C5b-9. Several surface-expressed and intracellular proteins that protect cells from complement-dependent cytotoxicity (CDC) have been identified. In this study, we investigated the function of heat shock protein 90 (Hsp90), an essential and ubiquitously expressed chaperone, overexpressed in cancer cells, in C5b-9-induced cell death. As shown, inhibition of Hsp90 with geldanamycin or radicicol is enhancing sensitivity of K562 erythroleukemia cells to CDC. Similarly, Hsp90 inhibition confers in Ramos B cell lymphoma cells elevated sensitivity to treatment with rituximab and complement. C5b-9 deposition is elevated on geldanamycin-treated cells. Purified Hsp90 binds directly to C9 and inhibits zinc-induced C9 polymerization, indicating that Hsp90 may act directly on the C5b-9 complex. Mortalin, also known as stress protein 70 or GRP75, is a mitochondrial chaperone that confers resistance to CDC. The postulated cooperation between Hsp90 and mortalin in protection from CDC was tested. Geldanamycin failed to sensitize toward CDC cells with knocked down mortalin. Direct binding of Hsp90 to mortalin was shown by co-immunoprecipitation in cell extracts after triggering with complement as well as by using purified recombinant proteins. These results provide an insight into the protective mechanisms utilized by cancer cells to evade CDC. They suggest that Hsp90 protects cells from CDC by inhibiting, together with mortalin, C5b-9 assembly and/or stability at the plasma membrane.

Gamendazole, an orally active indazole carboxylic acid male contraceptive agent, targets HSP90AB1 (HSP90BETA) and EEF1A1 (eEF1A), and stimulates Il1a transcription in rat Sertoli cells.

PubMed

Tash, Joseph S; Chakrasali, Ramappa; Jakkaraj, Sudhakar R; Hughes, Jennifer; Smith, S Kendall; Hornbaker, Kaori; Heckert, Leslie L; Ozturk, Sedide B; Hadden, M Kyle; Kinzy, Terri Goss; Blagg, Brian S J; Georg, Gunda I

2008-06-01

Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1. Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.

Combined pharmacophore and structure-guided studies to identify diverse HSP90 inhibitors.

PubMed

Sanam, Ramadevi; Tajne, Sunita; Gundla, Rambabu; Vadivelan, S; Machiraju, Pavan Kumar; Dayam, Raveendra; Narasu, Lakshmi; Jagarlapudi, Sarma; Neamati, Nouri

2010-02-26

Heat Shock Protein 90 (HSP90), an ATP-dependent molecular chaperone, has emerged as a promising target in the treatment of cancer. Inhibition of HSP90 represents a new target of antitumor therapy, since it may influence many specific signaling pathways. Many HSP90 inhibitors bind to the ATP-binding pocket, inhibit chaperone function, resulting in cell death. Recent clinical trials for treatment of cancer have put HSP90's importance into focus and have highlighted the need for full scale research into HSP90 related pathways. Here we report five novel HSP90 inhibitors which were identified by using pharmacophore models and docking studies. We used highly discriminative pharmacophore model as a 3D query to search against database of approximately 1 M compounds and cluster analysis results yielded 455 compounds which were further subjected for docking. Glide docking studies suggested 122 compounds as in silico hits and these compounds were further selected for the cytotoxicity assay in the HSP90-over expressing SKBr3 cell line. Of the 122 compounds tested, 5 compounds inhibited cell growth with an IC(50) value less than 50 microM. Copyright 2009 Elsevier Inc. All rights reserved.

HSP27 knockdown produces synergistic induction of apoptosis by HSP90 and kinase inhibitors in glioblastoma multiforme.

PubMed

Belkacemi, Louiza; Hebb, Matthew O

2014-09-01

The heat-shock proteins HSP27 and HSP90 perpetuate the malignant nature of glioblastoma multiforme (GBM) and offer promise as targets for novel cancer therapeutics. The present study sought to define synergistic antitumor benefits of concurrent HSP27-knockdown and the HSP90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) or, comparatively, the non-selective kinase inhibitor, staurosporine, in GBM cells. Dose-response relations were determined for 17-AAG and staurosporine in three GBM cell lines. HSP27-targeted siRNA was administered alone or in combination with subtherapeutic concentrations of each drug and cells were evaluated for viability, proliferation and apoptosis. Adjuvant HSP27 knockdown with 17-AAG or staurosporine produced marked and synergistic decrease in GBM cell viability and proliferation, with robust elevation of apoptotic fractions and caspase-3 activation. HSP27 knockdown confers potent chemosensitization of GBM cells. These novel data support the development of HSP-targeting strategies and, specifically, anti-HSP27 agents for the treatment of GBM. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Clinical Development of Gamitrinib, a Novel Mitochondrial-Targeted Small Molecule Hsp90 Inhibitor

DTIC Science & Technology

2015-09-01

Group 2 and Group 3 animals examined at the end of the 7-repeated doses was comparable to those in control Group 1 animals (Figure 2). (7) Despite... posttest (for more than two- group comparisons) using a GraphPad software package (Prism 6.0) for Windows. Data are expressed as mean ± SD or mean ± SEM...Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. J R Stat Soc Series B Stat

Anticancer Inhibitors of Hsp90 Function: Beyond the Usual Suspects

PubMed Central

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

2018-01-01

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

Symmetry broken and rebroken during the ATP hydrolysis cycle of the mitochondrial Hsp90 TRAP1

PubMed Central

Elnatan, Daniel; Betegon, Miguel; Liu, Yanxin; Ramelot, Theresa; Kennedy, Michael A; Agard, David A

2017-01-01

Hsp90 is a homodimeric ATP-dependent molecular chaperone that remodels its substrate ‘client’ proteins, facilitating their folding and activating them for biological function. Despite decades of research, the mechanism connecting ATP hydrolysis and chaperone function remains elusive. Particularly puzzling has been the apparent lack of cooperativity in hydrolysis of the ATP in each protomer. A crystal structure of the mitochondrial Hsp90, TRAP1, revealed that the catalytically active state is closed in a highly strained asymmetric conformation. This asymmetry, unobserved in other Hsp90 homologs, is due to buckling of one of the protomers and is most pronounced at the broadly conserved client-binding region. Here, we show that rather than being cooperative or independent, ATP hydrolysis on the two protomers is sequential and deterministic. Moreover, dimer asymmetry sets up differential hydrolysis rates for each protomer, such that the buckled conformation favors ATP hydrolysis. Remarkably, after the first hydrolysis, the dimer undergoes a flip in the asymmetry while remaining in a closed state for the second hydrolysis. From these results, we propose a model where direct coupling of ATP hydrolysis and conformational flipping rearranges client-binding sites, providing a paradigm of how energy from ATP hydrolysis can be used for client remodeling. DOI: http://dx.doi.org/10.7554/eLife.25235.001 PMID:28742020

Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata

PubMed Central

Singh-Babak, Sheena D.; Babak, Tomas; Diezmann, Stephanie; Hill, Jessica A.; Xie, Jinglin Lucy; Chen, Ying-Lien; Poutanen, Susan M.; Rennie, Robert P.; Heitman, Joseph; Cowen, Leah E.

2012-01-01

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential. PMID:22615574

Hsp90 C-Terminal Inhibitors Exhibit Antimigratory Activity by Disrupting the Hsp90α/Aha1 Complex in PC3-MM2 Cells

PubMed Central

2015-01-01

Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure–function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors. PMID:25402753

Quantitative proteomics reveals molecular mechanism of gamabufotalin and its potential inhibition on Hsp90 in lung cancer.

PubMed

Zhang, Liyuan; Yu, Zhenlong; Wang, Yan; Wang, Xiaobo; Zhang, Lianru; Wang, Chao; Yue, Qingxi; Wang, Xun; Deng, Sa; Huo, Xiaokui; Tian, Xiangge; Huang, Shanshan; Zhang, Baojing; Ma, Xiaochi

2016-11-22

Gamabufotalin (CS-6) is a major bufadienolide of Chansu, which shows desirable metabolic stability and less adverse effect in cancer therapy. CS-6 treatment inhibited the proliferation of NSCLC in a nanomolar range. And CS-6 could induce G2/M cell cycle arrest and apoptosis in A549 cells. However, its molecular mechanism in antitumor activity remains poorly understood. We employed a quantitative proteomics approach to identify the potential cellular targets of CS-6, and found 38 possible target-related proteins. Among them, 31 proteins were closely related in the protein-protein interaction network. One of the regulatory nodes in key pathways was occupied by Hsp90. Molecular docking revealed that CS-6 interacted with the ATP-binding sites of Hsp90. In addition, CS-6 inhibited the chaperone function of Hsp90 and reduced expression of Hsp90-dependent client proteins. Moreover, CS-6 markedly down-regulated the protein level of Hsp90 in tumor tissues of the xenograft mice. Taken together, our results suggest that CS-6 might be a novel inhibitor of Hsp90, and the possible network associated with CS-6 target-related proteins was constructed, which provided experimental evidence for the preclinical value of using CS-6 as an effective antitumor agent in treatment of NSCLC.

Characterization of HSP90 isoforms in transformed bovine leukocytes infected with Theileria annulata

PubMed Central

Kinnaird, Jane H.; Singh, Meetali; Gillan, Victoria; Weir, William; Calder, Ewen D. D.; Hostettler, Isabel; Shiels, Brian R.

2016-01-01

Summary HSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata‐infected counterpart was utilized to test the effects of geldanamycin and the derivative 17‐AAG. T. annulata‐infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17‐AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation. PMID:27649068

Acquired resistance to the Hsp90 inhibitor, ganetespib in KRAS mutant NSCLC is mediated via reactivation of the ERK–p90RSK–mTOR signaling network

PubMed Central

Chatterjee, Suman; Huang, Eric H.-B.; Christie, Ian; Kurland, Brenda F.; Burns, Timothy F.

2017-01-01

Approximately 25% of non-small cell lung cancer (NSCLC) patients have KRAS mutations and no effective therapeutic strategy exists for these patients. The use of Heat shock protein 90 (Hsp90) inhibitors in KRAS mutant NSCLC appeared to be a promising approach since these inhibitors target many KRAS downstream effectors, however, limited clinical efficacy has been observed due to resistance. Here, we examined the mechanism(s) of acquired resistance to the Hsp90 inhibitor, ganetespib, and identified novel and rationally devised Hsp90 inhibitor combinations which may prevent and overcome resistance to Hsp90 inhibitors. We derived KRAS mutant NSCLC ganetespib resistant (GR) cell lines to identify the resistance mechanism(s) and identified hyperactivation of RAF/MEK/ERK/RSK and PI3K/AKT/mTOR pathways as key resistance mechanisms. Furthermore, we found that GR cells are “addicted” to these pathways as ganetespib resistance lead to synthetic lethality to a dual PI3K/mTOR, a PI3K, or an ERK inhibitor. Interestingly, the levels and activity of a key activator of the mTOR pathway and an ERK downstream target, p90 ribosomal S6 kinase (RSK) were also increased in the GR cells. Genetic or pharmacologic inhibition of p90RSK in GR cells restored sensitivity to ganetespib, whereas p90RSK overexpression induced ganetespib resistance in naïve cells, validating p90RSK as a mediator of resistance and a novel therapeutic target. Our studies offer a way forward for Hsp90 inhibitors through the rational design of Hsp90 inhibitor combinations that may prevent and/or overcome resistance to Hsp90 inhibitors providing an effective therapeutic strategy for KRAS mutant NSCLC. PMID:28167505

Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90β

PubMed Central

Yim, Kendrick H.; Prince, Thomas L.; Qu, Shiwei; Bai, Fang; Jennings, Patricia A.; Onuchic, José N.; Theodorakis, Emmanuel A.; Neckers, Leonard

2016-01-01

Because of their importance in maintaining protein homeostasis, molecular chaperones, including heat-shock protein 90 (Hsp90), represent attractive drug targets. Although a number of Hsp90 inhibitors are in preclinical/clinical development, none strongly differentiate between constitutively expressed Hsp90β and stress-induced Hsp90α, the two cytosolic paralogs of this molecular chaperone. Thus, the importance of inhibiting one or the other paralog in different disease states remains unknown. We show that the natural product, gambogic acid (GBA), binds selectively to a site in the middle domain of Hsp90β, identifying GBA as an Hsp90β-specific Hsp90 inhibitor. Furthermore, using computational and medicinal chemistry, we identified a GBA analog, referred to as DAP-19, which binds potently and selectively to Hsp90β. Because of its unprecedented selectivity for Hsp90β among all Hsp90 paralogs, GBA thus provides a new chemical tool to study the unique biological role of this abundantly expressed molecular chaperone in health and disease. PMID:27466407

Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin.

PubMed

Peyretaillade, E; Broussolle, V; Peyret, P; Méténier, G; Gouy, M; Vivarès, C P

1998-06-01

An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.

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

PubMed Central

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

2016-01-01

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

Hsp90 molecular chaperone inhibitors: Are we there yet?

PubMed Central

Neckers, Len; Workman, Paul

2011-01-01

Heat shock protein (Hsp) 90 is an ATP-dependent molecular chaperone exploited by malignant cells to support activated oncoproteins, including many cancer-associated kinases and transcription factors, and is essential for oncogenic transformation. Originally viewed with skepticism, Hsp90 inhibitors are now actively pursued by the pharmaceutical industry, with 17 agents having entered clinical trials. Hsp90’s druggability was established using the natural products geldanamycin and radicicol which mimic the unusual ATP structure adopted in the chaperone’s N-terminal nucleotide-binding pocket and cause potent and selective blockade of ATP binding/hydrolysis, inhibit chaperone function, deplete oncogenic clients, and demonstrate antitumor activity. Preclinical data with these natural products have heightened interest in Hsp90 as a drug target, and 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) has demonstrated clinical activity (as defined by RECIST criteria) in HER2+ breast cancer. Many optimized synthetic small molecule Hsp90 inhibitors from diverse chemotypes are now in clinical trials. We review the discovery and development of Hsp90 inhibitors and assess their future potential. There has been significant learning from experience in both the basic biology and the translational drug development around Hsp90, enhanced by the use of Hsp90 inhibitors as chemical probes. Success will likely lie in treating cancers addicted to particular driver oncogene products, such as HER2, ALK, EGFR and BRAF, that are sensitive Hsp90 clients, as well as in malignancies, especially multiple myeloma, where buffering of proteotoxic stress is critical for survival. We discuss approaches to enhancing the effectiveness of Hsp90 inhibitors and highlight new chaperone and stress response pathway targets, including HSF1 and Hsp70. PMID:22215907

Molecular cloning, organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii.

PubMed

Toursel, C; Dzierszinski, F; Bernigaud, A; Mortuaire, M; Tomavo, S

2000-12-01

The obligate intracellular protozoan parasite Toxoplasma gondii has a single tubular mitochondrion. During infection, it recruits the host cell's mitochondria abutting to the intracellular vacuole, that contains the parasites. The respective contribution of host and parasitic mitochondria in the intracellular growth of T. gondii remains unknown. Heat shock protein, HSP60 has been reported in all eukaryotes examined, as an essential chaperone required for the folding and multimeric complex assembly of mitochondrial proteins. Here, we report the isolation and molecular characterization of two cDNAs corresponding to a single T. gondii gene coding for HSP60. Using a model fusion protein, preHSP60-chloramphenicol acetyl transferase (CAT), we demonstrate that the classical 22 amino acid mitochondrial presequence and the adjacent 32 amino acids of the mature protein are both required for the in vivo import into T. gondii mitochondria. The T. gondii HSP60 gene composed of five introns and six exons is transcribed into two related but differently spliced transcripts. Whereas the two transcripts can be detected in both developmental stages within the intermediate host, their levels are significantly increased in bradyzoites when compared to tachyzoites. By immunoblot analysis, the predicted 60-kDa protien corresponding to HSP60 was detected in both tachyzoite and bradyzoite forms. Using immunofluorescence assays. the polyclonal antibodies specific to T. gondii HSP60 recognized the mitochondrion in tachyzoites, as expected. In contrast, these antibodies reacted against two unknown vesicular bodies which are distinct from the classical mitochondrial pattern in bradyzoites. Taken together. these expression patterns of mitochondrial chaperone HSP60 suggests stage-specific induction of the respiratory pathway in the protozoan parasite T. gondii.

Approaches for Defining the Hsp90-dependent Proteome

PubMed Central

Hartson, Steven D.; Matts, Robert L.

2011-01-01

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

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

PubMed Central

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

2013-01-01

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

Hsp90: a novel target for the disruption of multiple signaling cascades.

PubMed

Bishop, Stephanie C; Burlison, Joseph A; Blagg, Brian S J

2007-06-01

The 90 kDa heat shock proteins (Hsp90) are proving to be an excellent target for the development of novel anti-cancer agents designed to selectively block the growth and proliferation of tumor cells. Since Hsp90 is a molecular chaperone and is responsible for folding numerous oncogenic proteins, its inhibition represents a novel approach toward the simultaneous disruption of multiple signaling cascades. This review summarizes recent literature implicating Hsp90 as a key facilitator for the maturation of proteins represented in all six hallmarks of cancer: 1) growth signal self-sufficiency, 2) anti-growth signal insensitivity, 3) evasion of apoptosis, 4) unlimited replicative potential, 5) metastasis and tissue invasion, and 6) sustained angiogenesis. Also described are recent advances towards the development of novel Hsp90 inhibitors via structure-based drug design that have contributed to the number of compounds undergoing clinical development.

Cold acclimation increases levels of some heat shock protein and sirtuin isoforms in threespine stickleback.

PubMed

Teigen, Laura E; Orczewska, Julieanna I; McLaughlin, Jessica; O'Brien, Kristin M

2015-10-01

Molecular chaperones [heat shock proteins (HSPs)] increase in response to rapid changes in temperatures, but long-term acclimation to cold temperature may also warrant elevations in HSPs. In fishes, cold acclimation increases mitochondrial density and oxidative stress in some tissues, which may increase demand for HSPs. We hypothesized that levels of HSPs, as well as sirtuins (SIRTs), NAD-dependent deacetylases that mediate changes in metabolism and responses to oxidative stress (including increases in HSPs), would increase during cold acclimation of threespine stickleback (Gasterosteus aculeatus). Transcript levels of hsp70, hsc70, hsp60 and hsp90-α, sirts1-4, as well as protein levels of HSP60, HSP90 and HSC70 were quantified in liver and pectoral adductor muscle of stickleback during cold acclimation from 20 °C to 8 °C. In liver, cold acclimation stimulated a transient increase in mRNA levels of hsp60 and hsc70. Transcript levels of sirt1 and sirt2 also increased in response to cold acclimation and remained elevated. In pectoral muscle, mRNA levels of hsp60, hsp90-α, hsc70 and sirt1 all transiently increased in response to cold acclimation, while levels of sirts2-4 remained constant or declined. Similar to transcript levels, protein levels of HSC70 increased in both liver and pectoral muscle. Levels of HSP90 also increased in liver after 4 weeks at 8 °C. HSP60 remained unchanged in both tissues, as did HSP90 in pectoral muscle. Our results indicate that while both HSPs and SIRTs increase in response to cold acclimation in stickleback, the response is tissue and isoform specific, likely reflecting differences in metabolism and oxidative stress. Copyright © 2015 Elsevier Inc. All rights reserved.

PPAR-γ Regulates Carnitine Homeostasis and Mitochondrial Function in a Lamb Model of Increased Pulmonary Blood Flow

PubMed Central

Rafikov, Ruslan; Kumar, Sanjiv; Hou, Yali; Oishi, Peter E.; Datar, Sanjeev A.; Raff, Gary; Fineman, Jeffrey R.; Black, Stephen M.

2012-01-01

Objective Carnitine homeostasis is disrupted in lambs with endothelial dysfunction secondary to increased pulmonary blood flow (Shunt). Our recent studies have also indicated that the disruption in carnitine homeostasis correlates with a decrease in PPAR-γ expression in Shunt lambs. Thus, this study was carried out to determine if there is a causal link between loss of PPAR-γ signaling and carnitine dysfunction, and whether the PPAR-γ agonist, rosiglitazone preserves carnitine homeostasis in Shunt lambs. Methods and Results siRNA-mediated PPAR-γ knockdown significantly reduced carnitine palmitoyltransferases 1 and 2 (CPT1 and 2) and carnitine acetyltransferase (CrAT) protein levels. This decrease in carnitine regulatory proteins resulted in a disruption in carnitine homeostasis and induced mitochondrial dysfunction, as determined by a reduction in cellular ATP levels. In turn, the decrease in cellular ATP attenuated NO signaling through a reduction in eNOS/Hsp90 interactions and enhanced eNOS uncoupling. In vivo, rosiglitazone treatment preserved carnitine homeostasis and attenuated the development of mitochondrial dysfunction in Shunt lambs maintaining ATP levels. This in turn preserved eNOS/Hsp90 interactions and NO signaling. Conclusion Our study indicates that PPAR-γ signaling plays an important role in maintaining mitochondrial function through the regulation of carnitine homeostasis both in vitro and in vivo. Further, it identifies a new mechanism by which PPAR-γ regulates NO signaling through Hsp90. Thus, PPAR-γ agonists may have therapeutic potential in preventing the endothelial dysfunction in children with increased pulmonary blood flow. PMID:22962578

Molecular Mechanisms of Prostate Cancer Progression

DTIC Science & Technology

2003-01-01

other drugs ( novobiocin and related hsp90 inhibitors have been shown to bind to the N-ter- coumarins) that are reported to target hsp90 are now be...undesirable for an indirect method of telomerase inhibition (data not shown). However, radicicol, which binds in the ATP- binding pocket of hsp90 and...compounds (e.g. novobiocin ) to block chaperone function using a totally different mechanism of hsp90 inhbition, as well as innovative genetic approaches

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

PubMed Central

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.

2013-01-01

Abstract 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

Identification of epipolythiodioxopiperazines HDN-1 and chaetocin as novel inhibitor of heat shock protein 90

PubMed Central

Song, Xiaoping; Zhao, Zhimin; Qi, Xin; Tang, Shuai; Wang, Qiang; Zhu, Tianjiao; Gu, Qianqun; Liu, Ming; Li, Jing

2015-01-01

The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an important target for cancer treatment. HDN-1, an epipolythiopiperazine-2, 5-diones (ETPs) compound, was here identified as a new Hsp90 inhibitor. HDN-1 bound directly to C-terminus of Hsp90α, resulting in a potential conformational change that interfered with the binding of 17-AAG and novobiocin to Hsp90α. In contrast, association of 17-AAG, novobiocin or ATP with Hsp90α did not prevent the binding HDN-1 to Hsp90α. HDN-1 in combination with 17-AAG exhibited an enhanced inhibitory effect on non-small lung cancer cell proliferation. Molecular docking analyses revealed that HDN-1 bound to Hsp90α at C-terminal 526–570 region. In addition, HDN-1 degraded multiple oncoproteins and promoted EGF-induced wild type and mutated EGFR downregulation. Notably, chaetocin, used as a SUV39H1 inhibitor with similar structure to HDN-1, bound to Hsp90 and degraded Hsp90 client proteins and SUV39H1 as did HDN-1. These results indicate that HDN-1 and chaetocin are inhibitors of Hsp90 and that SUV39H1 is a novel client protein of Hsp90. PMID:25742791

Targeting HSP90 dimerization via the C-terminus is effective in imatinib resistant CML and lacks heat shock response.

PubMed

Bhatia, Sanil; Diedrich, Daniela; Frieg, Benedikt; Ahlert, Heinz; Stein, Stefan; Bopp, Bertan; Lang, Franziska; Zang, Tao; Kröger, Tobias; Ernst, Thomas; Kögler, Gesine; Krieg, Andreas; Lüdeke, Steffen; Kunkel, Hana; Rodrigues Moita, Ana J; Kassack, Matthias U; Marquardt, Viktoria; Opitz, Friederike V; Oldenburg, Marina; Remke, Marc; Babor, Florian; Grez, Manuel; Hochhaus, Andreas; Borkhardt, Arndt; Groth, Georg; Nagel-Steger, Luitgard; Jose, Joachim; Kurz, Thomas; Gohlke, Holger; Hansen, Finn K; Hauer, Julia

2018-05-03

Heat shock protein 90 (HSP90) stabilizes many client proteins including BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of CML in which treatment-free remission (TFR) is limited with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics, which synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain (NTD) of HSP90 are under investigation; however, side effects such as induction of heat shock response (HSR) and toxicity have so far precluded their FDA approval. We have developed a novel inhibitor (referred to as aminoxyrone) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain (CTD). This was achieved by structure-based molecular design, chemical synthesis, and functional pre-clinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. Aminoxyrone (AX) is a promising potential candidate, which induces apoptosis in leukemic stem cells (LSCs) fraction (CD34 + CD38 - ) as well as the leukemic bulk (CD34 + CD38 + ) of primary CML and in TKI-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated and targeting HSP90 C-terminus by AX does not induce HSR in vitro and in vivo. We also probed the potential of AX in other therapy refractory leukemia such as BCR-ABL1+ BCP-ALL, FLT3-ITD+ AML and Ph-like BCP-ALL. Therefore, AX is the first peptidometic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other therapy-refractory leukemia, due to its low toxicity profile and lack of HSR. Copyright © 2018 American Society of Hematology.

Targeted cancer therapy through 17-DMAG as an Hsp90 inhibitor: Overview and current state of the art.

PubMed

Mellatyar, Hassan; Talaei, Sona; Pilehvar-Soltanahmadi, Younes; Barzegar, Abolfazl; Akbarzadeh, Abolfazl; Shahabi, Arman; Barekati-Mowahed, Mazyar; Zarghami, Nosratollah

2018-06-01

Heat shock protein 90 (Hsp90) is an evolutionary preserved molecular chaperone which mediates many cellular processes such as cell transformation, proliferation, and survival in normal and stress conditions. Hsp90 plays an important role in folding, maturation, stabilization and activation of Hsp90 client proteins which all contribute to the development, and proliferation of cancer as well as other inflammatory diseases. Functional inhibition of Hsp90 can have a massive effect on various oncogenic and inflammatory pathways, and will result in the degradation of their client proteins. This turns it into an interesting target in the treatment of different malignancies. 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) as a semi-synthetic derivative of geldanamycin, has several advantages over 17-Allylamino-17-demethoxygeldanamycin (17-AAG) such as higher water solubility, good bioavailability, reduced metabolism, and greater anti-tumour capability. 17-DMAG binds to the Hsp90, and inhibits its function which eventually results in the degradation of Hsp90 client proteins. Here, we reviewed the pre-clinical data and clinical trial data on 17-DMAG as a single agent, in combination with other agents and loaded on nanomaterials in various cancers and inflammatory diseases. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Targeting HSF1 disrupts HSP90 chaperone function in chronic lymphocytic leukemia.

PubMed

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

2015-10-13

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

HSP-enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells.

PubMed

Ono, Kisho; Eguchi, Takanori; Sogawa, Chiharu; Calderwood, Stuart K; Futagawa, Junya; Kasai, Tomonari; Seno, Masaharu; Okamoto, Kuniaki; Sasaki, Akira; Kozaki, Ken-Ichi

2018-05-16

Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90β, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC. © 2018 Wiley Periodicals, Inc.

Effectiveness of hsp90 inhibitors as anti-cancer drugs.

PubMed

Xiao, Li; Lu, Xiangyi; Ruden, Douglas M

2006-10-01

Hsp90 is a chaperone with over 100 identified client proteins. What makes Hsp90 especially promising as a target for anti-cancer drugs is that many of its client proteins are in signaling and chromatin-remodeling pathways, and these pathways are often disrupted in many types of cancers. Recently, it was determined that Hsp90 bound to a client protein in a co-chaperone complex has a higher ATPase activity and binds to the geldanamycin inhibitor with over 100-fold higher affinity than the low-ATPase form. Consequently, despite Hsp90 being an abundant protein in most cell types, Hsp90 inhibitors accumulate at high levels primarily in tumor cells because tumor cells are "oncogene addicted" and require especially high levels of the high-ATPase form of Hsp90. Numerous classes of Hsp90 inhibitors have recently been developed, such as the anasamysin geldanamycin and derivatives 17-AAG and 17-DMAG; the macrolide radicicol and derivatives; purine-scaffold derivatives; pyrazoles; and shepherdins that bind to the N-terminal high-affinity ATP-binding domain of Hsp90. Other inhibitors have recently been shown to bind to the C-terminal dimerization domain of Hsp90, such as cisplatin and novobiocin, or modify Hsp90 postranslationally, such as histone deacetylase or proteasome inhibitors. In this mini-review, we present hypothetical mechanisms for Hsp90 inhibitors in treating cancers, preliminary studies in early clinical trials, and potential tumor-killing and tumor-promoting activities of Hsp90 inhibitors.

The topoisomerase II-Hsp90 complex: a new chemotherapeutic target?

PubMed

Barker, Catherine R; Hamlett, Jane; Pennington, Stephen R; Burrows, Francis; Lundgren, Karen; Lough, Rachel; Watson, Alastair J M; Jenkins, John R

2006-06-01

The modulation of DNA topology by topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and has thus become a highly attractive target for chemotherapeutic drugs. However, these drugs are highly toxic, and so new approaches are required. One such strategy is to target topoisomerase II-interacting proteins. Here we report the identification of potential topoisomerase II-associated proteins using immunoprecipitation, followed by 1-D and 2-D gel electrophoresis and MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIalpha-associated proteins by coimmunoprecipitation and Western blot. Six of the interacting proteins were cellular chaperones, including 3 members of the heat shock protein-90 (Hsp90) family, and so the effect of Hsp90 modulation on the antitumor activity of topoisomerase II drugs was tested using the sulforhodamine B assay, clonogenic assays and a xenograft model. The Hsp90 inhibitors geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and radicicol significantly enhanced the activity of the topoisomerase II poisons etoposide and mitoxantrone in vitro and in vivo. Thus, our method of identifying topoisomerase II-interacting proteins appears to be effective, and at least 1 novel topoisomerase IIalpha-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy.

Systemic and mucosal immunization with Candida albicans hsp90 elicits hsp90-specific humoral response in vaginal mucosa which is further enhanced during experimental vaginal candidiasis.

PubMed

Raska, Milan; Belakova, Jana; Horynova, Milada; Krupka, Michal; Novotny, Jiri; Sebestova, Martina; Weigl, Evzen

2008-08-01

The Candida albicans heat shock protein 90 kDa (hsp90-CA) is an important target for protective antibodies in disseminated candidiasis of experimental mice and humans. Hsp90-CA is present in the cell wall of Candida pseudohyphae or hyphae--typical pathogenic morphotypes in both mucosal and systemic Candida infections. However, the potential protective effects of hsp90-CA-specific antibodies in vaginal candidiasis has not yet been reported. In the present study we used various vaccine formulations (recombinant hsp90-CA protein and hsp90-CA-encoding DNA vaccine) and routes of administration (intradermal, intranasal, and intravenous) to induce both hsp90-CA-specific systemic and vaginal mucosa immune responses in experimental BALB/c mice. The results showed that intradermal recombinant hsp90-CA protein priming, followed by intranasal or intradermal recombinant hsp90-CA protein boosting induced significant increases in both serum and vaginal hsp90-CA-specific IgG and IgA antibodies compared to the control group, as well as enhanced hsp90-CA-specific splenocyte responses in vitro. In the intradermally boosted group, subsequent experimental vaginal Candida infection induced additional increases in the hsp90-CA specific IgG isotype, suggesting that Candida has the ability to induce a local hsp90-specific antibody (IgG) response during vulvovaginal candidiasis. Further work is required to elucidate the importance of immunity to highly conserved antigens during infection of the human female reproductive tract where a balance between immunity to and tolerance for commonly antigens such as hsp90 is necessary for the maintenance of fertility.

Targets Fishing and Identification of Calenduloside E as Hsp90AB1: Design, Synthesis, and Evaluation of Clickable Activity-Based Probe

PubMed Central

Wang, Shan; Tian, Yu; Zhang, Jing-Yi; Xu, Hui-Bo; Zhou, Ping; Wang, Min; Lu, Sen-Bao; Luo, Yun; Wang, Min; Sun, Gui-Bo; Xu, Xu-Dong; Sun, Xiao-Bo

2018-01-01

Calenduloside E (CE), a natural triterpenoid compound isolated from Aralia elata, can protect against ox-LDL-induced human umbilical vein endothelial cell (HUVEC) injury in our previous reports. However, the exact targets and mechanisms of CE remain elusive. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. Based on the previous studies of the structure-activity relationship (SAR), we introduced an alkyne moiety at the C-28 carboxylic group of CE, which kept the protective and anti-apoptosis activity. Via proteomic approach, one of the potential proteins bound to CE-P was identified as Hsp90AB1, and further verification was performed by pure recombinant Hsp90AB1 and competitive assay. These results demonstrated that CE could bind to Hsp90AB1. We also found that CE could reverse the Hsp90AB1 decrease after ox-LDL treatment. To make our results more convincing, we performed SPR analysis and the affinity kinetic assay showed that CE/CE-P could bind to Hsp90AB1 in a dose-dependent manner. Taken together, our research showed CE could probably bind to Hsp90AB1 to protect the cell injury, which might provide the basis for the further exploration of its cardiovascular protective mechanisms. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. PMID:29875664

Disruption of Heat Shock Protein 90 (Hsp90)-Protein Kinase Cδ (PKCδ) Interaction by (−)-Maackiain Suppresses Histamine H1 Receptor Gene Transcription in HeLa Cells*

PubMed Central

Nariai, Yuki; Mizuguchi, Hiroyuki; Ogasawara, Takeyasu; Nagai, Hiroaki; Sasaki, Yohei; Okamoto, Yasunobu; Yoshimura, Yoshiyuki; Kitamura, Yoshiaki; Nemoto, Hisao; Takeda, Noriaki; Fukui, Hiroyuki

2015-01-01

The histamine H1 receptor (H1R) gene is an allergic disease sensitive gene, and its expression level is strongly correlated with the severity of allergic symptoms. (−)-Maackiain was identified as a Kujin-derived anti-allergic compound that suppresses the up-regulation of the H1R gene. However, the underlying mechanism of H1R gene suppression remains unknown. Here, we sought to identify a target protein of (−)-maackiain and investigate its mechanism of action. A fluorescence quenching assay and immunoblot analysis identified heat shock protein 90 (Hsp90) as a target protein of (−)-maackiain. A pull-down assay revealed that (−)-maackiain disrupted the interaction of Hsp90 with PKCδ, resulting in the suppression of phorbol 12-myristate 13-acetate (PMA)-induced up-regulation of H1R gene expression in HeLa cells. Additional Hsp90 inhibitors, including 17-(allylamino)-17-demethoxygeldanamycin, celastrol, and novobiocin also suppressed PMA-induced H1R gene up-regulation. 17-(Allylamino)-17-demethoxygeldanamycin inhibited PKCδ translocation to the Golgi and phosphorylation of Tyr311 on PKCδ. These data suggest that (−)-maackiain is a novel Hsp90 pathway inhibitor. The underlying mechanism of the suppression of PMA-induced up-regulation of H1R gene expression by (−)-maackiain and Hsp90 inhibitors is the inhibition of PKCδ activation through the disruption of Hsp90-PKCδ interaction. Involvement of Hsp90 in H1R gene up-regulation suggests that suppression of the Hsp90 pathway could be a novel therapeutic strategy for allergic rhinitis. PMID:26391399

Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization.

PubMed

Allan, Rudi K; Mok, Danny; Ward, Bryan K; Ratajczak, Thomas

2006-03-17

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.

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

[Pancreatic acinar neoplasms : Comparative molecular characterization].

PubMed

Bergmann, F

2016-11-01

Pancreatic acinar cell carcinomas are biologically aggressive neoplasms for which treatment options are very limited. The molecular mechanisms of tumor initiation and progression are largely not understood and precursor lesions have not yet been identified. In this study, pancreatic acinar cell carcinomas were cytogenetically characterized as well as by molecular and immunohistochemical analyses. Corresponding investigations were carried out on pancreatic ductal adenocarcinomas and pancreatic neuroendocrine neoplasms augmented by functional analyses. We show that pancreatic acinar cell carcinomas display a microsatellite stable, chromosomal unstable genotype, characterized by recurrent chromosomal imbalances that clearly discriminate them from pancreatic ductal adenocarcinomas and neuroendocrine neoplasms. Based on findings obtained from comparative genomic hybridization, candidate genes could be identified, such as deleted in colorectal cancer (DCC) and c-MYC. Furthermore, several therapeutic targets were identified in acinar cell carcinomas and other pancreatic neoplasms, including epidermal growth factor receptor (EGFR), L1 cell adhesion molecule (L1CAM) and heat shock protein 90 (HSP90). Moreover, L1CAM was shown to play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma. Functional analyses in cell lines derived from pancreatic neuroendocrine neoplasms revealed promising anti-tumorigenic effects using EGFR and HSP90 inhibitors affecting the cell cycle and in the case of HSP90, regulating several other oncogenes. Finally, based on mutational analyses of mitochondrial DNA, molecular evidence is provided that acinar cell cystadenomas (or better cystic acinar transformation) represent non-clonal lesions, suggesting an inflammatory reactive non-neoplastic nature.

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.

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

Cloning, purification and characterization of a 90kDa heat shock protein from Citrus sinensis (sweet orange).

PubMed

Mendonça, Yuri A; Ramos, Carlos H I

2012-01-01

Protein misfolding is stimulated by stress, such as heat, and heat shock proteins (Hsps) are the first line of defense against these undesirable situations. Plants, which are naturally sessile, are perhaps more exposed to stress factors than some other organisms, and consequently, the role of Hsps is crucial to maintain homeostasis. Hsp90, because of its key role in infection and other stresses, is targeted in therapies that improve plant production by increasing resistance to both biotic and abiotic stress. In addition, Hsp90 is a primary factor in the maintenance of homeostasis in plants. Therefore, we cloned and purified Hsp90 from Citrus sinensis (sweet orange). Recombinant C. sinensis Hsp90 (rCsHsp90) was produced and measured by circular dichroism (CD), intrinsic fluorescence spectroscopy and dynamic light scattering. rCsHsp90 formed a dimer in solution with a Stokes radius of approximately 62Å. In addition, it was resistant to thermal unfolding, was able to protect citrate synthase from aggregation, and Western blot analysis demonstrated that CsHsp90 was constitutively expressed in C. sinensis cells. Our analysis indicated that CsHsp90 is conformationally similar to that of yeast Hsp90, for which structural information is available. Therefore, we showed that C. sinensis expresses an Hsp90 chaperone that has a conformation and function similar to other Hsp90s. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2012-01-01

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients. PMID:22624053

Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

PubMed

Munday, Diane C; Howell, Gareth; Barr, John N; Hiscox, Julian A

2015-03-01

The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology. © 2014 Royal Pharmaceutical Society.

Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail Theba pisana in relation to genetic structure.

PubMed

Mizrahi, Tal; Goldenberg, Shoshana; Heller, Joseph; Arad, Zeev

2016-03-01

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.

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.

Hsp90: a novel target for cancer therapy.

PubMed

Solit, David B; Rosen, Neal

2006-01-01

Hsp90 is a molecular chaperone required for the stress-survival response, protein refolding, and the conformational maturation of a variety of signaling proteins. Natural products that bind selectively to Hsp90 and inhibit its function have been used to determine its biologic role. Experiments with these drugs have shown that Hsp90 is required for maintaining the malignant phenotype of cancer cells. Studies in vivo show that Hsp90 inhibitors have antitumor activity when given alone and in combination with cytotoxics. The basis for the therapeutic index (selective toxicity to cancer cells) of Hsp90 inhibitors is complex and may have to do with induction of degradation of mutant oncoproteins and other proteins necessary for their proliferation and survival as well as to an enhanced requirement of these cells for Hsp90 stress-survival functions. Based on these data, 17-AAG, an ansamycin antibiotic inhibitor of Hsp90, is being tested extensively in clinical trials in patients with advanced cancer. These trials demonstrate that the biologic function of Hsp90 can be inhibited in patients and antitumor activity has been noted in patients with breast cancer, multiple myeloma and other cancers. These data and the physicochemical properties of 17-AAG that limit its use as a drug, have led to broad efforts to develop improved and novel Hsp90 inhibitors. This article will review the preclinical data which supports the testing of Hsp90 inhibitors as cancer drugs and update the reader on the current status of the ongoing clinical trials of Hsp90 inhibitors.

Mitochondrial heat shock protein (Hsp) 70 and Hsp10 cooperate in the formation of Hsp60 complexes.

PubMed

Böttinger, Lena; Oeljeklaus, Silke; Guiard, Bernard; Rospert, Sabine; Warscheid, Bettina; Becker, Thomas

2015-05-01

Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cotargeting HSP90 and Its Client Proteins for Treatment of Prostate Cancer.

PubMed

Chen, Long; Li, Jie; Farah, Elia; Sarkar, Sukumar; Ahmad, Nihal; Gupta, Sanjay; Larner, James; Liu, Xiaoqi

2016-09-01

Castration-resistant prostate cancer (CRPC) is the later stage of prostate cancer when the disease has stopped responding to androgen deprivation therapy (ADT). It has been established that androgen receptor (AR) reactivation is responsible for the recurrence of prostate cancer after ADT. Thus, targeting different pathways that regulate AR stability and activity should be a promising strategy for treatment of CRPC. Heat shock proteins (HSP) are chaperones that modify stability and activity of their client proteins. HSP90, a major player in the HSP family, regulates stability of many proteins, including AR and Polo-like kinase 1 (Plk1), a critical regulator of many cell-cycle events. Further, HSP90 is overexpressed in different cancers, including prostate cancer. Herein, we show that cotreatment of prostate cancer with AR antagonist enzalutamide and HSP90 inhibitor leads to more severe cell death due to a synergistic reduction of AR protein. Interestingly, we show that overexpression of Plk1 rescued the synergistic effect and that cotargeting HSP90 and Plk1 also leads to more severe cell death. Mechanistically, we show that E3 ligase CHIP, in addition to targeting AR, is responsible for the degradation of Plk1 as well. These findings suggest that cotargeting HSP90 and some of its client proteins may be a useful strategy in treatment of CRPC. Mol Cancer Ther; 15(9); 2107-18. ©2016 AACR. ©2016 American Association for Cancer Research.

Docosahexaenoic Acid-mediated Inhibition of Heat Shock Protein 90-p23 Chaperone Complex and Downstream Client Proteins in Lung and Breast Cancer.

PubMed

Mouradian, Michael; Ma, Irvin V; Vicente, Erika D; Kikawa, Keith D; Pardini, Ronald S

2017-01-01

The molecular chaperone, heat shock protein 90 (Hsp90), is a critical regulator for the proper folding and stabilization of several client proteins, and is a major contributor to carcinogenesis. Specific Hsp90 inhibitors have been designed to target the ATP-binding site in order to prevent Hsp90 chaperone maturation. The current study investigated the effects of docosahexaenoic acid (DHA; C22:6 n-3) on Hsp90 function and downstream client protein expression. In vitro analyses of BT-474 human breast carcinoma and A549 human lung adenocarcinoma cell lines revealed dose-dependent decreases in intracellular ATP levels by DHA treatment, resulting in a significant reduction of Hsp90 and p23 association in both cell lines. Attenuation of the Hsp90-p23 complex led to the inhibition of Hsp90 client proteins, epidermal growth factor receptor 2 (ErbB2), and hypoxia-inducible factor 1α (HIF-1α). Similar results were observed when employing 2-deoxyglucose (2-DG), confirming that DHA and 2-DG, both independently and combined, can disturb Hsp90 molecular chaperone function. In vivo A549 xenograft analysis also demonstrated decreased expression levels of Hsp90-p23 association and diminished protein levels of ErbB2 and HIF-1α in mice supplemented with dietary DHA. These data support a role for dietary intervention to improve cancer therapy in tumors overexpressing Hsp90 and its client proteins.

Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Robert Y.L., E-mail: yuwang@mail.cgu.edu.tw; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333 Taiwan; Kuo, Rei-Lin

2013-09-01

Molecular chaperones are reported to be crucial for virus propagation, but are not yet addressed in Human Enterovirus 71 (EV71). Here we describe the specific association of heat shock protein-90-beta (Hsp90β), but not alpha form (Hsp90α), with EV71 viral particles by the co-purification with virions using sucrose density gradient ultracentrifugation, and by the colocalization with viral particles, as assessed by immunogold electron microscopy. The reduction of the Hsp90β protein using RNA interference decreased the correct assembly of viral particles, without affecting EV71 replication levels. Tracking ectopically expressed Hsp90β protein associated with EV71 virions revealed that Hsp90β protein was transmitted tomore » new host cells through its direct association with infectious viral particles. Our findings suggest a new antiviral strategy in which extracellular Hsp90β protein is targeted to decrease the infectivity of EV71 and other enteroviruses, without affecting the broader functions of this constitutively expressed molecular chaperone. - Highlights: • Hsp90β is associated with EV71 virion and is secreted with the release virus. • Hsp90β effects on the correct assembly of viral particles. • Viral titer of cultured medium was reduced in the presence of geldanamycin. • Viral titer was also reduced when Hsp90β was suppressed by siRNA treatment. • The extracellular Hsp90β was also observed in other RNA viruses-infected cells.« less

Mitochondrial deficits and abnormal mitochondrial retrograde axonal transport play a role in the pathogenesis of mutant Hsp27-induced Charcot Marie Tooth Disease.

PubMed

Kalmar, Bernadett; Innes, Amy; Wanisch, Klaus; Kolaszynska, Alicia Koyen; Pandraud, Amelie; Kelly, Gavin; Abramov, Andrey Y; Reilly, Mary M; Schiavo, Giampietro; Greensmith, Linda

2017-09-01

Mutations in the small heat shock protein Hsp27, encoded by the HSPB1 gene, have been shown to cause Charcot Marie Tooth Disease type 2 (CMT-2) or distal hereditary motor neuropathy (dHMN). Protein aggregation and axonal transport deficits have been implicated in the disease. In this study, we conducted analysis of bidirectional movements of mitochondria in primary motor neuron axons expressing wild type and mutant Hsp27. We found significantly slower retrograde transport of mitochondria in Ser135Phe, Pro39Leu and Arg140Gly mutant Hsp27 expressing motor neurons than in wild type Hsp27 neurons, although anterograde movement velocities remained normal. Retrograde transport of other important cargoes, such as the p75 neurotrophic factor receptor was minimally altered in mutant Hsp27 neurons, implicating that axonal transport deficits primarily affect mitochondria and the axonal transport machinery itself is less affected. Investigation of mitochondrial function revealed a decrease in mitochondrial membrane potential in mutant Hsp27 expressing motor axons, as well as a reduction in mitochondrial complex 1 activity, increased vulnerability of mitochondria to mitochondrial stressors, leading to elevated superoxide release and reduced mitochondrial glutathione (GSH) levels, although cytosolic GSH remained normal. This mitochondrial redox imbalance in mutant Hsp27 motor neurons is likely to cause low level of oxidative stress, which in turn will contribute to, and indeed may be the underlying cause of the deficits in mitochondrial axonal transport. Together, these findings suggest that the mitochondrial abnormalities in mutant Hsp27-induced neuropathies may be a primary cause of pathology, leading to further deficits in the mitochondrial axonal transport and onset of disease. © The Author 2017. Published by Oxford University Press.

Mitochondrial deficits and abnormal mitochondrial retrograde axonal transport play a role in the pathogenesis of mutant Hsp27-induced Charcot Marie Tooth Disease

PubMed Central

Innes, Amy; Wanisch, Klaus; Kolaszynska, Alicia Koyen; Pandraud, Amelie; Kelly, Gavin; Abramov, Andrey Y.; Reilly, Mary M.; Schiavo, Giampietro; Greensmith, Linda

2017-01-01

Abstract Mutations in the small heat shock protein Hsp27, encoded by the HSPB1 gene, have been shown to cause Charcot Marie Tooth Disease type 2 (CMT-2) or distal hereditary motor neuropathy (dHMN). Protein aggregation and axonal transport deficits have been implicated in the disease. In this study, we conducted analysis of bidirectional movements of mitochondria in primary motor neuron axons expressing wild type and mutant Hsp27. We found significantly slower retrograde transport of mitochondria in Ser135Phe, Pro39Leu and Arg140Gly mutant Hsp27 expressing motor neurons than in wild type Hsp27 neurons, although anterograde movement velocities remained normal. Retrograde transport of other important cargoes, such as the p75 neurotrophic factor receptor was minimally altered in mutant Hsp27 neurons, implicating that axonal transport deficits primarily affect mitochondria and the axonal transport machinery itself is less affected. Investigation of mitochondrial function revealed a decrease in mitochondrial membrane potential in mutant Hsp27 expressing motor axons, as well as a reduction in mitochondrial complex 1 activity, increased vulnerability of mitochondria to mitochondrial stressors, leading to elevated superoxide release and reduced mitochondrial glutathione (GSH) levels, although cytosolic GSH remained normal. This mitochondrial redox imbalance in mutant Hsp27 motor neurons is likely to cause low level of oxidative stress, which in turn will contribute to, and indeed may be the underlying cause of the deficits in mitochondrial axonal transport. Together, these findings suggest that the mitochondrial abnormalities in mutant Hsp27-induced neuropathies may be a primary cause of pathology, leading to further deficits in the mitochondrial axonal transport and onset of disease. PMID:28595321

The Noncompetitive Effect of Gambogic Acid Displaces Fluorescence-Labeled ATP but Requires ATP for Binding to Hsp90/HtpG.

PubMed

Yue, Qing; Stahl, Frank; Plettenburg, Oliver; Kirschning, Andreas; Warnecke, Athanasia; Zeilinger, Carsten

2018-05-08

The heat shock protein 90 (Hsp90) family plays a critical role in maintaining the homeostasis of the intracellular environment for human and prokaryotic cells. Hsp90 orthologues were identified as important target proteins for cancer and plant disease therapies. It was shown that gambogic acid (GBA) has the potential to inhibit human Hsp90. However, it is unknown whether it is also able to act on the bacterial high-temperature protein (HtpG) analogue. In this work, we screened GBA and nine other novel potential Hsp90 inhibitors using a miniaturized high-throughput protein microarray-based assay and found that GBA shows an inhibitory effect on different Hsp90s after dissimilarity analysis of the protein sequence alignment. The dissociation constant of GBA and HtpG Xanthomonas (XcHtpG) computed from microscale thermophoresis is 682.2 ± 408 μM in the presence of ATP, which is indispensable for the binding of GBA to XcHtpG. Our results demonstrate that GBA is a promising Hsp90/HtpG inhibitor. The work further demonstrates that our assay concept has great potential for finding new potent Hsp/HtpG inhibitors.

Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

PubMed Central

Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

2015-01-01

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

A model in which heat shock protein 90 targets protein-folding clefts: rationale for a new approach to neuroprotective treatment of protein folding diseases.

PubMed

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

2014-11-01

In an EBM Minireview published in 2010, we proposed that the heat shock protein (Hsp)90/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. The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand-binding clefts. 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. 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. © 2014 by the Society for Experimental Biology and Medicine.

Transcriptional requirements of the distal heavy-strand promoter of mtDNA

PubMed Central

Zollo, Ornella; Tiranti, Valeria; Sondheimer, Neal

2012-01-01

The heavy strand of mtDNA contains two promoters with nonoverlapping functions. The role of the minor heavy-strand promoter (HSP2) is controversial, because the promoter has been difficult to activate in an in vitro system. We have isolated HSP2 by excluding its interaction with the more powerful HSP1 promoter, and we find that it is transcribed efficiently by recombinant mtRNA polymerase and mitochondrial transcription factor B2. The mitochondrial transcription factor A is not required for initiation, but it has the ability to alternatively activate and repress the HSP2 transcriptional unit depending on the ratio between mitochondrial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current understanding of HSP2 activity in vivo. Serial deletion of HSP2 shows that only proximal sequences are required. Several mutations, including the disruption of a polycytosine track upstream of the HSP2 initiation site, influence transcriptional activity. Transcription from HSP2 is also observed when HeLa cell mitochondrial extract is used as the source of mitochondrial polymerase, and this transcription is maintained when HSP2 is provided in proper spacing and context to the HSP1 promoter. Studies of the linked heavy-strand promoters show that they are differentially regulated by ATP dosage. We conclude that HSP2 is transcribed and has features that allow it to regulate mitochondrial mRNA synthesis. PMID:22454497

Eukaryotic Hsp70 chaperones in the intermembrane space of chloroplasts.

PubMed

Bionda, Tihana; Gross, Lucia E; Becker, Thomas; Papasotiriou, Dimitrios G; Leisegang, Matthias S; Karas, Michael; Schleiff, Enrico

2016-03-01

Multiple eukaryotic Hsp70 typically localized in the cytoplasm are also distributed to the intermembrane space of chloroplasts and might thereby represent the missing link in energizing protein translocation. Protein translocation into organelles is a central cellular process that is tightly regulated. It depends on signals within the preprotein and on molecular machines catalyzing the process. Molecular chaperones participate in transport and translocation of preproteins into organelles to control folding and to provide energy for the individual steps. While most of the processes are explored and the components are identified, the transfer of preproteins into and across the intermembrane space of chloroplasts is not yet understood. The existence of an energy source in this compartment is discussed, because the required transit peptide length for successful translocation into chloroplasts is shorter than that found for mitochondria where energy is provided exclusively by matrix chaperones. Furthermore, a cytosolic-type Hsp70 homologue was proposed as component of the chloroplast translocon in the intermembrane space energizing the initial translocation. The molecular identity of such intermembrane space localized Hsp70 remained unknown, which led to a controversy concerning its existence. We identified multiple cytosolic Hsp70s by mass spectrometry on isolated, thermolysin-treated Medicago sativa chloroplasts. The localization of these Hsp70s of M. sativa or Arabidopsis thaliana in the intermembrane space was confirmed by a self-assembly GFP-based in vivo system. The localization of cytosolic Hsp70s in the stroma of chloroplasts or different mitochondrial compartments could not be observed. Similarly, we could not identify any cytosolic Hsp90 in the intermembrane space of chloroplast. With respect to our results we discuss the possible targeting and function of the Hsp70 found in the intermembrane space.

[Effects of Electroacupuncture and Moxibustion Pretreatment on Expressions of HSP 27, HSP 70, HSP 90 at Different Time-points in Rabbits with Myocardial Ischemia-reperfusion Injury].

PubMed

Tan, Cheng-Fu; Yan, Jie; Wang, Chao; Chang, Xiao-Rong; Xie, Wen-Juan; Yang, Jing-Jing; Liu, Mi; Lin, Hai-Bo; He, Xiang-Chang

2017-02-25

To observe the effect of electroacupuncture (EA) and moxibustion (Moxi) pretreatment on expression of myocardial heat shock protein (HSP) in acute myocardial ischemia-reperfusion injury (MIRI) rabbits. A total of 72 New Zealand rabbits were randomly divided into 4 groups:sham operation, MIRI model, EA pretreatment and Moxi pretreatment ( n =18 rabbits in each group) which were further divided into 0, 24 and 48 h (time-point) subgroups ( n =6 in each). The MIRI model was established by occlusion of the anterior descending branch (ADB) of the left coronary artery for 40 min and reperfusion for 60 min. EA and Moxi stimulation was respectively applied to bilateral "Neiguan"(PC 6) for 20 min, once daily for 5 days before ADB occlusion. The expressions of myocardial HSP 27, HSP 70 and HSP 90 were detected by immunohistochemistry. The pathological and ultrastructural changes of left ventricular ischemia tissue were observed under light and transmission electronic microscope (TEM), respectively. Outcomes of H.E. staining and ultrastructure showed that MIRI-induced changes of disordered arrangement of cardiomyocytes, vague myocardial transverse striation, inflammatory infiltration, cardiac myofibre necrosis and fibrolysis (light microscope), and myofiber atrophy, vague and disorder in the arrangement of myofiber, myofilament necrosis, interstitial edema, mitochondrial swelling, microvessel expansion, etc. (TEM) were relatively milder in both EA and Moxi pretreatment groups (48 h). In comparison with the sham group, the expression levels of myocardial HSP 27, HSP 70 and HSP 90 had no significant changes after MIRI at the 3 time-points ( P >0.05). In the pretreatment groups, the expression levels of HSP 27 at 24 and 48 h in both EA and Moxi groups, HSP 70 at 48 h in both groups, HSP 70 at 0 and 24 h in the Moxi group were significantly up-regulated compared with the model group ( P <0.05, P <0.01). No significant changes were found in the expression of HSP 90 at the 3 time-points in the EA and Moxi pretreatment groups ( P >0.05). No significant differences were found between EA and Moxi in up-regulating expressions of myocardial HSP 27, HSP 70 and HSP 90 proteins at the 3 time-points ( P >0.05) except HSP 70 at 24 h (Moxi being stronger relative to EA, P <0.05). EA and Moxi pretreatment has a protective effect on ischemic myocardium in MIRI rabbits, which Feb be associated with their actions in up-regulating myocardial HSP 27 and HSP 70 expression.

Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans.

PubMed

David, Cynthia L; Smith, Harold E; Raynes, Deborah A; Pulcini, Elizabeth J; Whitesell, Luke

2003-01-01

In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol. Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide-binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases. To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake. Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span. However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90. Consistent with this observation, we found that solid phase-immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system. Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA. Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background. It heterodimerized with its vertebrate counterpart and showed no evidence of compromising its essential cellular functions. Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant. These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that target-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.

The Heat Shock Protein 90 Inhibitor, Epigallocatechin Gallate, has Anti-Cancer Activity in a Novel Human Prostate Cancer Progression Model

PubMed Central

Moses, Michael A.; Henry, Ellen C.; Ricke, William A.; Gasiewicz, Thomas A.

2015-01-01

(−)-Epigallocatechin gallate (EGCG), a major tea polyphenol, elicits anti-cancer effects. However, the mechanism of action is not fully understood. Our laboratory previously showed that EGCG inhibits heat shock protein 90 (HSP90). We utilized non-tumorigenic (NT), tumorigenic, and metastatic cancer cells from a novel human prostate cancer (PRCA) progression model to test the hypotheses that certain stages are more or less sensitive to EGCG and that sensitivity is related to HSP90 inhibition. Treatment of cells with EGCG, novobiocin (NB), or 17-AAG resulted in more potent cytotoxic effects on tumorigenic and metastatic cells than NT cells. When tumorigenic or metastatic cells were grown in vivo, mice supplemented with 0.06% EGCG in drinking water developed significantly smaller tumors than untreated mice. Furthermore, EGCG prevented malignant transformation in vivo using the full PRCA model. To elucidate the mechanism of EGCG action, we performed binding assays with EGCG-Sepharose, a C-terminal HSP90 antibody, and HSP90 mutants. These experiments revealed that EGCG-Sepharose bound more HSP90 from metastatic cells compared to NT cells and binding occurred through the HSP90 C-terminus. Additionally, EGCG bound HSP90 mutants that mimic both complexed and uncomplexed HSP90. Consistent with HSP90 inhibitory activity, EGCG, NB, and 17-AAG induced changes in HSP90-client proteins in NT cells and larger differences in metastatic cells. These data suggest that EGCG may be efficacious for the treatment of PRCA because it preferentially targets cancer cells and inhibits a molecular chaperone supportive of the malignant phenotype. PMID:25604133

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression and prognostic examination of heat shock proteins (HSP 27, HSP 70, and HSP 90) in medulloblastoma.

PubMed

Hauser, Péter; Hanzély, Zoltán; Jakab, Zsuzsanna; Oláh, Lászlóné; Szabó, Erika; Jeney, András; Schuler, Dezso; Fekete, Gyoörgy; Bognár, László; Garami, Miklós

2006-07-01

Expression of heat shock proteins (HSPs) is of prognostic significance in several tumor types. HSP expression levels were determined in medulloblastomas and tested whether HSPs expression was associated with prognostic parameters. Expression of antiapoptotic HSP 27, HSP 70, and HSP 90 was investigated by immunohistochemistry, on paraffin-embedded sections from 65 patients. Expression of HSPs was validated on internal vascular controls and by Western blotting analysis. Sample evaluation was based on the estimated percentage of HSP positive tumor cells. For survival analysis Kaplan-Meier method, for statistical analysis chi2 test, univariate analysis, and log rank test were applied. Expression of HSPs varied in medulloblastomas. On the basis of the average expression rate of HSPs, at HSP 27 and HSP 90 with a 10% cut off, and at HSP 70 with a 70% cut off 2 groups were created. The amount of expression of any of the HSP types was not significantly associated with known prognostic factors (age of patient, extent of resection, presence of metastasis) and histologic subtype. After an average follow-up period of 4.30 years, no significant difference was observed in survival depending on the expression of HSP 27 or HSP 70 or HSP 90. The high expression of HSPs indicates that these proteins are potential therapeutic targets.

Hsp90 Governs Dispersion and Drug Resistance of Fungal Biofilms

PubMed Central

Nett, Jeniel; Rajendran, Ranjith; Ramage, Gordon; Lopez-Ribot, Jose L.; Andes, David; Cowen, Leah E.

2011-01-01

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections. PMID:21931556

Allosteric Regulation of the Hsp90 Dynamics and Stability by Client Recruiter Cochaperones: Protein Structure Network Modeling

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins. PMID:24466147

Allosteric regulation of the Hsp90 dynamics and stability by client recruiter cochaperones: protein structure network modeling.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Chunlan; Department of Physiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058; Oh, Joon Seok

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 significantlymore » 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 protective autophagy in Hep3B cells.« less

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. © 2016 Elsevier Inc. All rights reserved.

FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation

PubMed Central

2014-01-01

Background Heat shock protein 90 (Hsp90) is a promising therapeutic target and inhibition of Hsp90 will presumably result in suppression of multiple signaling pathways. FW-04-806, a bis-oxazolyl macrolide compound extracted from China-native Streptomyces FIM-04-806, was reported to be identical in structure to the polyketide Conglobatin. Methods We adopted the methods of chemproteomics, computational docking, immunoprecipitation, siRNA gene knock down, Quantitative Real-time PCR and xenograft models on the research of FW-04-806 antitumor mechanism, through the HER2-overexpressing breast cancer SKBR3 and HER2-underexpressing breast cancer MCF-7 cell line. Results We have verified the direct binding of FW-04-806 to the N-terminal domain of Hsp90 and found that FW-04-806 inhibits Hsp90/cell division cycle protein 37 (Cdc37) chaperone/co-chaperone interactions, but does not affect ATP-binding capability of Hsp90, thereby leading to the degradation of multiple Hsp90 client proteins via the proteasome pathway. In breast cancer cell lines, FW-04-806 inhibits cell proliferation, caused G2/M cell cycle arrest, induced apoptosis, and downregulated Hsp90 client proteins HER2, Akt, Raf-1 and their phosphorylated forms (p-HER2, p-Akt) in a dose and time-dependent manner. Importantly, FW-04-806 displays a better anti-tumor effect in HER2-overexpressed SKBR3 tumor xenograft model than in HER2-underexpressed MCF-7 model. The result is consistent with cell proliferation assay and in vitro apoptosis assay applied for SKBR-3 and MCF-7. Furthermore, FW-04-806 has a favorable toxicity profile. Conclusions As a novel Hsp90 inhibitor, FW-04-806 binds to the N-terminal of Hsp90 and inhibits Hsp90/Cdc37 interaction, resulting in the disassociation of Hsp90/Cdc37/client complexes and the degradation of Hsp90 client proteins. FW-04-806 displays promising antitumor activity against breast cancer cells both in vitro and in vivo, especially for HER2-overexpressed breast cancer cells. PMID:24927996

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians☆

PubMed Central

Arisue, Nobuko; Sánchez, Lidya B.; Weiss, Louis M.; Müller, Miklós; Hashimoto, Tetsuo

2011-01-01

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past. PMID:11880223

Blocking the chaperone kinome pathway: Mechanistic insights into a novel dual inhibition approach for supra-additive suppression of malignant tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grover, Abhinav; Shandilya, Ashutosh; Agrawal, Vibhuti

2011-01-07

Research highlights: {yields} Withaferin A and 17-DMAG synergistically inhibit the Hsp90-Cdc37 chaperone pair. {yields} Binding of WA to Cdc37 cleft suppresses its kinase binding activity. {yields} 17-DMAG binding to the association complex results in H-bonds with 60% clustering. {yields} The ligands' bound complex was found structurally and thermodynamically stable. -- Abstract: The chaperone Hsp90 is involved in regulating the stability and activation state of more than 200 'client' proteins and takes part in the cancer diseased states. The major clientele-protein kinases depend on Hsp90 for their proper folding and functioning. Cdc37, a kinase targeting co-chaperone of Hsp90, mediates the interactionsmore » between Hsp90 and protein kinases. Targeting of Cdc37 has the prospect of delivering predominantly kinase-selective molecular responses as compared to the current pharmacologic Hsp90 inhibitors. The present work reports a bio-computational study carried out with the aim of exploring the dual inhibition of Hsp90/Cdc37 chaperone/co-chaperone association complex by the naturally occurring drug candidates withaferin A and 17-DMAG along with their possible modes of action. Our molecular docking studies reveal that withaferin A in combination with 17-DMAG can act as potent chaperone system inhibitors. The structural and thermodynamic stability of the ligands' bound complex was also observed from molecular dynamics simulations in water. Our results suggest a novel tumor suppressive action mechanism of herbal ligands which can be looked forward for further clinical investigations for possible anticancer drug formulations.« less

HSP27, 70 and 90, anti-apoptotic proteins, in clinical cancer therapy (Review).

PubMed

Wang, Xiaoxia; Chen, Meijuan; Zhou, Jing; Zhang, Xu

2014-07-01

Among the heat shock proteins (HSP), HSP27, HSP70 and HSP90 are the most studied stress-inducible HSPs, and are induced in response to a wide variety of physiological and environmental insults, thus allowing cells to survive to lethal conditions based on their powerful cytoprotective functions. Different functions of HSPs have been described to explain their cytoprotective functions, including their most basic role as molecular chaperones, that is to regulate protein folding, transport, translocation and assembly, especially helping in the refolding of misfolded proteins, as well as their anti-apoptotic properties. In cancer cells, the expression and/or activity of the three HSPs is abnormally high, and is associated with increased tumorigenicity, metastatic potential of cancer cells and resistance to chemotherapy. Associating with key apoptotic factors, they are powerful anti-apoptotic proteins, having the capacity to block the cell death process at different levels. Altogether, the properties suggest that HSP27, HSP70 and HSP90 are appropriate targets for modulating cell death pathways. In this review, we summarize the role of HSP90, HSP70 and HSP27 in apoptosis and the emerging strategies that have been developed for cancer therapy based on the inhibition of the three HSPs.

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 and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90. PMID:23977182

Synthesis and Evaluation of the Tumor Cell Growth Inhibitory Potential of New Putative HSP90 Inhibitors.

PubMed

Bizarro, Ana; Sousa, Diana; Lima, Raquel T; Musso, Loana; Cincinelli, Raffaella; Zuco, Vantina; De Cesare, Michelandrea; Dallavalle, Sabrina; Vasconcelos, M Helena

2018-02-13

Heat shock protein 90 (HSP90) is a well-known target for cancer therapy. In a previous work, some of us have reported a series of 3-aryl-naphtho[2,3- d ]isoxazole-4,9-diones as inhibitors of HSP90. In the present work, various compounds with new chromenopyridinone and thiochromenopyridinone scaffolds were synthesized as potential HSP90 inhibitors. Their binding affinity to HSP90 was studied in vitro. Selected compounds ( 5 and 8 ) were further studied in various tumor cell lines regarding their potential to cause cell growth inhibition, alter the cell cycle profile, inhibit proliferation, and induce apoptosis. Their effect on HSP90 client protein levels was also confirmed in two cell lines. Finally, the antitumor activity of compound 8 was studied in A431 squamous cell carcinoma xenografts in nude mice. Our results indicated that treatment with compounds 5 and 8 decreased the proliferation of tumor cell lines and compound 8 induced apoptosis. In addition, these two compounds were able to downregulate selected proteins known as "clients" of HSP90. Finally, treatment of xenografted mice with compound 5 resulted in a considerable dose-dependent inhibition of tumor growth. Our results show that two new compounds with a chromenopyridinone and thiochromenopyridinone scaffold are promising putative HSP90 inhibitors causing tumor cell growth inhibition.

Hsp90 activator Aha1 drives production of pathological tau aggregates

PubMed Central

Shelton, Lindsey B.; Baker, Jeremy D.; Zheng, Dali; Sullivan, Leia E.; Solanki, Parth K.; Webster, Jack M.; Sun, Zheying; Sabbagh, Jonathan J.; Nordhues, Bryce A.; Koren, John; Ghosh, Suman; Blagg, Brian S. J.; Dickey, Chad A.

2017-01-01

The microtubule-associated protein tau (MAPT, tau) forms neurotoxic aggregates that promote cognitive deficits in tauopathies, the most common of which is Alzheimer’s disease (AD). The 90-kDa heat shock protein (Hsp90) chaperone system affects the accumulation of these toxic tau species, which can be modulated with Hsp90 inhibitors. However, many Hsp90 inhibitors are not blood–brain barrier-permeable, and several present associated toxicities. Here, we find that the cochaperone, activator of Hsp90 ATPase homolog 1 (Aha1), dramatically increased the production of aggregated tau. Treatment with an Aha1 inhibitor, KU-177, dramatically reduced the accumulation of insoluble tau. Aha1 colocalized with tau pathology in human brain tissue, and this association positively correlated with AD progression. Aha1 overexpression in the rTg4510 tau transgenic mouse model promoted insoluble and oligomeric tau accumulation leading to a physiological deficit in cognitive function. Overall, these data demonstrate that Aha1 contributes to tau fibril formation and neurotoxicity through Hsp90. This suggests that therapeutics targeting Aha1 may reduce toxic tau oligomers and slow or prevent neurodegenerative disease progression. PMID:28827321

Activating HSP72 in rodent skeletal muscle increases mitochondrial number and oxidative capacity and decreases insulin resistance.

PubMed

Henstridge, Darren C; Bruce, Clinton R; Drew, Brian G; Tory, Kálmán; Kolonics, Attila; Estevez, Emma; Chung, Jason; Watson, Nadine; Gardner, Timothy; Lee-Young, Robert S; Connor, Timothy; Watt, Matthew J; Carpenter, Kevin; Hargreaves, Mark; McGee, Sean L; Hevener, Andrea L; Febbraio, Mark A

2014-06-01

Induction of heat shock protein (HSP)72 protects against obesity-induced insulin resistance, but the underlying mechanisms are unknown. Here, we show that HSP72 plays a pivotal role in increasing skeletal muscle mitochondrial number and oxidative metabolism. Mice overexpressing HSP72 in skeletal muscle (HSP72Tg) and control wild-type (WT) mice were fed either a chow or high-fat diet (HFD). Despite a similar energy intake when HSP72Tg mice were compared with WT mice, the HFD increased body weight, intramuscular lipid accumulation (triacylglycerol and diacylglycerol but not ceramide), and severe glucose intolerance in WT mice alone. Whole-body VO2, fatty acid oxidation, and endurance running capacity were markedly increased in HSP72Tg mice. Moreover, HSP72Tg mice exhibited an increase in mitochondrial number. In addition, the HSP72 coinducer BGP-15, currently in human clinical trials for type 2 diabetes, also increased mitochondrial number and insulin sensitivity in a rat model of type 2 diabetes. Together, these data identify a novel role for activation of HSP72 in skeletal muscle. Thus, the increased oxidative metabolism associated with activation of HSP72 has potential clinical implications not only for type 2 diabetes but also for other disorders where mitochondrial function is compromised. © 2014 by the American Diabetes Association.

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage.

PubMed

Barker, Catherine R; McNamara, Anne V; Rackstraw, Stephen A; Nelson, David E; White, Mike R; Watson, Alastair J M; Jenkins, John R

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90-topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage

PubMed Central

Barker, Catherine R.; McNamara, Anne V.; Rackstraw, Stephen A.; Nelson, David E.; White, Mike R.; Watson, Alastair J. M.; Jenkins, John R.

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90–topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death. PMID:16504968

HSP90 regulates cell survival via inositol hexakisphosphate kinase-2

PubMed Central

Chakraborty, Anutosh; Koldobskiy, Michael A.; Sixt, Katherine M.; Juluri, Krishna R.; Mustafa, Asif K.; Snowman, Adele M.; van Rossum, Damian B.; Patterson, Randen L.; Snyder, Solomon H.

2008-01-01

Heat-shock proteins (HSPs) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. Inositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate IP7 [diphosphoinositol pentakisphosphate (5-PP-IP5)], mediates apoptosis. Increased IP6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds IP6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90–IP6K2 binding elicit activation of IP6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy. PMID:18195352

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

PubMed

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

2014-02-28

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

Macrocycles that inhibit the binding between heat shock protein 90 and TPR-containing proteins

PubMed Central

Ardi, Veronica C.; Alexander, Leslie D.; Johnson, Victoria; McAlpine, Shelli R.

2011-01-01

Heat shock protein 90 (Hsp90) accounts for 1–2% of the total proteins in normal cells and functions as a molecular chaperone that folds, assembles, and stabilizes client proteins. Hsp90 is over-expressed (3–6-fold increase) in stressed cells, including cancer cells, and regulates over 200 client and co-chaperone proteins. Hsp90 client proteins are involved in a plethora of cellular signaling events including numerous growth and apoptotic pathways. Since pathway-specific inhibitors can be problematic in drug-resistant cancers, shutting down multiple pathways at once is a promising approach when developing new therapeutics. Hsp90’s ability to modulate many growth and signaling pathways simultaneously makes this protein an attractive target in the field of cancer therapeutics. Herein we present evidence that a small molecule modulates Hsp90 via binding between the N and middle domain and allosterically inhibiting the binding interaction between Hsp90 and four C-terminal binding client proteins: IP6K2, FKBP38, FKBP52, and HOP. These last three clients contain a tetratricopeptide-repeat (TPR) region, which is known to interact with the MEEVD sequence on the C-terminus of Hsp90. Thus, this small molecule modulates the activity between co-chaperones that contain TPR motifs and Hsp90’s MEEVD region. This mechanism of action is unique from that of all Hsp90 inhibitors currently in clinical trials where these molecules have no effect on proteins that bind to the C-terminus of Hsp90. Further, our small molecule induces a Caspase-3 dependent apoptotic event. Thus, we describe the mechanism of a novel scaffold that is a useful tool for studying cell-signaling events that result when blocking the MEEVD-TPR interaction between Hsp90 and co-chaperone proteins. PMID:21950602

KU135, a Novel Novobiocin-Derived C-Terminal Inhibitor of the 90-kDa Heat Shock Protein, Exerts Potent Antiproliferative Effects in Human Leukemic Cells

PubMed Central

Shelton, Shary N.; Shawgo, Mary E.; Matthews, Shawna B.; Lu, Yuanming; Donnelly, Alison C.; Szabla, Kristen; Tanol, Mehmet; Vielhauer, George A.; Rajewski, Roger A.; Matts, Robert L.; Blagg, Brian S. J.

2009-01-01

The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Consequently, there is considerable interest in developing chemotherapeutic drugs that specifically disrupt the function of Hsp90. Here, we investigated the extent to which a novel novobiocin-derived C-terminal Hsp90 inhibitor, designated KU135, induced antiproliferative effects in Jurkat T-lymphocytes. The results indicated that KU135 bound directly to Hsp90, caused the degradation of known Hsp90 client proteins, and induced more potent antiproliferative effects than the established N-terminal Hsp90 inhibitor 17-allylamino-demethoxygeldanamycin (17-AAG). Closer examination of the cellular response to KU135 and 17-AAG revealed that only 17-AAG induced a strong up-regulation of Hsp70 and Hsp90. In addition, KU135 caused wild-type cells to undergo G2/M arrest, whereas cells treated with 17-AAG accumulated in G1. Furthermore, KU135 but not 17-AAG was found to be a potent inducer of mitochondria-mediated apoptosis as evidenced, in part, by the fact that cell death was inhibited to a similar extent by Bcl-2/Bcl-xL overexpression or the depletion of apoptotic protease-activating factor-1 (Apaf-1). Together, these data suggest that KU135 inhibits cell proliferation by regulating signaling pathways that are mechanistically different from those targeted by 17-AAG and as such represents a novel opportunity for Hsp90 inhibition. PMID:19741006

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

Evidence for loss of mitochondria in Microsporidia from a mitochondrial-type HSP70 in Nosema locustae.

PubMed

Germot, A; Philippe, H; Le Guyader, H

1997-08-01

In molecular phylogenies based on ribosomal RNA, three amitochondriate protist lineages, Microsporidia, Metamonada (including diplomonads) and Parabasala (including trichomonads), are the earliest offshoots of the eukaryotic tree. As an explantation for the lack of mitochondria in these organisms, the hypothesis that they have diverged before the mitochondrial endosymbiosis is preferred to the less parsimonious hypothesis of several independent losses of the organelle. Nevertheless, if they had descended from mitochondrion-containing ancestors, it may be possible to find in their nuclear DNA genes that derive from the endosymbiont which gave rise to mitochondria. Based on similar evidence, secondary losses of mitochondria have recently been suggested for Entamoeba histolytica and for Trichomonas vaginalis. In this study, we have isolated a gene encoding a chaperone protein (HSP70, 70 kDa heat shock protein) from the microspordian Nosema locustae. In phylogenetic trees, this HSP70 was located within a group of sequences that in other lineages is targetted to the mitochondrial compartment, itself included in the proteobacterial clade. In addition, the N. locustae protein contained the GDAW(V) motif shared by mitochondrial and proteobacterial sequences, with only one conservative substitution. Moreover, microsporidia, a phylum which was assumed to emerge close to the base of the eukaryotic tree, appears as the sister-group of fungi in the HSP70 phylogeny, in agreement with some ultrastructural characters and phylogenies based on alpha- and beta-tubulins. Loss of mitochondria, now demonstrated for several amitochondriate groups, indicates that the common ancestor of all the extant eukaryotic species could have been a mitochondriate eukaryote.

Acetylation of androgen receptor by ARD1 promotes dissociation from HSP90 complex and prostate tumorigenesis

PubMed Central

Zhang, Guanyi; Qian, Chiping; Zhang, Haitao; Zabaleta, Jovanny; Liu, Wanguo

2016-01-01

Prostate cancer is an androgen receptor (AR)-driven disease and post-translational modification of AR is critical for AR activation. We previously reported that Arrest-defective protein 1 (ARD1) is an oncoprotein in prostate cancer. It acetylates and activates AR to promote prostate tumorigenesis. However, the ARD1-targeted residue within AR and the mechanisms of the acetylation event in prostate tumorigenesis remained unknown. In this study, we show that ARD1 acetylates AR at lysine 618 (K618) in vitro and in vivo. An AR construct with the charged lysine substitution by arginine (AR-618R) reduces RNA Pol II binding, AR transcriptional activity, prostate cancer cell growth, and xenograft tumor formation due to attenuation of AR nuclear translocation, whereas, construct mimicking neutral polar substitution acetylation at K618 by glutamine (AR-618Q) enhanced these effects beyond that of the wild-type AR. Mechanistically, ARD1 forms a ternary complex with AR and HSP90 in vitro and in vivo. Expression of ARD1 increases levels of AR acetylation and AR-HSP90 dissociation in a dose dependent manner. Moreover, the AR acetylation defective K618R mutant is unable to dissociate from HSP90 while the HSP90-dissociated AR is acetylated following ligand exposure. This work identifies a new mechanism for ligand-induced AR-HSP90 dissociation and AR activation. Targeting ARD1-mediated AR acetylation may be a potent intervention for AR-dependent prostate cancer therapy. PMID:27659526

Antimyeloma activity of heat shock protein-90 inhibition.

PubMed

Mitsiades, Constantine S; Mitsiades, Nicholas S; McMullan, Ciaran J; Poulaki, Vassiliki; Kung, Andrew L; Davies, Faith E; Morgan, Gareth; Akiyama, Masaharu; Shringarpure, Reshma; Munshi, Nikhil C; Richardson, Paul G; Hideshima, Teru; Chauhan, Dharminder; Gu, Xuesong; Bailey, Charles; Joseph, Marie; Libermann, Towia A; Rosen, Neal S; Anderson, Kenneth C

2006-02-01

We show that multiple myeloma (MM), the second most commonly diagnosed hematologic malignancy, is responsive to hsp90 inhibitors in vitro and in a clinically relevant orthotopic in vivo model, even though this disease does not depend on HER2/neu, bcr/abl, androgen or estrogen receptors, or other hsp90 chaperoning clients which are hallmarks of tumor types traditionally viewed as attractive clinical settings for use of hsp90 inhibitors, such as the geldanamycin analog 17-AAG. This class of agents simultaneously suppresses in MM cells the expression and/or function of multiple levels of insulin-like growth factor receptor (IGF-1R) and interleukin-6 receptor (IL-6R) signaling (eg, IKK/NF-kappaB, PI-3K/Akt, and Raf/MAPK) and downstream effectors (eg, proteasome, telomerase, and HIF-1alpha activities). These pleiotropic proapoptotic effects allow hsp90 inhibitors to abrogate bone marrow stromal cell-derived protection on MM tumor cells, and sensitize them to other anticancer agents, including cytotoxic chemotherapy and the proteasome inhibitor bortezomib. These results indicate that hsp90 can be targeted therapeutically in neoplasias that may not express or depend on molecules previously considered to be the main hsp90 client proteins. This suggests a more general role for hsp90 in chaperoning tumor- or tissue-type-specific constellations of client proteins with critical involvement in proliferative and antiapoptotic cellular responses, and paves the way for more extensive future therapeutic applications of hsp90 inhibition in diverse neoplasias, including MM.

Novobiocin and additional inhibitors of the Hsp90 C-terminal nucleotide-binding pocket.

PubMed

Donnelly, Alison; Blagg, Brian S J

2008-01-01

The 90 kDa heat shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site ( approximately 700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones. Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-linked DNA, which prohibits rapidly dividing cells from duplicating DNA for mitosis. Itoh and co-workers reported that cisplatin decreases the chaperone activity of Hsp90. This group applied bovine brain cytosol to a cisplatin affinity column, eluted with cisplatin and detected Hsp90 in the eluent. Subsequent experiments indicated that cisplatin exhibits high affinity for Hsp90. Moreover Csermely and co-workers determined that the cisplatin binding site is located proximal to the C-terminal ATP binding site. EGCG is one of the active ingredients found in green tea. EGCG is known to inhibit the activity of many Hsp90-dependent client proteins, including telomerase, several kinases, and the aryl hydrocarbon receptor (AhR). Recently Gasiewicz and co-workers reported that EGCG manifests its antagonistic activity against AhR through binding Hsp90. Similar to novobiocin, EGCG was shown to bind the C-terminus of Hsp90. Unlike previously identified N-terminal Hsp90 inhibitors, EGCG does not appear to prevent Hsp90 from forming multiprotein complexes. Studies are currently underway to determine whether EGCG competes with novobiocin or cisplatin binding. Taxol, a well-known drug for the treatment of cancer, is responsible for the stabilization of microtubules and the inhibition of mitosis. Previous studies have shown that taxol induces the activation of kinases and transcription factors, and mimics the effect of bacterial lipopolysaccharide (LPS), an attribute unrelated to its tubulin-binding properties. Rosen and co-workers prepared a biotinylated taxol derivative and performed affinity chromatography experiments with lysates from both mouse brain and macrophage cell lines. These studies led to identification of two chaperones, Hsp70 and Hsp90, by mass spectrometry. In contrast to typical Hsp90-binding drugs, taxol exhibits a stimulatory response. Recently it was reported that the geldanamycin derivative 17-AAG behaves synergistically with taxol-induced apoptosis. This review describes the different C-terminal inhibitors of Hsp90, with specific emphasis on structure-activity relationship studies of novobiocin and their effects on anti-proliferative activity.

Novobiocin and Additional Inhibitors of the Hsp90 C-Terminal Nucleotide-binding Pocket

PubMed Central

Donnelly, Alison; Blagg, Brian S. J.

2009-01-01

The 90 kDa heal shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site (~700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones. Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-linked DNA, which prohibits rapidly dividing cells from duplicating DNA for mitosis. Itoh and co-workers reported that cisplatin decreases the chaperone activity of Hsp90. This group applied bovine brain cytosol to a cisplatin affinity column, eluted with cisplatin and detected Hsp90 in the eluent. Subsequent experiments indicated that cisplatin exhibits high affinity for Hsp90. Moreover Csermely and co-workers determined that the cisplatin binding site is located proximal to the C-terminal ATP binding site. EGCG is one of the active ingredients found in green tea EGCG is known to inhibit the activity of many Hsp90-dependent client proteins, including telomerase, several kinases, and the aryl hydrocarbon receptor (AhR). Recently Gasiewicz and co-workers reported that EGCG manifests its antagonistic activity against AhR through binding Hsp90. Similar to novobiocin, EGCG was shown to bind the C-terminus of Hsp90. Unlike previously identified N-terminal Hsp90 inhibitors, EGCG does not appear to prevent Hsp90 from forming multiprotein complexes. Studies are currently underway to determine whether EGCG competes with novobiocin or cisplatin binding. Taxol, a well-known drug for the treatment of cancer, is responsible for the stabilization of microtubules and the inhibition of mitosis. Previous studies have shown that taxol induces the activation of kinases and transcription factors, and mimies the effect of bacterial lipopolysaccharide (LPS), an attribute unrelated to its tubulin-binding properties. Rosen and co-workers prepared a biotinylated taxol derivative and performed affinity chromatography experiments with lysates from both mouse brain and macrophage cell lines. These studies led to identification of two chaperones. Hsp70 and Hsp90, by mass spectrometry. In contrast to typical Hsp90-binding drugs, taxol exhibits a stimulatory response. Recently it was reported that the geldanamycin derivative 17-AAG behaves synergistically with taxol-induced apoptosis. This review describes the different C-terminal inhibitors of Hsp90, with specific emphasis on structure-activity relationship studies of novobiocin and their effects on anti-proliferative activity. PMID:18991631

Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors.

PubMed

Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine; Ahlbrecht, Christin; Stahl, Frank; Scheper, Thomas; Preller, Matthias; Surup, Frank; Stadler, Marc; Kirschning, Andreas; Zeilinger, Carsten

2017-12-15

A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC 50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein-protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Heat-shock protein 27 (HSP27, HSPB1) is synthetic lethal to cells with oncogenic activation of MET, EGFR and BRAF.

PubMed

Konda, John D; Olivero, Martina; Musiani, Daniele; Lamba, Simona; Di Renzo, Maria F

2017-06-01

The small heat-shock protein of 27 kDa (HSP27) is highly expressed in many cancers and is associated with aggressive tumour behaviour, metastasis, poor prognosis and resistance to chemotherapy. We aimed at assessing the role of HSP27 in modulating responses to target therapies. We selected several oncogene-addicted cancer cell lines, which undergo either cell cycle blockade or cell death in response to agents that target the specific oncogene. Surprisingly, HSP27 suppression alone resulted in the apoptotic death of MET-addicted EBC-1 lung cancer cells, epidermal growth factor receptor (EGFR)-addicted colorectal carcinoma (CRC) DiFi cells and BRAF-addicted CRC COLO205 and OXCO-1 and melanoma COLO741 cells, all of which also undergo death when treated with the specific targeted agent. In other cell lines, such as MET-addicted gastric carcinoma MKN45 and EGFR-addicted CRC SW48 lines, where oncogene inhibition only blocked proliferation, HSP27 knockdown made targeted agents switch from cytostatic to cytotoxic activity. Mechanistically, the more the cells were susceptible to HSP27 suppression, the more they were primed for death, as demonstrated by increased levels of mitochondrial outer membrane permeabilization. Priming for death was accompanied by the increase in pro-apoptotic proteins of the BCL2 family and of active caspase-3 and lamin B. Together, these data suggest that oncogene-addicted cells require HSP27 for survival and that HSP27 might interfere with the effectiveness of targeted agents. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

IMPACT (Imaging and Molecular Markers for Patients with Lung Cancer: Approaches with Molecular Targets and Complementary, Innovative Treatments and Therapeutic Modalities)

DTIC Science & Technology

2010-02-01

in t heir o ptimal g rowth media a nd h arvested an d l ysed t hen analyzed by Western bl otting f or t he expression of H sp90, Hsp27 , Hsp60...analyzed by western blotting for the expression of Hsp27 , 70 and 90. The same membrane was probed with actin as a control for protein loading

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Dan, E-mail: DZhou@syntapharma.com; Liu, Yuan; Ye, Josephine

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 andmore » 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 elimination rate are linked to toxicity potential. • Rat retinotoxic responses to individual Hsp90 inhibitors reflect clinical profiles. • Rodent modeling may be used to assess ocular risks of targeted Hsp90 compounds.« less

17-DMAG induces heat shock protein 90 functional impairment in human bladder cancer cells: knocking down the hallmark traits of malignancy.

PubMed

Karkoulis, Panagiotis K; Stravopodis, Dimitrios J; Voutsinas, Gerassimos E

2016-05-01

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains the structural and functional integrity of various protein clients involved in multiple oncogenic signaling pathways. Hsp90 holds a prominent role in tumorigenesis, as numerous members of its broad clientele are involved in the generation of the hallmark traits of cancer. 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) specifically targets Hsp90 and interferes with its function as a molecular chaperone, impairing its intrinsic ATPase activity and undermining proper folding of multiple protein clients. In this study, we have examined the effects of 17-DMAG on the regulation of Hsp90-dependent tumorigenic signaling pathways directly implicated in cell cycle progression, survival, and motility of human urinary bladder cancer cell lines. We have used MTT-based assays, FACS analysis, Western blotting, semiquantitative PCR (sqPCR), immunofluorescence, and scratch-wound assays in RT4 (p53(wt)), RT112 (p53(wt)), T24 (p53(mt)), and TCCSUP (p53(mt)) human urinary bladder cancer cell lines. We have demonstrated that, upon exposure to 17-DMAG, bladder cancer cells display prominent cell cycle arrest and commitment to apoptotic and autophagic cell death, in a dose-dependent manner. Furthermore, 17-DMAG administration induced pronounced downregulation of multiple Hsp90 protein clients and other downstream oncogenic effectors, therefore causing inhibition of cell proliferation and decline of cell motility due to the molecular "freezing" of critical cytoskeletal components. In toto, we have clearly demonstrated the dose-dependent and cell type-specific effects of 17-DMAG on the hallmark traits of cancer, appointing Hsp90 as a key molecular component in bladder cancer targeted therapy.

Targeting Heat Shock Protein 90 Overrides the Resistance of Lung Cancer Cells by Blocking Radiation-induced Stabilization of Hypoxia-inducible Factor 1α

PubMed Central

Kim, Woo-Young; Oh, Seung Hyun; Woo, Jong-Kyu; Hong, Waun Ki; Lee, Ho-Young

2008-01-01

Hypoxia-inducible factor-1 (HIF-1) has been suggested to play a major role in tumor radioresistance. However, the mechanisms through which irradiation regulates HIF-1α expression remain unclear. The purpose of this study was to investigate the mechanisms that mediate HIF-1 activation and thus radioresistance. Here we show that irradiation induces survival and angiogenic activity in a subset of radioresistant lung cancer cell lines by elevating HIF-1α protein expression. Radiation induced HIF-1α protein expression mainly through two distinct pathways, including an increase in de novo protein synthesis via activation of PI3K/Akt/mTOR and stabilization of HIF-1α protein via augmenting the interaction between heat shock protein 90 (Hsp90) and HIF-1α protein. While the PI3K/Akt/mTOR pathway was activated by irradiation in all the lung cancer cells examined, the HSP90-HIF-1α interaction was enhanced in the resistant cells only. Inhibition of Hsp90 function by 17-AAG or deguelin, a novel natural inhibitor of HSP90, suppressed increases in HIF-1α/Hsp90 interaction and HIF-1α expression in radioresistant cells. Furthermore, combined treatment of radiation with deguelin significantly decreased the survival and angiogenic potential of radioresistant lung cancer cells in vitro. We finally determined in vivo that systemic administration of deguelin resulted in profound inhibition of tumor growth and angiogenesis when combined with radiation. These results provide a strong rationale to target Hsp90 as a means to block radiation-induced HIF-1α and thus to circumvent radioresistance in lung cancer cells. PMID:19176399

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

PubMed Central

2011-01-01

Background The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. Methods PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. Results KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. Conclusions Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer. PMID:22039910

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

PubMed Central

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.

2013-01-01

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, 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. We report here 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. PMID:23511419

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis

PubMed Central

Guo, Yang; Ziesch, Andreas; Hocke, Sandra; Kampmann, Eric; Ochs, Stephanie; De Toni, Enrico N; Göke, Burkhard; Gallmeier, Eike

2015-01-01

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients. PMID:25331547

Dual targeting of mitochondrial function and mTOR pathway as a therapeutic strategy for diffuse intrinsic pontine glioma

PubMed Central

Tsoli, Maria; Liu, Jie; Franshaw, Laura; Shen, Han; Cheng, Cecilia; Jung, MoonSun; Joshi, Swapna; Ehteda, Anahid; Khan, Aaminah; Montero-Carcabosso, Angel; Dilda, Pierre J.; Hogg, Philip; Ziegler, David S.

2018-01-01

Diffuse Intrinsic Pontine Gliomas (DIPG) are the most devastating of all pediatric brain tumors. They mostly affect young children and, as there are no effective treatments, almost all patients with DIPG will die of their tumor within 12 months of diagnosis. A key feature of this devastating tumor is its intrinsic resistance to all clinically available therapies. It has been shown that glioma development is associated with metabolic reprogramming, redox state disruption and resistance to apoptotic pathways. The mitochondrion is an attractive target as a key organelle that facilitates these critical processes. PENAO is a novel anti-cancer compound that targets mitochondrial function by inhibiting adenine nucleotide translocase (ANT). Here we found that DIPG neurosphere cultures express high levels of ANT2 protein and are sensitive to the mitochondrial inhibitor PENAO through oxidative stress, while its apoptotic effects were found to be further enhanced upon co-treatment with mTOR inhibitor temsirolimus. This combination therapy was found to act through inhibition of PI3K/AKT/mTOR pathway, HSP90 and activation of AMPK. In vivo experiments employing an orthotopic model of DIPG showed a marginal anti-tumour effect likely due to poor penetration of the inhibitors into the brain. Further testing of this anti-DIPG strategy with compounds that penetrate the BBB is warranted. PMID:29484131

N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting.

PubMed

Lin, Pei-Hsuan; Lin, Hsien-Yi; Kuo, Cheng-Chin; Yang, Liang-Tung

2015-06-24

The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3's action remain unanswered. We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants. Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on mitochondria.

Plasmodium falciparum Hop (PfHop) Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity.

PubMed

Zininga, Tawanda; Makumire, Stanely; Gitau, Grace Wairimu; Njunge, James M; Pooe, Ofentse Jacob; Klimek, Hanna; Scheurr, Robina; Raifer, Hartmann; Prinsloo, Earl; Przyborski, Jude M; Hoppe, Heinrich; Shonhai, Addmore

2015-01-01

Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally eluted as a protein species of twice its predicted size, suggesting that it may occur as a dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition, the protein is capable of self-association. The findings suggest that PfHop serves as a module that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this study are important towards the development of possible antimalarial inhibitors targeting the cooperation of these two chaperones.

Sulforaphane Potentiates the Efficacy of 17-Allylamino 17-Demethoxygeldanamycin Against Pancreatic Cancer Through Enhanced Abrogation of Hsp90 Chaperone Function

PubMed Central

Li, Yanyan; Zhang, Tao; Schwartz, Steven J.; Sun, Duxin

2013-01-01

Heat shock protein 90 (Hsp90), an essential molecular chaperone that regulates the stability of a wide range of oncogenic proteins, is a promising target for cancer therapeutics. We investigated the combination efficacy and potential mechanisms of sulforaphane, a dietary component from broccoli and broccoli sprouts, and 17-allylamino 17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, in pancreatic cancer. MTS assay demonstrated that sulforaphane sensitized pancreatic cancer cells to 17-AAG in vitro. Caspase-3 was activated to 6.4-fold in response to simultaneous treatment with sulforaphane and 17-AAG, whereas 17-AAG alone induced caspase-3 activity to 2-fold compared to control. ATP binding assay and coimmunoprecipitation revealed that sulforaphane disrupted Hsp90-p50Cdc37 interaction, whereas 17-AAG inhibited ATP binding to Hsp90. Concomitant use of sulforaphane and 17-AAG synergistically downregulated Hsp90 client proteins in Mia Paca-2 cells. Co-administration of sulforaphane and 17-AAG in pancreatic cancer xenograft model led to more than 70% inhibition of the tumor growth, whereas 17-AAG alone only suppressed the tumor growth by 50%. Our data suggest that sulforaphane potentiates the efficacy of 17-AAG against pancreatic cancer through enhanced abrogation of Hsp90 function. These findings provide a rationale for further evaluation of broccoli/broccoli sprout preparations combined with 17-AAG for better efficacy and lower dose-limiting toxicity in pancreatic cancer. PMID:21875325

Targeting SRC Family Kinases in HSP90 in Lung Cancer

DTIC Science & Technology

2015-10-01

shock protein ( HSP ) 90 are both associated with cancer progression, invasion, tumor angiogenesis and drug-resistance, and both are targets of...vitro, with an IC50 of 10 nM, and reducing the viability by 70 % at 30-50 nM. 2) Dasatinib was less effective in these cells, with an IC50 of 100 nM...3) Ganetespib treatment reduced the rate of growth of tumors in this model. Although the average degree of reduction was marked ( 70 -80% less in

Preclinical Study of AUY922, a Novel Hsp90 Inhibitor, in the Treatment of Esophageal Adenocarcinoma.

PubMed

Kosovec, Juliann E; Zaidi, Ali H; Kelly, Lori A; Rotoloni, Christina L; Vytlacil, Christopher; DiCarlo, Christina; Matsui, Daisuke; Komatsu, Yoshihiro; Boyd, Natalie H; Omstead, Ashten; Kolano, Elena L; Biederman, Robert W W; Finley, Gene; Silverman, Jan F; Landreneau, Rodney J; Jobe, Blair A

2016-08-01

To assess the efficacy of heat-shock protein 90 (Hsp90) inhibitor, NVP-AUY922-AG (AUY922), in the treatment of esophageal adenocarcinoma (EAC) in vitro and in vivo. EAC is a leading cause of cancer death, and current treatment options are limited. Hsp90, a chaperone protein that regulates several oncoproteins, is upregulated in EAC, and may be a novel target for therapy. In vitro, EAC cell lines were utilized to evaluate AUY922, alone and in combination with 5-fluorouracil (5-FU) and cisplatin. BrdU ELISA and flow cytometry were used to assess proliferation and measure apoptosis, respectively. Western blot and RT-PCR were performed to quantitate Hsp90 pathway expression. In vivo, esophagojejunostomy was performed on rats and treatment animals received AUY922 32 to 40 weeks postoperatively. Drug efficacy was evaluated with magnetic resonance imaging (MRI), endoscopic biopsy, gross histological evaluation, and Hsp90 pathway expression. In vitro, AUY922 demonstrated antiproliferative activity in both cell lines and showed enhanced efficacy with cisplatin and 5-FU. Western Blot and RT-PCR demonstrated downregulation of CDK1 and CDK4 and upregulation of Hsp72. In vivo, AUY922 showed decrease in tumor volume in 36.4% of rats (control = 9.4%), increase in 9.1% (control = 37.5%), and stable disease in 54.5% (control = 43.7%). Necropsy confirmed the presence of EAC in 50% of treatment animals and 75% of control animals. mRNA expression, pre- and posttreatment, demonstrated significant downregulation of MIF, Hsp70, Hsp90β, and CDK4, and upregulation of Hsp72. AUY922 exhibits antitumor efficacy in vitro and in vivo for EAC, suggesting the need for human clinical trials.

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-hepatocellular carcinoma therapeutic agent in the future.

The heat shock protein 60 promotes progesterone synthesis in mitochondria of JEG-3 cells.

PubMed

Monreal-Flores, Jessica; Espinosa-García, María Teresa; García-Regalado, Alejandro; Arechavaleta-Velasco, Fabian; Martínez, Federico

2017-06-01

Progesterone synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental progesterone synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in progesterone synthesis. Here, we examined the role of HSP60 in progesterone synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes progesterone synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes progesterone synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial progesterone synthesis. These findings provide novel insights into progesterone synthesis in the human placenta and its role in maintaining pregnancy. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Inhibition of heat-shock protein 90 sensitizes liver cancer stem-like cells to magnetic hyperthermia and enhances anti-tumor effect on hepatocellular carcinoma-burdened nude mice

PubMed Central

Yang, Rui; Tang, Qiusha; Miao, Fengqin; An, Yanli; Li, Mengfei; Han, Yong; Wang, Xihui; Wang, Juan; Liu, Peidang; Chen, Rong

2015-01-01

Purpose To explore the thermoresistance and expression of heat-shock protein 90 (HSP90) in magnetic hyperthermia-treated human liver cancer stem-like cells (LCSCs) and the effects of a heat-shock protein HSP90 inhibitor 17-allylamino-17-demethoxgeldanamycin (17-AAG) on hepatocellular carcinoma-burdened nude mice. Methods CD90+ LCSCs were isolated by magnetic-activated cell sorting from BEL-7404. Spheroid formation, proliferation, differentiation, drug resistance, and tumor formation assays were performed to identify stem cell characteristics. CD90-targeted thermosensitive magnetoliposomes (TMs)-encapsulated 17-AAG (CD90@17-AAG/TMs) was prepared by reverse-phase evaporation and its characteristics were studied. Heat tolerance in CD90+ LCSCs and the effect of CD90@17-AAG/TMs-mediated heat sensitivity were examined in vitro and in vivo. Results CD90+ LCSCs showed significant stem cell-like properties. The 17-AAG/TMs were successfully prepared and were spherical in shape with an average size of 128.9±7.7 nm. When exposed to magnetic hyperthermia, HSP90 was up-regulated in CD90+ LCSCs. CD90@17-AAG/TMs inhibited the activity of HSP90 and increased the sensitivity of CD90+ LCSCs to magnetic hyperthermia. Conclusion The inhibition of HSP90 could sensitize CD90+ LCSCs to magnetic hyperthermia and enhance its anti-tumor effects in vitro and in vivo. PMID:26677324

Hsf1 in Her2-Positive Breast Cancer

DTIC Science & Technology

2012-10-01

to heat shock proteins (Hsps) such as Hsp70, Hsp90, and Hsp27 , though besides Hsps, Hsf1 regulates hundreds of other targets (32). Initially, Hsps...phos- phorylation of p38MAPK and phosphorylation of its substrate Hsp27 (Fig. 2A). Activation of p38MAPK led to the activation of its down- stream...IR as demonstrated by suppression of Hsp27 phosphorylation and IL-6 and IL-8 transcription (Fig. 2A; Fig. S10B). Importantly, inhibition of p38MAPK

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy

PubMed Central

Datta, Ritwik; Bansal, Trisha; Rana, Santanu; Datta, Kaberi; Datta Chaudhuri, Ratul; Chawla-Sarkar, Mamta

2016-01-01

ABSTRACT Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats (Rattus norvegicus) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy. PMID:28031326

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy.

PubMed

Datta, Ritwik; Bansal, Trisha; Rana, Santanu; Datta, Kaberi; Datta Chaudhuri, Ratul; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

2017-03-15

Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats ( Rattus norvegicus ) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy. Copyright © 2017 American Society for Microbiology.

Overexpression of GmHsp90s, a Heat Shock Protein 90 (Hsp90) Gene Family Cloning from Soybean, Decrease Damage of Abiotic Stresses in Arabidopsis thaliana

PubMed Central

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

2013-01-01

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

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60.

PubMed

Yamamoto, Soh; Okamoto, Tomoya; Ogasawara, Noriko; Hashimoto, Shin; Shiraishi, Tsukasa; Sato, Toyotaka; Yamamoto, Keisuke; Tsutsumi, Hiroyuki; Takano, Kenichi; Himi, Testuo; Itoh, Hideaki; Yokota, Shin-Ichi

2017-02-12

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2. Copyright © 2016 Elsevier Inc. All rights reserved.

The novel Hsp90 inhibitor NXD30001 induces tumor regression in a genetically engineered mouse model of glioblastoma multiforme.

PubMed

Zhu, Haihao; Woolfenden, Steve; Bronson, Roderick T; Jaffer, Zahara M; Barluenga, Sofia; Winssinger, Nicolas; Rubenstein, Allan E; Chen, Ruihong; Charest, Al

2010-09-01

Glioblastoma multiforme (GBM) has an abysmal prognosis. We now know that the epidermal growth factor receptor (EGFR) signaling pathway and the loss of function of the tumor suppressor genes p16Ink4a/p19ARF and PTEN play a crucial role in GBM pathogenesis: initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. We have recently shown that this genetic combination is sufficient to promote the development of GBM in adult mice. Therapeutic agents raised against single targets of the EGFR signaling pathway have proven rather inefficient in GBM therapy, showing the need for combinatorial therapeutic approaches. An effective strategy for concurrent disruption of multiple signaling pathways is via the inhibition of the molecular chaperone heat shock protein 90 (Hsp90). Hsp90 inhibition leads to the degradation of so-called client proteins, many of which are key effectors of GBM pathogenesis. NXD30001 is a novel second generation Hsp90 inhibitor that shows improved pharmacokinetic parameters. Here we show that NXD30001 is a potent inhibitor of GBM cell growth in vitro consistent with its capacity to inhibit several key targets and regulators of GBM biology. We also show the efficacy of NXD30001 in vivo in an EGFR-driven genetically engineered mouse model of GBM. Our findings establish that the Hsp90 inhibitor NXD30001 is a therapeutically multivalent molecule, whose actions strike GBM at the core of its drivers of tumorigenesis and represent a compelling rationale for its use in GBM treatment.

Drug sensitivity profiling identifies potential therapies for lymphoproliferative disorders with overactive JAK/STAT3 signaling

PubMed Central

Kuusanmäki, Heikki; Dufva, Olli; Parri, Elina; van Adrichem, Arjan J.; Rajala, Hanna; Majumder, Muntasir M.; Yadav, Bhagwan; Parsons, Alun; Chan, Wing C.; Wennerberg, Krister; Mustjoki, Satu; Heckman, Caroline A.

2017-01-01

Constitutive JAK/STAT3 signaling contributes to disease progression in many lymphoproliferative disorders. Recent genetic analyses have revealed gain-of-function STAT3 mutations in lymphoid cancers leading to hyperactivation of STAT3, which may represent a potential therapeutic target. Using a functional reporter assay, we screened 306 compounds with selective activity against various target molecules to identify drugs capable of inhibiting the cellular activity of STAT3. Top hits were further validated with additional models including STAT3-mutated natural killer (NK)-cell leukemia/lymphoma cell lines and primary large granular lymphocytic (LGL) leukemia cells to assess their ability to inhibit STAT3 phosphorylation and STAT3 dependent cell viability. We identified JAK, mTOR, Hsp90 and CDK inhibitors as potent inhibitors of both WT and mutant STAT3 activity. The Hsp90 inhibitor luminespib was highly effective at reducing the viability of mutant STAT3 NK cell lines and LGL leukemia patient samples. Luminespib decreased the phosphorylation of mutant STAT3 at Y705, whereas JAK1/JAK2 inhibitor ruxolitinib had reduced efficacy on mutant STAT3 phosphorylation. Additionally, combinations involving Hsp90, JAK and mTOR inhibitors were more effective at reducing cell viability than single agents. Our findings show alternative approaches to inhibit STAT3 activity and suggest Hsp90 as a therapeutic target in lymphoproliferative disorders with constitutively active STAT3. PMID:29228628

Atomistic simulations and network-based modeling of the Hsp90-Cdc37 chaperone binding with Cdk4 client protein: A mechanism of chaperoning kinase clients by exploiting weak spots of intrinsically dynamic kinase domains

PubMed Central

Czemeres, Josh; Buse, Kurt

2017-01-01

A fundamental role of the Hsp90 and Cdc37 chaperones in mediating conformational development and activation of diverse protein kinase clients is essential in signal transduction. There has been increasing evidence that the Hsp90-Cdc37 system executes its chaperoning duties by recognizing conformational instability of kinase clients and modulating their folding landscapes. The recent cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have combined atomistic simulations with protein stability and network-based rigidity decomposition analyses to characterize dynamic factors underlying allosteric mechanism of the chaperone-kinase cycle and identify regulatory hotspots that control client recognition. Through comprehensive characterization of conformational dynamics and systematic identification of stabilization centers in the unbound and client- bound Hsp90 forms, we have simulated key stages of the allosteric mechanism, in which Hsp90 binding can induce instability and partial unfolding of Cdk4 client. Conformational landscapes of the Hsp90 and Cdk4 structures suggested that client binding can trigger coordinated dynamic changes and induce global rigidification of the Hsp90 inter-domain regions that is coupled with a concomitant increase in conformational flexibility of the kinase client. This process is allosteric in nature and can involve reciprocal dynamic exchanges that exert global effect on stability of the Hsp90 dimer, while promoting client instability. The network-based rigidity analysis and emulation of thermal unfolding of the Cdk4-cyclin D complex and Hsp90-Cdc37-Cdk4 complex revealed weak spots of kinase instability that are present in the native Cdk4 structure and are targeted by the chaperone during client recruitment. Our findings suggested that this mechanism may be exploited by the Hsp90-Cdc37 chaperone to recruit and protect intrinsically dynamic kinase clients from degradation. The results of this investigation are discussed and interpreted in the context of diverse experimental data, offering new insights into mechanisms of chaperone regulation and binding. PMID:29267381

Discovery and development of pyrazole-scaffold Hsp90 inhibitors.

PubMed

McDonald, Edward; Jones, Keith; Brough, Paul A; Drysdale, Martin J; Workman, Paul

2006-01-01

This review explains why the chaperone Hsp90 is an exciting protein target for the discovery of new drugs to treat cancer in the clinic, and summarises the properties of natural product derived inhibitors before relating the discovery and current state of development of synthetic pyrazole compounds. Blockade of Hsp90 results in reduced cellular levels of several proteins implicated in cancer including CDK4, ERBB2 and C-RAF, and causes simultaneous inhibition of cancer cell proliferation in culture and of tumor xenograft growth in vivo. Hsp90 has an ATPase domain that is necessary for its Hsp chaperone function, and X-ray crystallography has shown that natural product inhibitors (geldanamycin, radicicol) of Hsp90 function bind to this domain. High throughput assays focusing on the ATPase activity of Hsp90 were developed and used to discover novel chemical starting points for cancer drug discovery. The discovery, synthesis and SAR of 3,4-diaryl pyrazoles is described. X-Ray crystallography of protein-inhibitor complexes revealed important interactions involving the resorcinol substituent at C-3, and these X-ray structures strongly influenced subsequent medicinal chemistry research that has resulted in highly potent inhibitors with sub-micromolar activity in cells. SAR and X-ray data are summarised for analogues in which the 4-phenyl substituent is replaced by amides or piperazine derivatives. Prospects for the pyrazoles as they progress towards clinical development are discussed in relation to current Phase I trials with derivatives of geldanamycin.

In Vitro Antifungal Activity and Mode of Action of 2',4'-Dihydroxychalcone against Aspergillus fumigatus

PubMed Central

Seo, Young Ho; Kim, Sung-Su

2015-01-01

2',4'-Dihydroxychalcone (2',4'-DHC) was identified from a heat shock protein 90 (Hsp90)-targeting library as a compound with Hsp90 inhibitory and antifungal effects. In the presence of 2',4'-DHC (8 µg/mL), radial growth of Aspergillus fumigatus was inhibited 20% compared to the control, and green pigmentation was completely blocked. The expression of the conidiation-associated genes abaA, brlA, and wetA was significantly decreased (approximately 3- to 5-fold) by treatment with 2',4'-DHC. The expression of calcineurin signaling components, cnaA and crzA, was also significantly reduced. The inhibitory effects of 2',4'-DHC on metabolic activity and mycelial growth were significantly enhanced by combination treatment with itraconazole and caspofungin. Docking studies indicated that 2',4'-DHC bind to the ATPase domain of Hsp90. These results suggest that 2',4'-DHC act as an Hsp90-calcinurin pathway inhibitor. PMID:26190922

In Vitro Antifungal Activity and Mode of Action of 2',4'-Dihydroxychalcone against Aspergillus fumigatus.

PubMed

Seo, Young Ho; Kim, Sung-Su; Shin, Kwang-Soo

2015-06-01

2',4'-Dihydroxychalcone (2',4'-DHC) was identified from a heat shock protein 90 (Hsp90)-targeting library as a compound with Hsp90 inhibitory and antifungal effects. In the presence of 2',4'-DHC (8 µg/mL), radial growth of Aspergillus fumigatus was inhibited 20% compared to the control, and green pigmentation was completely blocked. The expression of the conidiation-associated genes abaA, brlA, and wetA was significantly decreased (approximately 3- to 5-fold) by treatment with 2',4'-DHC. The expression of calcineurin signaling components, cnaA and crzA, was also significantly reduced. The inhibitory effects of 2',4'-DHC on metabolic activity and mycelial growth were significantly enhanced by combination treatment with itraconazole and caspofungin. Docking studies indicated that 2',4'-DHC bind to the ATPase domain of Hsp90. These results suggest that 2',4'-DHC act as an Hsp90-calcinurin pathway inhibitor.

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. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis.

PubMed

Guo, Yang; Ziesch, Andreas; Hocke, Sandra; Kampmann, Eric; Ochs, Stephanie; De Toni, Enrico N; Göke, Burkhard; Gallmeier, Eike

2015-02-01

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27-mediated through both the intrinsic and the extrinsic apoptotic pathways-at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Hsp90 Inhibition Results in Glucocorticoid Receptor Degradation in Association with Increased Sensitivity to Paclitaxel in Triple-Negative Breast Cancer.

PubMed

Agyeman, Abena S; Jun, Wesley J; Proia, David A; Kim, Caroline R; Skor, Maxwell N; Kocherginsky, Masha; Conzen, Suzanne D

2016-04-01

Targetable molecular drivers for triple-negative breast cancer (TNBC) have been difficult to identify; therefore, standard treatment remains limited to conventional chemotherapy. Recently, new-generation small-molecule Hsp90 inhibitors (e.g., ganetespib and NVP-AUY922) have demonstrated improved safety and activity profiles over the first-generation ansamycin class. In breast cancer, clinical responses have been observed in a subset of TNBC patients following ganetespib monotherapy; however, the underlying biology of Hsp90 inhibitor treatment and tumor response is not well understood. Glucocorticoid receptor (GR) activity in TNBC is associated with chemotherapy resistance. Here, we find that treatment of TNBC cell lines with ganetespib resulted in GR degradation and decreased GR-mediated gene expression. Ganetespib-associated GR degradation also sensitized TNBC cells to paclitaxel-induced cell death both in vitro and in vivo. The beneficial effect of the Hsp90 inhibitor on paclitaxel-induced cytotoxicity was reduced when GR was depleted in TNBC cells but could be recovered with GR overexpression. These findings suggest that GR-regulated anti-apoptotic and pro-proliferative signaling networks in TNBC are disrupted by Hsp90 inhibitors, thereby sensitizing TNBC to paclitaxel-induced cell death. Thus, GR+ TNBC patients may be a subgroup of breast cancer patients who are most likely to benefit from adding an Hsp90 inhibitor to taxane therapy.

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

PubMed

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

2012-01-18

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

Chaperone substrate provides missing link for cancer drug discovery.

PubMed

Byrd, Katherine M; Blagg, Brian S J

2018-02-16

Both Hsp70 and Hsp90 chaperones are overexpressed in cancer, making them relevant targets for the development of cancer chemotherapeutics, but a lack of biomolecular readouts for Hsp70 inhibition has limited the pursuit of specific inhibitors for this enzyme. A new study from Cesa et al. identifies two inhibitors of apoptosis proteins (IAPs) as specific client substrates of Hsp70. These results establish biomarkers that can be utilized to monitor Hsp70 inhibition and provide a framework for future efforts to deconvolute chaperone networks. © 2018 Byrd and Blagg.

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. PMID:27818721

Targeting Hsp70: A possible therapy for cancer

PubMed Central

Kumar, Sanjay; Stokes, James; Singh, Udai P.; Gunn, Karyn Scissum; Acharya, Arbind; Manne, Upender; Mishra, Manoj

2017-01-01

In all organisms, heat-shock proteins (HSPs) provide an ancient defense system. These proteins act as molecular chaperones by assisting proper folding and refolding of misfolded proteins and aid in the elimination of old and damaged cells. HSPs include Hsp100, Hsp90, Hsp70, Hsp40, and small HSPs. Through its substrate-binding domains, Hsp70 interacts with wide spectrum of molecules, ranging from unfolded to natively folded and aggregated proteins, and provides cytoprotective role against various cellular stresses. Under pathophysiological conditions, the high expression of Hsp70 allows cells to survive with lethal injuries. Increased Hsp70, by interacting at several points on apoptotic signaling pathways, leads to inhibition of apoptosis. Elevated expression of Hsp70 in cancer cells may be responsible for tumorigenesis and for tumor progression by providing resistance to chemotherapy. In contrast, inhibition or knockdown of Hsp70 reduces the size of tumors and can cause their complete regression. Moreover, extracellular Hsp70 acts as an immunogen that participates in cross presentation of MHC-I molecules. The goals of this review are to examine the roles of Hsp70 in cancer and to present strategies targeting Hsp70 in the development of cancer therapeutics. PMID:26898980

Overcoming HSP27-mediated resistance by altered dimerization of HSP27 using small molecules.

PubMed

Kim, Jee Hye; Jung, Ye Jin; Choi, Byeol; Lee, Na Lim; Lee, Hae Jun; Kwak, Soo Yeon; Kwon, Youngjoo; Na, Younghwa; Lee, Yun-Sil

2016-08-16

Heat shock protein 27 (HSP27, HSPB1) is an anti-apoptotic protein characterized for its tumorigenic and metastatic properties, and now referenced as a major therapeutic target in many types of cancer. The biochemical properties of HSP27 rely on a structural oligomeric and dynamic organization that is important for its chaperone activity. Down-regulation by small interfering RNA or inhibition with a dominant-negative mutant efficiently counteracts the anti-apoptotic and protective properties of HSP27. However, unlike other HSPs such as HSP90 and HSP70, small molecule approaches for neutralization of HSP27 are not well established because of the absence of an ATP binding domain. Previously, we found that a small molecule, zerumbone (ZER), induced altered dimerization of HSP27 by cross linking the cysteine residues required to build a large oligomer, led to sensitization in combination with radiation. In this study, we identified another small molecule, a xanthone compound, more capable of altering dimeric HSP27 than ZER and yielding sensitization in human lung cancer cells when combined with HSP90 inhibitors or standard anticancer modalities such as irradiation and cytotoxic anticancer drugs. Therefore, altered dimerization of HSP27 represents a good strategy for anticancer therapy in HSP27-overexpressing cancer cells.

Overcoming HSP27-mediated resistance by altered dimerization of HSP27 using small molecules

PubMed Central

Choi, Byeol; Lee, Na Lim; Lee, Hae Jun; Kwak, Soo Yeon; Kwon, Youngjoo; Na, Younghwa; Lee, Yun-Sil

2016-01-01

Heat shock protein 27 (HSP27, HSPB1) is an anti-apoptotic protein characterized for its tumorigenic and metastatic properties, and now referenced as a major therapeutic target in many types of cancer. The biochemical properties of HSP27 rely on a structural oligomeric and dynamic organization that is important for its chaperone activity. Down-regulation by small interfering RNA or inhibition with a dominant-negative mutant efficiently counteracts the anti-apoptotic and protective properties of HSP27. However, unlike other HSPs such as HSP90 and HSP70, small molecule approaches for neutralization of HSP27 are not well established because of the absence of an ATP binding domain. Previously, we found that a small molecule, zerumbone (ZER), induced altered dimerization of HSP27 by cross linking the cysteine residues required to build a large oligomer, led to sensitization in combination with radiation. In this study, we identified another small molecule, a xanthone compound, more capable of altering dimeric HSP27 than ZER and yielding sensitization in human lung cancer cells when combined with HSP90 inhibitors or standard anticancer modalities such as irradiation and cytotoxic anticancer drugs. Therefore, altered dimerization of HSP27 represents a good strategy for anticancer therapy in HSP27-overexpressing cancer cells. PMID:27449291

Anticancer activity of celastrol in combination with ErbB2-targeted therapeutics for treatment of ErbB2-overexpressing breast cancers

PubMed Central

Clubb, Robert J; Ortega-Cava, Cesar; Williams, Stetson H; Bailey, Tameka A; Duan, Lei; Zhao, Xiangshan; Reddi, Alagarasamy L; Nyong, Abijah M; Natarajan, Amarnath; Band, Vimla

2011-01-01

The receptor tyrosine kinase ErbB2 is overexpressed in up to a third of breast cancers, allowing targeted therapy with ErbB2-directed humanized antibodies such as Trastuzumab. Concurrent targeting of ErbB2 stability with HSP90 inhibitors is synergistic with Trastuzumab, suggesting that pharmacological agents that can inhibit HSP90 as well as signaling pathways activated by ErbB2 could be useful against ErbB2-overexpressing breast cancers. The triterpene natural product Celastrol inhibits HSP90 and several pathways relevant to ErbB2-dependent oncogenesis including the NFκB pathway and the proteasome, and has shown promising activity in other cancer models. Here, we demonstrate that Celastrol exhibits in vitro antitumor activity against a panel of human breast cancer cell lines with selectivity towards those overexpressing ErbB2. Celastrol strongly synergized with ErbB2-targeted therapeutics Trastuzumab and Lapatinib, producing higher cytotoxicity with substantially lower doses of Celastrol. Celastrol significantly retarded the rate of growth of ErbB2-overexpressing human breast cancer cells in a mouse xenograft model with only minor systemic toxicity. Mechanistically, Celastrol not only induced the expected ubiquitinylation and degradation of ErbB2 and other HSP90 client proteins, but it also increased the levels of reactive oxygen species (ROS). Our studies show that the Michael Acceptor functionality in Celastrol is important for its ability to destabilize ErbB2 and exert its bioactivity against ErbB2-overexpressing breast cancer cells. These studies suggest the potential use of Michael acceptor-containing molecules as novel therapeutic modalities against ErbB2-driven breast cancer by targeting multiple biological attributes of the driver oncogene. PMID:21088503

Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

PubMed

Thériault, Jimmy R; Lambert, Herman; Chávez-Zobel, Aura T; Charest, Gabriel; Lavigne, Pierre; Landry, Jacques

2004-05-28

Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinson's disease.

PubMed

Goswami, Arvind Vittal; Samaddar, Madhuja; Sinha, Devanjan; Purushotham, Jaya; D'Silva, Patrick

2012-08-01

Parkinson's disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multifactorial in nature, a recent genetic screen involving PD patients identified two mitochondrial Hsp70 variants (P509S and R126W) that are suggested in PD pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD variants are centrally involved in PD progression is totally elusive. In this article, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD variants. Biochemically, the R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, the P509S variant exhibits significantly enhanced interaction with J-protein cochaperones involved in folding and import machinery, thus altering the overall regulation of chaperone-mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD mutations at the cellular level, we developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with 'mitochondrial dysfunction', including compromised growth, impairment in protein translocation, reduced functional mitochondrial mass, mitochondrial DNA loss, respiratory incompetency and increased susceptibility to oxidative stress. In addition to that, R103W protein is prone to aggregate in vivo due to reduced stability, whereas P486S showed enhanced interaction with J-proteins, thus remarkably recapitulating the cellular defects that are observed in human PD variants. Taken together, our findings provide evidence in favor of direct involvement of mtHsp70 as a susceptibility factor in PD.

The HSP90 inhibitor 17-PAG effectively inhibits the proliferation and migration of androgen-independent prostate cancer cells

PubMed Central

Peng, Ruixian; Li, Zhenyu; Lin, Zhiyuan; Wang, Yang; Wang, Wei; Hu, Bo; Wang, Xilong; Zhang, Jun; Wang, Yangyun; Zhou, Renyuan; Lu, Chunhua; Shen, Yuemao; Wang, Jifeng; Shi, Guowei

2015-01-01

Castration-resistant prostate cancer (CRPC) ultimately occurs after a period of treatment with androgen deprivation therapy. Furthermore, CRPC patients can only derive limited survival benefits from traditional cytotoxic drugs. HSP90, which is a molecular chaperone, plays a vital role in client protein processing and maintaining the function of cells. HSP90 is usually overexpressed in prostate cancer tissues, which makes it a potential target for managing prostate cancer. Geldanamycin (GA), which was recognized as the first natural HSP90 inhibitor, has demonstrated potent anti-tumor efficacy in large-scale pre-clinical studies, but its application in the clinic is not permitted due to its liver toxicity and unstable physical properties. In this study, we report a new GA derivative, 17-PAG (17-(propynylamino)-17-demethoxygeldanamycin), which demonstrates highly effective anti-tumor activity against androgen-independent prostate cancer cells. Treating cells with 17-PAG dose-dependently suppressed proliferation, reduced colony formation and induced apoptosis of DU-145/C4-2B cells. Moreover, 17-PAG suppressed the migration and invasion of DU-145/C4-2B cells by regulating epithelial mesenchymal transition (EMT). 17-PAG also downregulated the HSP90 client proteins, including Her2, EGFR, C-Raf, AKT, p-AKT, and CDK4. Animal assays confirmed that 17-PAG shows strong anti-tumor effects with no obvious organ toxicity in DU-145 cell xenografted nude mice. These results provide us with a potential target for treating androgen-independent prostate cancer in a safe and effective manner. PMID:26693070

Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation

PubMed Central

Yang, Chunzhang; Wang, Herui; Zhu, Dongwang; Hong, Christopher S.; Dmitriev, Pauline; Zhang, Chao; Li, Yan; Ikejiri, Barbara; Brady, Roscoe O.; Zhuang, Zhengping

2015-01-01

Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation. PMID:25583479

Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation.

PubMed

Yang, Chunzhang; Wang, Herui; Zhu, Dongwang; Hong, Christopher S; Dmitriev, Pauline; Zhang, Chao; Li, Yan; Ikejiri, Barbara; Brady, Roscoe O; Zhuang, Zhengping

2015-01-27

Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation.

Imaging Heat Shock Protein 90 (Hsp90) Activity in Hormone-Refractory Prostate Cancer

DTIC Science & Technology

2011-01-01

according to the approximate relative molecular weights of their encoded proteins, including HSP10, HSP27 , HSP40, HSP60, HSP70, HSP90 and HSP110...theHSPcohort,which recognizesdenaturedproteins through the holding properties of HSP27 , HSP70 and HSP90, and subsequently refolds them with the aid of

Heat shock proteins and chronic fatigue in primary Sjögren's syndrome.

PubMed

Bårdsen, Kjetil; Nilsen, Mari Mæland; Kvaløy, Jan Terje; Norheim, Katrine Brække; Jonsson, Grete; Omdal, Roald

2016-04-01

Fatigue occurs frequently in patients with cancer, neurological diseases and chronic inflammatory diseases, but the biological mechanisms that lead to and regulate fatigue are largely unknown. When the innate immune system is activated, heat shock proteins (HSPs) are produced to protect cells. Some extracellular HSPs appear to recognize cellular targets in the brain, and we hypothesize that fatigue may be generated by specific HSPs signalling through neuronal or glial cells in the central nervous system. From a cohort of patients with primary Sjögren's syndrome, 20 patients with high and 20 patients with low fatigue were selected. Fatigue was evaluated with a fatigue visual analogue scale. Plasma concentrations of HSP32, HSP60, HSP72 and HSP90α were measured and analysed to determine if there were associations with the level of fatigue. Plasma concentrations of HSP90α were significantly higher in patients with high fatigue compared with those with low fatigue, and there was a tendency to higher concentrations of HSP72 in patients with high fatigue compared with patients with low fatigue. There were no differences in concentrations of HSP32 and HSP60 between the high- and low-fatigue groups. Thus, extracellular HSPs, particularly HSP90α, may signal fatigue in chronic inflammation. This supports the hypothesis that fatigue is generated by cellular defence mechanisms. © The Author(s) 2016.

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

PubMed

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

2016-01-01

Histone deacetylase (HDAC)6 is a unique isoenzyme targeting specific substrates including α-tubulin and heat shock protein (HSP)90. HDAC6 is involved in protein trafficking and degradation, cell shape and migration. Deregulation of HDAC6 activity is associated with a variety of diseases including cancer leading to a growing interest for developing HDAC6 inhibitors. Here, we identified two new structurally related 4-hydroxybenzoic acids as selective HDAC6 inhibitors reducing proliferation, colony and spheroid formation as well as viability of prostate cancer cells. Both compounds strongly enhanced α-tubulin acetylation leading to remodeling of microtubular organization. Furthermore, 4-hydroxybenzoic acids decreased HSP90α regulation of the human androgen receptor in prostate cancer cells by increasing HSP90α acetylation levels. Collectively, our data support the potential of 4-hydroxybenzoic acid derivatives as HDAC6-specific inhibitors with anti-cancer properties. Copyright © 2015 Elsevier Inc. All rights reserved.

Exploiting cancer cell vulnerabilities to develop a combination therapy for Ras-driven tumors

PubMed Central

De Raedt, Thomas; Walton, Zandra; Yecies, Jessica L.; Li, Danan; Chen, Yimei; Malone, Clare F.; Maertens, Ophelia; Jeong, Seung Min; Bronson, Roderick T.; Lebleu, Valerie; Kalluri, Raghu; Normant, Emmanuel; Haigis, Marcia C.; Manning, Brendan D.; Wong, Kwok-Kin; Macleod, Kay F; Cichowski, Karen

2011-01-01

Summary Ras-driven tumors are often refractory to conventional therapies. Here we identify a promising targeted therapeutic strategy for two Ras-driven cancers: Nf1-deficient malignancies and KRas/p53-mutant lung cancer. We show that agents that enhance proteotoxic stress, including the HSP90 inhibitor IPI-504, induce tumor regression in aggressive mouse models, but only when combined with rapamycin. These agents synergize by promoting irresolvable ER stress, resulting in catastrophic ER and mitochondrial damage. This process is fueled by oxidative stress, which is caused by IPI-504-dependent production of reactive oxygen species, and the rapamycin-dependent suppression of glutathione, an important endogenous antioxidant. Notably, the mechanism by which these agents cooperate reveals a therapeutic paradigm that can be expanded to develop additional combinations. PMID:21907929

Modulating molecular chaperones improves sensory fiber recovery and mitochondrial function in diabetic peripheral neuropathy

PubMed Central

Urban, Michael J.; Pan, Pan; Farmer, Kevin L.; Zhao, Huiping; Blagg, Brian S.J.; Dobrowsky, Rick T.

2012-01-01

Quantification of intra-epidermal nerve fibers (iENFs) is an important approach to stage diabetic peripheral neuropathy (DPN) and is a promising clinical endpoint for identifying beneficial therapeutics. Mechanistically, diabetes decreases neuronal mitochondrial function and enhancing mitochondrial respiratory capacity may aid neuronal recovery from glucotoxic insults. We have proposed that modulating the activity and expression of heat shock proteins (Hsp) may be of benefit in treating DPN. KU-32 is a C-terminal Hsp90 inhibitor that improved thermal hypoalgesia in diabetic C57Bl/6 mice but it was not determined if this was associated with an increase in iENF density and mitochondrial function. After 16 weeks of diabetes, Swiss Webster mice showed decreased electrophysiological and psychosensory responses and a >30% loss of iENFs. Treatment of the mice with ten weekly doses of 20 mg/kg KU-32 significantly reversed pre-existing deficits in nerve conduction velocity and responses to mechanical and thermal stimuli. KU-32 therapy significantly reversed the pre-existing loss of iENFs despite the identification of a sub-group of drug-treated diabetic mice that showed improved thermal sensitivity but no increase in iENF density. To determine if the improved clinical indices correlated with enhanced mitochondrial activity, sensory neurons were isolated and mitochondrial bioenergetics assessed ex vivo using extracellular flux technology. Diabetes decreased maximal respiratory capacity in sensory neurons and this deficit was improved following KU-32 treatment. In conclusion, KU-32 improved physiological and morphologic markers of degenerative neuropathy and drug efficacy may be related to enhanced mitochondrial bioenergetics in sensory neurons. PMID:22465570

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

PubMed

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

2015-09-01

The ubiquitously expressed heat shock protein 90 is an encouraging target for the development of novel anticancer agents. In a program directed towards uncovering novel chemical scaffolds against Hsp90, we performed molecular docking studies using Tripos-Sybyl drug designing software by including the required conserved water molecules. The results of the docking studies predicted Mannich bases derived from 2,4-dihydroxy acetophenone/5-chloro 2,4-dihydroxy acetophenone as potential Hsp90 inhibitors. Subsequently, a few of them were synthesized (1-6) and characterized by IR, (1)H NMR, (13)C NMR and mass spectral analysis. The synthesized Mannich compounds were evaluated for their potential to suppress Hsp90 ATPase activity by the colorimetric Malachite green assay. Subsequently, the molecules were screened for their antiproilferative effect against PC3 pancreatic carcinoma cells by adopting the 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The activity profile of the identified derivatives correlated well with their docking results. Copyright © 2015 Elsevier B.V. All rights reserved.

Down-regulation of HSP60 Suppresses the Proliferation of Glioblastoma Cells via the ROS/AMPK/mTOR Pathway

PubMed Central

Tang, Haiping; Li, Jin; Liu, Xiaohui; Wang, Guihuai; Luo, Minkui; Deng, Haiteng

2016-01-01

Glioblastoma is a fatal and incurable cancer with the hyper-activated mTOR pathway. HSP60, a major chaperone for maintenance of mitochondrial proteostasis, is highly expressed in glioblastoma patients. To understand the effects of HSP60 on glioblastoma tumorigenesis and progression, we characterized the HSP60-knockdowned glioblastoma cells and revealed that HSP60 silencing markedly suppressed cell proliferation and promoted cell to undergo the epithelial-mesenchymal transition (EMT). Proteomic analysis showed that ribosomal proteins were significantly downregulated whereas EMT-associated proteins were up-regulated in HSP60-knockdowned U87 cells as confirmed by a distinct enrichment pattern in newly synthesized proteins with azido-homoalanine labeling. Biochemical analysis revealed that HSP60 knockdown increased reactive oxygen species (ROS) production that led to AMPK activation, similarly to the complex I inhibitor rotenone-induced AMPK activation. Activated AMPK suppressed mTORC1 mediated S6K and 4EBP1 phosphorylation to decrease protein translation, which slowed down cell growth and proliferation. On the other hand, high levels of ROS in HSP60 knockdowned or rotenone-treated U87 cells contributed to EMT. These results indicate that HSP60 silencing deactivates the mTOR pathway to suppress glioblastoma progression, suggesting that HSP60 is a potential therapeutic target for glioblastoma treatment. PMID:27325206

Split Renilla Luciferase Protein Fragment-assisted Complementation (SRL-PFAC) to Characterize Hsp90-Cdc37 Complex and Identify Critical Residues in Protein/Protein Interactions*

PubMed Central

Jiang, Yiqun; Bernard, Denzil; Yu, Yanke; Xie, Yehua; Zhang, Tao; Li, Yanyan; Burnett, Joseph P.; Fu, Xueqi; Wang, Shaomeng; Sun, Duxin

2010-01-01

Hsp90 requires cochaperone Cdc37 to load its clients to the Hsp90 superchaperone complex. The purpose of this study was to utilize split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) bioluminescence to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/Cdc37 interaction in living cells. SRL-PFAC showed that full-length human Hsp90/Cdc37 interaction restored dramatically high luciferase activity through Hsp90-Cdc37-assisted complementation of the N and C termini of luciferase (compared with the set of controls). Immunoprecipitation confirmed that the expressed fusion proteins (NRL-Hsp90 and Cdc37-CRL) preserved their ability to interact with each other and also with native Hsp90 or Cdc37. Molecular dynamic simulation revealed several critical residues in the two interaction patches (hydrophobic and polar) at the interface of Hsp90/Cdc37. Mutagenesis confirmed the critical residues for Hsp90-Cdc37 complex formation. SRL-PFAC bioluminescence evaluated the contributions of these critical residues in Hsp90/Cdc37 interaction. The results showed that mutations in Hsp90 (Q133A, F134A, and A121N) and mutations in Cdc37 (M164A, R167A, L205A, and Q208A) reduced the Hsp90/Cdc37 interaction by 70–95% as measured by the resorted luciferase activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A and S113A) and a mutation in Cdc37 (A204E) decreased the Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A, and C598A) and mutations in Cdc37 (C54S, C57S, and C64S) did not change Hsp90/Cdc37 interactions. The data suggest that single amino acid mutation in the interface of Hsp90/Cdc37 is sufficient to disrupt its interaction, although Hsp90/Cdc37 interactions are through large regions of hydrophobic and polar interactions. These findings provides a rationale to develop inhibitors for disruption of the Hsp90/Cdc37 interaction. PMID:20413594

Split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) to characterize Hsp90-Cdc37 complex and identify critical residues in protein/protein interactions.

PubMed

Jiang, Yiqun; Bernard, Denzil; Yu, Yanke; Xie, Yehua; Zhang, Tao; Li, Yanyan; Burnett, Joseph P; Fu, Xueqi; Wang, Shaomeng; Sun, Duxin

2010-07-02

Hsp90 requires cochaperone Cdc37 to load its clients to the Hsp90 superchaperone complex. The purpose of this study was to utilize split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) bioluminescence to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/Cdc37 interaction in living cells. SRL-PFAC showed that full-length human Hsp90/Cdc37 interaction restored dramatically high luciferase activity through Hsp90-Cdc37-assisted complementation of the N and C termini of luciferase (compared with the set of controls). Immunoprecipitation confirmed that the expressed fusion proteins (NRL-Hsp90 and Cdc37-CRL) preserved their ability to interact with each other and also with native Hsp90 or Cdc37. Molecular dynamic simulation revealed several critical residues in the two interaction patches (hydrophobic and polar) at the interface of Hsp90/Cdc37. Mutagenesis confirmed the critical residues for Hsp90-Cdc37 complex formation. SRL-PFAC bioluminescence evaluated the contributions of these critical residues in Hsp90/Cdc37 interaction. The results showed that mutations in Hsp90 (Q133A, F134A, and A121N) and mutations in Cdc37 (M164A, R167A, L205A, and Q208A) reduced the Hsp90/Cdc37 interaction by 70-95% as measured by the resorted luciferase activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A and S113A) and a mutation in Cdc37 (A204E) decreased the Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A, and C598A) and mutations in Cdc37 (C54S, C57S, and C64S) did not change Hsp90/Cdc37 interactions. The data suggest that single amino acid mutation in the interface of Hsp90/Cdc37 is sufficient to disrupt its interaction, although Hsp90/Cdc37 interactions are through large regions of hydrophobic and polar interactions. These findings provides a rationale to develop inhibitors for disruption of the Hsp90/Cdc37 interaction.

Targeting Hsp70: A possible therapy for cancer.

PubMed

Kumar, Sanjay; Stokes, James; Singh, Udai P; Scissum Gunn, Karyn; Acharya, Arbind; Manne, Upender; Mishra, Manoj

2016-04-28

In all organisms, heat-shock proteins (HSPs) provide an ancient defense system. These proteins act as molecular chaperones by assisting proper folding and refolding of misfolded proteins and aid in the elimination of old and damaged cells. HSPs include Hsp100, Hsp90, Hsp70, Hsp40, and small HSPs. Through its substrate-binding domains, Hsp70 interacts with wide spectrum of molecules, ranging from unfolded to natively folded and aggregated proteins, and provides cytoprotective role against various cellular stresses. Under pathophysiological conditions, the high expression of Hsp70 allows cells to survive with lethal injuries. Increased Hsp70, by interacting at several points on apoptotic signaling pathways, leads to inhibition of apoptosis. Elevated expression of Hsp70 in cancer cells may be responsible for tumorigenesis and for tumor progression by providing resistance to chemotherapy. In contrast, inhibition or knockdown of Hsp70 reduces the size of tumors and can cause their complete regression. Moreover, extracellular Hsp70 acts as an immunogen that participates in cross presentation of MHC-I molecules. The goals of this review are to examine the roles of Hsp70 in cancer and to present strategies targeting Hsp70 in the development of cancer therapeutics. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

[Expressions of heat shock protein (HSP) family HSP 60, 70 and 90alpha in colorectal cancer tissues and their correlations to pathohistological characteristics].

PubMed

Zhang, Wen-Li; Gao, Xue-Qin; Han, Jin-Xiang; Wang, Guo-Qiang; Yue, Long-Tao

2009-06-01

Colorectal cancer is the third common malignant tumor in the world. Heat shock protein (HSP) family has been reported to play an important role in carcinogenesis of various cancers. However, little is known about expressions of HSP60,HSP70 and HSP90alpha in colorectal cancer. This study was to investigate expressions of HSP 60, 70 and 90alpha, and analyzed their correlations to pathohistologic characteristics in colorectal cancer. Colorectal cancer tissues and adjacent normal tissues 2 cm away from the tumor focus were collected from 49 patients. Expressions of HSP60, HSP70 and HSP90alpha mRNA were detected by RT-PCR. The protein expressions of HSP60, HSP70 and HSP90alpha were determined by immunohistochemistry and western blot. The mRNA and protein levels of HSP60, HSP70 and HSP90alpha, as well as their positive rates were significantly increased in tumor tissues compared with those in para-cancerous tissues. The overexpression rates of HSP60, HSP70 and HSP90alpha were also significantly higher in the colorectal cancer tissues than those in the corresponding para-cancerous tissues. The positive and overexpression rates of HSP60, HSP70 and HSP90alpha in well, moderately and poorly differentiated colorectal cancer were not significantly different. HSP60, HSP70 and HSP90alpha may play important roles in the pathogenesis of colorectal cancer, although they are not correlated with the pathological grading.

Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90

PubMed Central

2011-01-01

Background Multiple myeloma (MM) is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. Results In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. Conclusions Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma. PMID:21871133

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

The ‘active life’ of Hsp90 complexes☆

PubMed Central

Prodromou, Chrisostomos

2012-01-01

Hsp90 forms a variety of complexes differing both in clientele and co-chaperones. Central to the role of co-chaperones in the formation of Hsp90 complexes is the delivery of client proteins and the regulation of the ATPase activity of Hsp90. Determining the mechanisms by which co-chaperones regulate Hsp90 is essential in understanding the assembly of these complexes and the activation and maturation of Hsp90's clientele. Mechanistically, co-chaperones alter the kinetics of the ATP-coupled conformational changes of Hsp90. The structural changes leading to the formation of a catalytically active unit involve all regions of the Hsp90 dimer. Their complexity has allowed different orthologues of Hsp90 to evolve kinetically in slightly different ways. The interaction of the cytosolic Hsp90 with a variety of co-chaperones lends itself to a complex set of different regulatory mechanisms that modulate Hsp90's conformation and ATPase activity. It also appears that the conformational switches of Hsp90 are not necessarily coupled under all circumstances. Here, I described different co-chaperone complexes and then discuss in detail the mechanisms and role that specific co-chaperones play in this. I will also discuss emerging evidence that post-translational modifications also affect the ATPase activity of Hsp90, and thus complex formation. Finally, I will present evidence showing how Hsp90's active site, although being highly conserved, can be altered to show resistance to drug binding, but still maintain ATP binding and ATPase activity. Such changes are therefore unlikely to significantly alter Hsp90's interactions with client proteins and co-chaperones. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90) PMID:21840346

Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51.

PubMed

Kumar, Rajnish; Moche, Martin; Winblad, Bengt; Pavlov, Pavel F

2017-10-27

FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.

Targeting c-KIT (CD117) by dasatinib and radotinib promotes acute myeloid leukemia cell death.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jeong, Yoo Kyung; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-11-10

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.

Development of Heat Shock Protein (Hsp90) Inhibitors To Combat Resistance to Tyrosine Kinase Inhibitors through Hsp90-Kinase Interactions.

PubMed

Wang, Meining; Shen, Aijun; Zhang, Chi; Song, Zilan; Ai, Jing; Liu, Hongchun; Sun, Liping; Ding, Jian; Geng, Meiyu; Zhang, Ao

2016-06-23

Heat shock protein 90 (Hsp90) is a ubiquitous chaperone of all of the oncogenic tyrosine kinases. Many Hsp90 inhibitors, alone or in combination, have shown significant antitumor efficacy against the kinase-positive naïve and mutant models. However, clinical trials of these inhibitors are unsuccessful due to insufficient clinical benefits and nonoptimal safety profiles. Recently, much progress has been reported on the Hsp90-cochaperone-client complex, which will undoubtedly assist in the understanding of the interactions between Hsp90 and its clients. Meanwhile, Hsp90 inhibitors have shown promise against patients' resistance caused by early generation tyrosine kinase inhibitors (TKIs), and at least 13 Hsp90 inhibitors are being reevaluated in the clinic. In this regard, the objectives of the current perspective are to summarize the structure and function of the Hsp90-cochaperone-client complex, to analyze the structural and functional insights into the Hsp90-client interactions to address several existing unresolved problems with Hsp90 inhibitors, and to highlight the preclinical and clinical studies of Hsp90 inhibitors as an effective treatment against resistance to tyrosine kinase inhibitors.

Tumor induces muscle wasting in mice through releasing extracellular Hsp70 and Hsp90.

PubMed

Zhang, Guohua; Liu, Zhelong; Ding, Hui; Zhou, Yong; Doan, Hoang Anh; Sin, Ka Wai Thomas; Zhu, Zhiren J; Flores, Rene; Wen, Yefei; Gong, Xing; Liu, Qingyun; Li, Yi-Ping

2017-09-19

Cachexia, characterized by muscle wasting, is a major contributor to cancer-related mortality. However, the key cachexins that mediate cancer-induced muscle wasting remain elusive. Here, we show that tumor-released extracellular Hsp70 and Hsp90 are responsible for tumor's capacity to induce muscle wasting. We detected high-level constitutive release of Hsp70 and Hsp90 associated with extracellular vesicles (EVs) from diverse cachexia-inducing tumor cells, resulting in elevated serum levels in mice. Neutralizing extracellular Hsp70/90 or silencing Hsp70/90 expression in tumor cells abrogates tumor-induced muscle catabolism and wasting in cultured myotubes and in mice. Conversely, administration of recombinant Hsp70 and Hsp90 recapitulates the catabolic effects of tumor. In addition, tumor-released Hsp70/90-expressing EVs are necessary and sufficient for tumor-induced muscle wasting. Further, Hsp70 and Hsp90 induce muscle catabolism by activating TLR4, and are responsible for elevation of circulating cytokines. These findings identify tumor-released circulating Hsp70 and Hsp90 as key cachexins causing muscle wasting in mice.Cachexia affects many cancer patients causing weight loss and increasing mortality. Here, the authors identify extracellular Hsp70 and Hsp90, either in soluble form or secreted as part of exosomes from tumor cells, to be responsible for tumor induction of cachexia.

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)*

PubMed Central

Shimamoto, Seiko; Kubota, Yasuo; Yamaguchi, Fuminori; Tokumitsu, Hiroshi; Kobayashi, Ryoji

2013-01-01

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca2+/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca2+/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca2+/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca2+-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway. PMID:23344957

Downregulation of HSP60 disrupts mitochondrial proteostasis to promote tumorigenesis and progression in clear cell renal cell carcinoma

PubMed Central

Tang, Haiping; Chen, Yuling; Liu, Xiaohui; Wang, Shiyu; Lv, Yang; Wu, Di; Wang, Qingtao; Luo, Minkui; Deng, Haiteng

2016-01-01

In the present study, we demonstrate that HSP60 is unequivocally downregulated in clear cell renal cell carcinoma (ccRCC) tissues compared to pericarcinous tissues. Overexpression of HSP60 in ccRCC cancer cells suppresses cell growth. HSP60 knockdown increases cell growth and proliferation in both cell culture and nude mice xenografts, and drives cells to undergo epithelial to mesenchymal transition (EMT). Our results propose that HSP60 silencing disrupts the integrity of the respiratory complex I and triggers the excessive ROS production, which promotes tumor progression in the following aspects: (1) ROS activates the AMPK pathway that promotes acquisition of the Warburg phenotype in HSP60-KN cells; (2) ROS generated by HSP60 knockdown or by rotenone inhibition drives cells to undergo EMT; and (3) the high level of ROS may also fragment the Fe-S clusters that up regulates ADHFe1 expression and the 2-hydroxygluterate (2-HG) production leading to changes in DNA methylation. These results suggest that the high level of ROS is needed for tumorigenesis and progression in tumors with the low HSP60 expression and HSP60 is a potential diagnostic biomarker as well as a therapeutic target in ccRCC. PMID:27246978

The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation.

PubMed

Uzarska, Marta A; Dutkiewicz, Rafal; Freibert, Sven-Andreas; Lill, Roland; Mühlenhoff, Ulrich

2013-06-01

The mitochondrial Hsp70 chaperone Ssq1 plays a dedicated role in the maturation of iron-sulfur (Fe/S) proteins, an essential process of mitochondria. Similar to its bacterial orthologue HscA, Ssq1 binds to the scaffold protein Isu1, thereby facilitating dissociation of the newly synthesized Fe/S cluster on Isu1 and its transfer to target apoproteins. Here we use in vivo and in vitro approaches to show that Ssq1 also interacts with the monothiol glutaredoxin 5 (Grx5) at a binding site different from that of Isu1. Grx5 binding does not stimulate the ATPase activity of Ssq1 and is most pronounced for the ADP-bound form of Ssq1, which interacts with Isu1 most tightly. The vicinity of Isu1 and Grx5 on the Hsp70 chaperone facilitates rapid Fe/S cluster transfer from Isu1 to Grx5. Grx5 and its bound Fe/S cluster are required for maturation of all cellular Fe/S proteins, regardless of the type of bound Fe/S cofactor and subcellular localization. Hence Grx5 functions as a late-acting component of the core Fe/S cluster (ISC) assembly machinery linking the Fe/S cluster synthesis reaction on Isu1 with late assembly steps involving Fe/S cluster targeting to dedicated apoproteins.

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space.

PubMed

Veri, Amanda O; Miao, Zhengqiang; Shapiro, Rebecca S; Tebbji, Faiza; O'Meara, Teresa R; Kim, Sang Hu; Colazo, Juan; Tan, Kaeling; Vyas, Valmik K; Whiteway, Malcolm; Robbins, Nicole; Wong, Koon Ho; Cowen, Leah E

2018-03-01

The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space

PubMed Central

Miao, Zhengqiang; Tan, Kaeling; Vyas, Valmik K.; Whiteway, Malcolm; Robbins, Nicole; Wong, Koon Ho; Cowen, Leah E.

2018-01-01

The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition. PMID:29590106

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. © 2016 Elsevier Inc. All rights reserved.

Mps1 Mediated Phosphorylation of Hsp90 Confers Renal Cell Carcinoma Sensitivity and Selectivity to Hsp90 Inhibitors.

PubMed

Woodford, Mark R; Truman, Andrew W; Dunn, Diana M; Jensen, Sandra M; Cotran, Richard; Bullard, Renee; Abouelleil, Mourad; Beebe, Kristin; Wolfgeher, Donald; Wierzbicki, Sara; Post, Dawn E; Caza, Tiffany; Tsutsumi, Shinji; Panaretou, Barry; Kron, Stephen J; Trepel, Jane B; Landas, Steve; Prodromou, Chrisostomos; Shapiro, Oleg; Stetler-Stevenson, William G; Bourboulia, Dimitra; Neckers, Len; Bratslavsky, Gennady; Mollapour, Mehdi

2016-02-02

The molecular chaperone Hsp90 protects deregulated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensitivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and disrupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensitizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modulation of ASK1 expression during overexpression of Trx and HSP70 in stressed fish liver mitochondria.

PubMed

Padmini, Ekambaram; Vijaya Geetha, Bose

2009-09-01

Mitochondrial heat shock protein 70 (mtHSP70) is found to play a primary role in cellular defense against physiological stress like exposure to environmental contaminants and helpful in the maintenance of cellular homeostasis by promoting the cell survival. In the present investigation, the environmental-stress-induced increase in mtHSP70 levels along with the quantification of apoptosis signal regulating kinase 1 (ASK1) and thioredoxin (Trx) were measured in the liver mitochondria of grey mullets (Mugil cephalus) collected from the polluted Ennore estuary and the unpolluted Kovalam estuary for a period of 2 years. The results showed elevated lipid peroxide (LPO) and decreased total antioxidant capacity along with the decrease in mitochondrial viability percentage. Mitochondrial HSP70, ASK1, and Trx levels were increased under this stress condition. A 42% increase in LPO levels and 18% decrease in mitochondrial survivality were observed in the polluted-site fish liver mitochondria when compared to the results of unpolluted estuary. We also report that, under observed oxidative stress condition in Ennore fish samples, the ASK1 levels are only moderately elevated (13% increase). This may be due to mitochondrial-HSP70-induced adaptive tolerance signaling for the activation of Trx (22% increase) which suppresses the ASK1 expression thereby promoting the cell survival that leads to the maintenance of the cellular homeostasis.

Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation.

PubMed

Henrich, C J; Brooks, A D; Erickson, K L; Thomas, C L; Bokesch, H R; Tewary, P; Thompson, C R; Pompei, R J; Gustafson, K R; McMahon, J B; Sayers, T J

2015-02-26

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects.

Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

PubMed Central

Henrich, C J; Brooks, A D; Erickson, K L; Thomas, C L; Bokesch, H R; Tewary, P; Thompson, C R; Pompei, R J; Gustafson, K R; McMahon, J B; Sayers, T J

2015-01-01

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects. PMID:25719250

[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.

Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90β) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C.

PubMed

Yan, Jie; Liang, Xiao; Zhang, Yin; Li, Yang; Cao, Xiaojuan; Gao, Jian

2017-07-01

Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full-length cDNA sequences of HSP70, HSP90α and HSP90β from loach Misgurnus anguillicaudatus were cloned. The full-length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90α and HSP90β were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio. The deduced amino acid sequences of HSP90α and HSP90β in loach both shared the highest identity with those of M. amblycephala. Their mRNA tissue expression results showed that the maximum expressions of HSP70, HSP90α and HSP90β were respectively present in the intestine, brain and kidney of loach. Quantitative real-time PCR was employed to analyze the temporal expressions of HSP70, HSP90α and HSP90β in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence that the novobiocin-sensitive ATP-binding site of the heat shock protein 90 (hsp90) is necessary for its autophosphorylation.

PubMed

Langer, T; Schlatter, H; Fasold, H

2002-01-01

The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced. It is assumed, that under these conditions Hsp90 is required to protect other proteins from aggregation. One property of Hsp90 is its ability to undergo autophosphorylation. The N-terminal domain of Hsp90 has been shown to contain an unusual ATP-binding site. A well-known inhibitor of Hsp90 function is geldanamycin binding to the N-terminal ATP-binding site with high affinity. Recently it was shown that Hsp90 possesses a second ATP-binding site in the C-terminal region, which can be competed with novobiocin. Autophosphorylation of Hsp90 was analysed by incubation with gamma(32)P-ATP. Addition of geldanamycin did not interfere with the capability for autophosphorylation, while novobiocin indeed did. These results suggest that the C-terminal ATP-binding site is required for autophosphorylation of Hsp90.

Characterization of a novel novobiocin analogue as a putative C-terminal inhibitor of heat shock protein 90 in prostate cancer cells.

PubMed

Matthews, Shawna B; Vielhauer, George A; Manthe, Craig A; Chaguturu, Vamsee K; Szabla, Kristen; Matts, Robert L; Donnelly, Alison C; Blagg, Brian S J; Holzbeierlein, Jeffrey M

2010-01-01

Hsp90 is important in the folding, maturation and stabilization of pro-tumorigenic client proteins and represents a viable drug target for the design of chemotherapies. Previously, we reported the development of novobiocin analogues designed to inhibit the C-terminal portion of Hsp90, which demonstrated the ability to decrease client protein expression. We now report the characterization of the novel novobiocin analogue, F-4, which demonstrates improved cytotoxicity in prostate cancer cell lines compared to the N-terminal inhibitor, 17-AAG. LNCaP and PC-3 cells were treated with 17-AAG or F-4 in anti-proliferative, apoptosis, cell cycle and cytotoxicity assays. Western blot and prostate specific antigen (PSA) ELISAs were used to determine client protein degradation, induction of Hsp90 and to assess the functional status of the androgen receptor (AR) in response to F-4 treatment. Surface plasmon resonance (SPR) was also used to determine the binding properties of F-4 to Hsp90. F-4 demonstrated improved potency and efficacy compared to novobiocin in anti-proliferative assays and decreased expression of client proteins. PSA secretion was inhibited in a dose-dependent manner that paralleled a decrease in AR expression. The binding of F-4 to Hsp90 was determined to be saturable with a binding affinity (K(d)) of 100 microM. In addition, superior efficacy was demonstrated by F-4 compared to 17-AAG in experiments measuring cytotoxicity and apoptosis. These data reveal distinct modes of action for N-terminal and C-terminal Hsp90 inhibitors, which may offer unique therapeutic benefits for the treatment of prostate cancer.

Characterization of a novel novobiocin analogue as a putative C-terminal inhibitor of heat shock protein 90 in prostate cancer cells

PubMed Central

Shawna, B. Comer; George, A. Vielhauer; Craig, A. Manthe; Vamsee, K. Chaguturu; Kristen, Szabla; Robert, L. Matts; Alison, C. Donnelly; Brian, S. J. Blagg; Jeffrey, M. Holzbeierlein

2009-01-01

Purpose Hsp90 is important in the folding, maturation and stabilization of pro-tumorigenic client proteins and represents a viable drug target for the design of chemotherapies. Previously, we reported the development of novobiocin analogues designed to inhibit the C-terminal portion of Hsp90, which demonstrated the ability to decrease client protein expression. We now report the characterization of the novel novobiocin analogue, F-4, which demonstrates improved cytotoxicity in prostate cancer cell lines compared to the N-terminal inhibitor, 17-AAG. Materials and Methods LNCaP and PC-3 cells were treated with 17-AAG or F-4 in anti-proliferative, apoptosis, cell cycle and cytotoxicity assays. Western blot and prostate specific antigen (PSA) ELISAs were used to determine client protein degradation, induction of Hsp90 and to assess the functional status of the androgen receptor (AR) in response to F-4 treatment. Surface Plasmon Resonance (SPR) was also used to determine the binding properties of F-4 to Hsp90. Results F-4 demonstrated improved potency and efficacy compared to novobiocin in anti-proliferative assays and decreased expression of client proteins. PSA secretion was inhibited in a dose-dependent manner that paralleled a decrease in AR expression. The binding of F-4 to Hsp90 was determined to be saturable with a binding affinity (Kd) of 100 µM. In addition, superior efficacy was demonstrated by F-4 compared to 17-AAG in experiments measuring cytotoxicity and apoptosis Conclusions These data reveal distinct modes of action for N-terminal and C-terminal Hsp90 inhibitors, which may offer unique therapeutic benefits for the treatment of prostate cancer. PMID:19739131

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

PubMed Central

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

2011-01-01

Background The molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro. Methodology Plant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction. Conclusions Our results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of a non-pathogen-derived ligand for TLRs. PMID:21701588

Cloning HSP70 and HSP90 genes of kaluga (Huso dauricus) and the effects of temperature and salinity stress on their gene expression.

PubMed

Peng, Guogan; Zhao, Wen; Shi, Zhenguang; Chen, Huirong; Liu, Yang; Wei, Jie; Gao, Fengying

2016-03-01

The genes encoding HSP70 and HSP90 proteins were isolated from kaluga by homologous cloning and rapid amplification of complementary DNA (cDNA) ends (RACE). HSP70 (GenBank accession no. KP050541) and HSP90 (GenBank accession no. KP050542) cDNAs were composed of 2275 and 2718 bp and encoded polypeptides of 650 and 725 amino acids, respectively. Basic Local Alignment Search Tool (BLAST) analysis showed that HSP70 and HSP90 of kaluga shared high identities with those of Acipenser ruthenus, Acipenser schrenckii, and Acipenser baerii (98-99 %). Fluorescent real-time RT-PCR under unstressed conditions revealed that HSP70 and HSP90 were expressed in 11 different tissues of kaluga. Messenger RNA (mRNA) expressions of both HSP70 and HSP90 were highest in the intestine and lowest in the muscle. In addition, the patterns of mRNA expression of HSP70 and HSP90 were similar, although the level of expression was more in HSP90 than in HSP70 (P < 0.05).We also analyzed patterns of HSP70 and HSP90 expression in the muscle, gill, and liver of kaluga under different combinations of temperature and salinity stress, including temperatures of 4,10, 25, and 28 °C at 0 ppt salinity, and salinities of 10, 20, 30, and 40 ppt at 16 °C, where 16 °C at 0 ppt (parts per thousand) served as the control. We found that levels of mRNA expression of both HSP70 and HSP90 were highest at 4 °C in the muscle, gill, and liver and changed little with salinity stress. These results increase understanding of the mechanisms of stress response of cold freshwater fish.

Synergistic role of HSP90α and HSP90β to promote myofibroblast persistence in lung fibrosis.

PubMed

Bellaye, Pierre-Simon; Shimbori, Chiko; Yanagihara, Toyoshi; Carlson, David A; Hughes, Philip; Upagupta, Chandak; Sato, Seidai; Wheildon, Nolan; Haystead, Timothy; Ask, Kjetil; Kolb, Martin

2018-02-01

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung parenchyma, causing significant morbidity through worsening dyspnoea and overall functional decline. IPF is characterised by apoptosis-resistant myofibroblasts, which are a major source for the excessive production of extracellular matrix (ECM) overtaking normal lung tissue. We sought to study the role of heat shock protein (HSP) isoforms HSP90α and HSP90β, whose distinct roles in lung fibrogenesis remain elusive.We determined the level of circulating HSP90α in IPF patients (n=31) and age-matched healthy controls (n=9) by ELISA. The release of HSP90α and HSP90β was evaluated in vitro in primary IPF and control lung fibroblasts and ex vivo after mechanical stretch on fibrotic lung slices from rats receiving adenovector-mediated transforming growth factor-β1.We demonstrate that circulating HSP90α is upregulated in IPF patients in correlation with disease severity. The release of HSP90α is enhanced by the increase in mechanical stress of the fibrotic ECM. This increase in extracellular HSP90α signals through low-density lipoprotein receptor-related protein 1 (LRP1) to promote myofibroblast differentiation and persistence. In parallel, we demonstrate that the intracellular form of HSP90β stabilises LRP1, thus amplifying HSP90α extracellular action.We believe that the specific inhibition of extracellular HSP90α is a promising therapeutic strategy to reduce pro-fibrotic signalling in IPF. Copyright ©ERS 2018.

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

Principal component analysis on molecular descriptors as an alternative point of view in the search of new Hsp90 inhibitors.

PubMed

Lauria, Antonino; Ippolito, Mario; Almerico, Anna Maria

2009-10-01

Inhibiting a protein that regulates multiple signal transduction pathways in cancer cells is an attractive goal for cancer therapy. Heat shock protein 90 (Hsp90) is one of the most promising molecular targets for such an approach. In fact, Hsp90 is a ubiquitous molecular chaperone protein that is involved in folding, activating and assembling of many key mediators of signal transduction, cellular growth, differentiation, stress-response and apoptothic pathways. With the aim to analyze which molecular descriptors have the higher importance in the binding interactions of these classes, we first performed molecular docking experiments on the 187 Hsp90 inhibitors included in the BindingDB, a public database of measured binding affinities. Further, for each frozen conformation obtained from the docking, a set of 250 molecular descriptors was calculated, and the resulting Structure/Descriptors matrix was submitted to Principal Component Analysis. From the factor scores it emerged a good clusterization among similar compounds both in terms of structural class and activity spectrum, while examination of the loadings of the first two factors also allowed to study the classes of descriptors which mainly contribute to each one.

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes

PubMed Central

Germot, Agnès; Philippe, Hervé; Le Guyader, Hervé

1996-01-01

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of α-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria. PMID:8962101

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes.

PubMed

Germot, A; Philippe, H; Le Guyader, H

1996-12-10

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of alpha-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.

Structural studies on the co-chaperone Hop and its complexes with Hsp90.

PubMed

Onuoha, S C; Coulstock, E T; Grossmann, J G; Jackson, S E

2008-06-13

The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90. Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure. Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, 'open' state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers.

Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.

PubMed

Pennisi, Rosa; Antoccia, Antonio; Leone, Stefano; Ascenzi, Paolo; di Masi, Alessandra

2017-08-01

The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair. © 2017 Federation of European Biochemical Societies.

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

Hsp70 Forms Antiparallel Dimers Stabilized by Post-translational Modifications to Position Clients for Transfer to Hsp90

PubMed Central

Morgner, Nina; Schmidt, Carla; Beilsten-Edmands, Victoria; Ebong, Ima-obong; Patel, Nisha A.; Clerico, Eugenia M.; Kirschke, Elaine; Daturpalli, Soumya; Jackson, Sophie E.; Agard, David; Robinson, Carol V.

2015-01-01

Summary Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90. PMID:25921532

Hsp90 Binds Directly to Fibronectin (FN) and Inhibition Reduces the Extracellular Fibronectin Matrix in Breast Cancer Cells

PubMed Central

Kenyon, Amy; Dhanani, Karim C. H.; Prinsloo, Earl; Edkins, Adrienne L.

2014-01-01

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis. PMID:24466266

Hsp90 binds directly to fibronectin (FN) and inhibition reduces the extracellular fibronectin matrix in breast cancer cells.

PubMed

Hunter, Morgan C; O'Hagan, Kyle L; Kenyon, Amy; Dhanani, Karim C H; Prinsloo, Earl; Edkins, Adrienne L

2014-01-01

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.

HSP90 Shapes the Consequences of Human Genetic Variation.

PubMed

Karras, Georgios I; Yi, Song; Sahni, Nidhi; Fischer, Máté; Xie, Jenny; Vidal, Marc; D'Andrea, Alan D; Whitesell, Luke; Lindquist, Susan

2017-02-23

HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 ≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeting the FKBP51/GR/Hsp90 Complex to Identify Functionally Relevant Treatments for Depression and PTSD.

PubMed

Sabbagh, Jonathan J; Cordova, Ricardo A; Zheng, Dali; Criado-Marrero, Marangelie; Lemus, Andrea; Li, Pengfei; Baker, Jeremy D; Nordhues, Bryce A; Darling, April L; Martinez-Licha, Carlos; Rutz, Daniel A; Patel, Shreya; Buchner, Johannes; Leahy, James W; Koren, John; Dickey, Chad A; Blair, Laura J

2018-06-19

Genetic and epigenetic alterations in FK506-binding protein 5 ( FKBP5) have been associated with increased risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can increase the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR. One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo brain slices and primary neurons from mice. In conclusion, we have identified a novel disruptor of the FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to developing treatments for disorders related to aberrant FKBP51 expression.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Xiaomin; Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084; Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084

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 andmore » 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.« less

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

PubMed Central

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

2014-01-01

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

Cucurbitacin D Is a Disruptor of the HSP90 Chaperone Machinery.

PubMed

Hall, Jessica A; Seedarala, Sahithi; Rice, Nichole; Kopel, Lucas; Halaweish, Fathi; Blagg, Brian S J

2015-04-24

Heat shock protein 90 (Hsp90) facilitates the maturation of many newly synthesized and unfolded proteins (clients) via the Hsp90 chaperone cycle, in which Hsp90 forms a heteroprotein complex and relies upon cochaperones, immunophilins, etc., for assistance in client folding. Hsp90 inhibition has emerged as a strategy for anticancer therapies due to the involvement of clients in many oncogenic pathways. Inhibition of chaperone function results in client ubiquitinylation and degradation via the proteasome, ultimately leading to tumor digression. Small molecule inhibitors perturb ATPase activity at the N-terminus and include derivatives of the natural product geldanamycin. However, N-terminal inhibition also leads to induction of the pro-survival heat shock response (HSR), in which displacement of the Hsp90-bound transcription factor, heat shock factor-1, translocates to the nucleus and induces transcription of heat shock proteins, including Hsp90. An alternative strategy for Hsp90 inhibition is disruption of the Hsp90 heteroprotein complex. Disruption of the Hsp90 heteroprotein complex is an effective strategy to prevent client maturation without induction of the HSR. Cucurbitacin D, isolated from Cucurbita texana, and 3-epi-isocucurbitacin D prevented client maturation without induction of the HSR. Cucurbitacin D also disrupted interactions between Hsp90 and two cochaperones, Cdc37 and p23.

Conformational and functional studies of a cytosolic 90 kDa heat shock protein Hsp90 from sugarcane.

PubMed

da Silva, Viviane C H; Cagliari, Thiago C; Lima, Tatiani B; Gozzo, Fábio C; Ramos, Carlos H I

2013-07-01

Hsp90s are involved in several cellular processes, such as signaling, proteostasis, epigenetics, differentiation and stress defense. Although Hsp90s from different organisms are highly similar, they usually have small variations in conformation and function. Thus, the characterization of different Hsp90s is important to gain insight into the structure-function relationship that makes these chaperones key regulators in protein homeostasis. This work describes the characterization of a cytosolic Hsp90 from sugarcane and its comparison with Hsp90s from other plants. Previous expressed sequence tag (EST) studies in Saccharum spp. (sugarcane) predicted the presence of an mRNA coding for a cytosolic Hsp90. The corresponding cDNA was cloned, and the recombinant protein was purified and its conformation and function characterized. The structural conformation of Hsp90 was assessed by chemical cross-linking and hydrogen/deuterium exchange using mass spectrometry and hydrodynamic assays, which revealed regions accessible to solvent and that Hsp90 is an elongated dimer in solution. The in vivo expression of Hsp90 in sugarcane leaves was confirmed by western blot, and in vitro functional characterization indicated that sugarcane Hsp90 has strong chaperone activity. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

CUCURBITACIN D IS A DISRUPTOR OF THE HSP90 CHAPERONE MACHINERY

PubMed Central

Hall, Jessica A.; Seedarala, Sahithi; Rice, Nichole; Kopel, Lucas; Halaweish, Fathi; Blagg, Brian S. J.

2018-01-01

Heat shock protein 90 (Hsp90) facilitates the maturation of many newly synthesized and unfolded proteins (clients) via the Hsp90 chaperone cycle, in which Hsp90 forms a heteroprotein complex and relies upon co-chaperones, immunophilins, etc. for assistance in client folding. Hsp90 inhibition has emerged as a strategy for anticancer therapies due to the involvement of clients in many oncogenic pathways. Inhibition of chaperone function results in client ubiquitinylation and degradation via the proteasome, ultimately leading to tumor digression. Small molecule inhibitors perturb ATPase activity at the N-terminus and include derivatives of the natural product geldanamycin. However, N-terminal inhibition also leads to induction of the pro-survival heat shock response (HSR), in which displacement of the Hsp90-bound transcription factor, Heat Shock Factor-1 translocates to the nucleus and induces transcription of heat shock proteins, including Hsp90. An alternative strategy for Hsp90 inhibition is disruption of the Hsp90 heteroprotein complex. Disruption of the Hsp90 heteroprotein complex is an effective strategy to prevent client maturation without induction of the HSR. Cucurbitacin D, isolated from Cucurbita texana, and 3-epi-isocucurbitacin D prevented client maturation without induction of the HSR. Cucurbitacin D also disrupted interactions between Hsp90 and two co-chaperones, Cdc37 and p23. PMID:25756299

Cloning of a heat shock protein 90 (HSP90) gene and expression analysis in the ridgetail white prawn Exopalaemon carinicauda.

PubMed

Li, Jitao; Han, Junying; Chen, Ping; Chang, Zhiqiang; He, Yuying; Liu, Ping; Wang, Qingyin; Li, Jian

2012-06-01

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. In this study, a heat shock protein 90 cDNA named EcHSP90 was cloned from the hepatopancreas of ridgetail white prawn Exopalaemon carinicauda by reverse transcription polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcHSP90 was of 2695 bp, including an open reading frame (ORF) of 2163 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 82.73 kDa and an estimated isoelectric point of 4.83. BLAST analysis revealed that the EcHSP90 shared high similarity (87.6%-75.24%) with other known HSP90s. The five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in EcHSP90, which indicated that EcHSP90 should be a cytosolic member of the HSP90 family. Quantitative real-time RT-PCR analysis revealed that EcHSP90 transcript could be detected in all the tested tissues, and strongly expressed in ovary of E. carinicauda. The transcript of EcHSP90 in hepatopancreas of E. carinicauda showed different expression profiles after pH and ammonia-N stresses. The results indicated that EcHSP90 was a constitutive and inducible expressed protein and could be induced by various stresses from environment. Copyright © 2012 Elsevier Ltd. All rights reserved.

Monitoring the change of mitochondrial morphology and its metabolism of the breast cancer cells with the treatment of Hsp70 inhibitor during heat shock by fluorescence imaging

NASA Astrophysics Data System (ADS)

Yu, Biying; Yang, Hongqin; Zhang, Xiaoman; Li, Hui

2016-10-01

Heat shock (HS) is one of the best-studied exogenous cellular stresses, and all cellular compartments and metabolic processes are involved in HS response. The heat shock proteins (Hsps) expression enhanced during HS mainly localized in subcellular compartments, such as cytosol, endoplasmic reticulum and mitochandria. The major inducible heat shock protein 70 (Hsp70) modulate cellular homeostasis and promote cellular survival by blocking a caspase independent cell death through its association with apoptosis inducing factor. Mitochondria as the critical elements of HS response that participate in key metabolic reactions, and the changes in mitochonrial morphology may impact on mitochondrial metabolism. In this paper, the changes of mitorchondrial morphology in breast cancer cell have been monitored in real time after heat shock (43 °) by the fluorescence imaging, and the influence of Hsp70 inhibitor on mitochandrial structures have also been investigated. Then the information of mitochondrial metabolism which can be characterized by the level of the mitochondrial membrane potential has also been obtained wihout/with the treatment of Hsp70 inhibitor. Our data indicated that the mitochandrial morphology were related with the mitochandrial membrane potential, and the mitochandrial membrane potential was influenced significantly with the treatment of Hsp70 inhibitor during HS.

Hsp90 inhibitors sensitise human colon cancer cells to topoisomerase I poisons by depletion of key anti-apoptotic and cell cycle checkpoint proteins.

PubMed

McNamara, Anne V; Barclay, Monica; Watson, Alastair J M; Jenkins, John R

2012-02-01

Hsp90 and topoisomerase I are both targets for chemotherapeutic agents. Topoisomerase I poisons are standard clinical treatments, whilst Hsp90 inhibitors are progressing through clinical trials. We have demonstrated that when an Hsp90 inhibitor and topoisomerase I poison are combined they produce a synergistic increase in apoptosis in both p53⁺/⁺ and p53⁻/⁻ HCT116 human colon cancer cells. Lack of p53 is associated with an increase in sensitivity to the combination treatment; p53⁺/⁺ cells treated with the topoisomerase I poison topotecan (TPT) arrest at G2, whereas in p53⁻/⁻ cells the additional presence of the Hsp90 inhibitor geldanamycin (GA) selectively abrogates the G2M checkpoint. More importantly we report that there is a common underlying p53-independent mechanism behind the observed synergistic combined drug effect. We show that concurrent treatment with GA and TPT is able to reverse TPT induced up-regulation of the anti-apoptotic protein Bcl2 in both p53⁺/⁺ and p53⁻/⁻ HCT116 cells. The data suggests that inhibition of Hsp90 mediates down-regulation of Bcl2 following the combination treatment and cause a synergistic increase in apoptosis in both p53⁺/⁺ and p53⁻/⁻ HCT116 cells; p53⁻/⁻ HCT116 cells are more sensitive to the treatment because they also fail to arrest at G2 in the cell cycle. Copyright © 2011 Elsevier Inc. All rights reserved.

Protein Analysis of Purified Respiratory Syncytial Virus Particles Reveals an Important Role for Heat Shock Protein 90 in Virus Particle Assembly*

PubMed Central

Radhakrishnan, Anuradha; Yeo, Dawn; Brown, Gaie; Myaing, Myint Zu; Iyer, Laxmi Ravi; Fleck, Roland; Tan, Boon-Huan; Aitken, Jim; Sanmun, Duangmanee; Tang, Kai; Yarwood, Andy; Brink, Jacob; Sugrue, Richard J.

2010-01-01

In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of β-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy. PMID:20530633

Hsp90 Promotes Kinase Evolution

PubMed Central

Lachowiec, Jennifer; Lemus, Tzitziki; Borenstein, Elhanan; Queitsch, Christine

2015-01-01

Heat-shock protein 90 (Hsp90) promotes the maturation and stability of its client proteins, including many kinases. In doing so, Hsp90 may allow its clients to accumulate mutations as previously proposed by the capacitor hypothesis. If true, Hsp90 clients should show increased evolutionary rate compared with nonclients; however, other factors, such as gene expression and protein connectivity, may confound or obscure the chaperone’s putative contribution. Here, we compared the evolutionary rates of many Hsp90 clients and nonclients in the human protein kinase superfamily. We show that Hsp90 client status promotes evolutionary rate independently of, but in a small magnitude similar to that of gene expression and protein connectivity. Hsp90’s effect on kinase evolutionary rate was detected across mammals, specifically relaxing purifying selection. Hsp90 clients also showed increased nucleotide diversity and harbored more damaging variation than nonclient kinases across humans. These results are consistent with the central argument of the capacitor hypothesis that interaction with the chaperone allows its clients to harbor genetic variation. Hsp90 client status is thought to be highly dynamic with as few as one amino acid change rendering a protein dependent on the chaperone. Contrary to this expectation, we found that across protein kinase phylogeny Hsp90 client status tends to be gained, maintained, and shared among closely related kinases. We also infer that the ancestral protein kinase was not an Hsp90 client. Taken together, our results suggest that Hsp90 played an important role in shaping the kinase superfamily. PMID:25246701

Posttranslational modification of mitochondrial transcription factor A in impaired mitochondria biogenesis: implications in diabetic retinopathy and metabolic memory phenomenon.

PubMed

Santos, Julia M; Mishra, Manish; Kowluru, Renu A

2014-04-01

Mitochondrial transcription factor A (TFAM) is one of the key regulators of the transcription of mtDNA. In diabetes, despite increase in gene transcripts of TFAM, its protein levels in the mitochondria are decreased and mitochondria copy numbers become subnormal. The aim of this study is to investigate the mechanism(s) responsible for decreased mitochondrial TFAM in diabetes. Using retinal endothelial cells, we have investigated the effect of overexpression of cytosolic chaperone, Hsp70, and TFAM on glucose-induced decrease in mitochondrial TFAM levels, and the transcription of mtDNA-encoded genes, NADH dehydrogenase subunit 6 (ND6) and cytochrome b (Cytb). To investigate the role of posttranslational modifications in subnormal mitochondrial TFAM, ubiquitination of TFAM was assessed, and the results were confirmed in the retina from streptozotocin-induced diabetic rats. While overexpression of Hsp70 failed to prevent glucose-induced decrease in mitochondrial TFAM and transcripts of ND6 and Cytb, overexpression of TFAM ameliorated decrease in its mitochondrial protein levels and transcriptional activity. TFAM was ubiquitinated by high glucose, and PYR-41, an inhibitor of ubiquitination, prevented TFAM ubiquitination and restored the transcriptional activity. Similarly, TFAM was ubiquitinated in the retina from diabetic rats, and it continued to be modified after reinstitution of normal glycemia. Our results clearly imply that the ubiquitination of TFAM impedes its transport to the mitochondria resulting in subnormal mtDNA transcription and mitochondria dysfunction, and inhibition of ubiquitination restores mitochondrial homeostasis. Reversal of hyperglycemia does not provide any benefit to TFAM ubiquitination. Thus, strategies targeting posttranslational modification could provide an avenue to preserve mitochondrial homeostasis, and inhibit the development/progression of diabetic retinopathy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cloning of HSP90, expression and localization of HSP70/90 in different tissues including lactating/non-lactating yak (Bos grunniens) breast tissue.

PubMed

Liu, Penggang; Yu, Sijiu; Cui, Yan; He, Junfeng; Yu, Chuan; Wen, Zexing; Pan, Yangyang; Yang, Kun; Song, Liangli; Yang, Xue

2017-01-01

The aim of this study is to investigate the expression and localization of HSP70/90 in different tissues and explore the regulation effects of HSP70/90 at lactation period of female yaks. HSP90 mRNA was cloned from the heart samples of female yaks, Quantitative real-time (qRT-PCR), Western blotting (WB), immunohistochemistry and immunofluorescence assays were utilized to analyze the expressions of HSP70/90 mRNA and protein in different tissues. Sequence analysis showed that HSP90 is a conserved molecular chaperone of female yaks. The qRT-PCR, WB results showed that the expressions of HSP70/90 mRNA and protein were significantly different in different tissues, and 3-fold higher expression during the lactation period than the non-lactation period of breast tissue (P < 0.01). Immunohistochemistry and immunofluorescence assays results showed that HSP70/90 were located in the cardiac muscle cells, cerebellar medulla, theca cells lining at the reproductive system, and the mammary epithelia of the breasts. In addition, the expression level of HSP70 was higher than those of HSP90 in all examined tissues. Therefore, our results strongly suggest that the expression and localization of HSP70/90 could provide significant evidence to further research in tissue specific expression, and lactation function of female yaks.

Hsp70 and Hsp90 Multichaperone Complexes Sequentially Regulate Thiazide-sensitive Cotransporter Endoplasmic Reticulum-associated Degradation and Biogenesis*

PubMed Central

Donnelly, Bridget F.; Needham, Patrick G.; Snyder, Avin C.; Roy, Ankita; Khadem, Shaheen; Brodsky, Jeffrey L.; Subramanya, Arohan R.

2013-01-01

The thiazide-sensitive NaCl cotransporter (NCC) is the primary mediator of salt reabsorption in the distal convoluted tubule and is a key determinant of the blood pressure set point. Given its complex topology, NCC is inefficiently processed and prone to endoplasmic reticulum (ER)-associated degradation (ERAD), although the mechanisms governing this process remain obscure. Here, we identify factors that impact the ER quality control of NCC. Analyses of NCC immunoprecipitates revealed that the cotransporter formed complexes with the core chaperones Hsp90, Hsp70, and Hsp40. Disruption of Hsp90 function accelerated NCC degradation, suggesting that Hsp90 promotes NCC folding. In addition, two cochaperones, the C terminus of Hsp70-interacting protein (CHIP) and the Hsp70/Hsp90 organizer protein, were associated with NCC. Although CHIP, an E3 ubiquitin ligase, promoted NCC ubiquitination and ERAD, the Hsp70/Hsp90 organizer protein stabilized NCC turnover, indicating that these two proteins differentially remodel the core chaperone systems to favor cotransporter degradation and biogenesis, respectively. Adjusting the folding environment in mammalian cells via reduced temperature enhanced NCC biosynthetic trafficking, increased Hsp90-NCC interaction, and diminished binding to Hsp70. In contrast, cotransporters harboring disease-causing mutations that impair NCC biogenesis failed to escape ERAD as efficiently as the wild type protein when cells were incubated at a lower temperature. Instead, these mutants interacted more strongly with Hsp70, Hsp40, and CHIP, consistent with a role for the Hsp70/Hsp40 system in selecting misfolded NCC for ERAD. Collectively, these observations indicate that Hsp70 and Hsp90 comprise two functionally distinct ER quality control checkpoints that sequentially monitor NCC biogenesis. PMID:23482560

The 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation*

PubMed Central

Weis, Felix; Moullintraffort, Laura; Heichette, Claire; Chrétien, Denis; Garnier, Cyrille

2010-01-01

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90. PMID:20110359

Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems.

PubMed

Tsuboyama, Kotaro; Tadakuma, Hisashi; Tomari, Yukihide

2018-05-17

Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form. Copyright © 2018 Elsevier Inc. All rights reserved.

Ammonia stress under high environmental ammonia induces Hsp70 and Hsp90 in the mud eel, Monopterus cuchia.

PubMed

Hangzo, Hnunlalliani; Banerjee, Bodhisattwa; Saha, Shrabani; Saha, Nirmalendu

2017-02-01

The obligatory air-breathing mud eel (Monopterus cuchia) is frequently being challenged with high environmental ammonia (HEA) exposure in its natural habitats. The present study investigated the possible induction of heat shock protein 70 and 90 (hsp70, hsc70, hsp90α and hsp90β) genes and more expression of Hsp70 and Hsp90 proteins under ammonia stress in different tissues of the mud eel after exposure to HEA (50 mM NH 4 Cl) for 14 days. HEA resulted in significant accumulation of toxic ammonia in different body tissues and plasma, which was accompanied with the stimulation of oxidative stress in the mud eel as evidenced by more accumulation of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) during exposure to HEA. Further, hyper-ammonia stress led to significant increase in the levels of mRNA transcripts for inducible hsp70 and hsp90α genes and also their translated proteins in different tissues probably as a consequence of induction of hsp70 and hsp90α genes in the mud eel. However, hyper-ammonia stress was neither associated with any significant alterations in the levels of mRNA transcripts for constitutive hsc70 and hsp90β genes nor their translated proteins in any of the tissues studied. More abundance of Hsp70 and Hsp90α proteins might be one of the strategies adopted by the mud eel to defend itself from the ammonia-induced cellular damages under ammonia stress. Further, this is the first report of ammonia-induced induction of hsp70 and hsp90α genes under hyper-ammonia stress in any freshwater air-breathing teleost.

KU675, a Concomitant Heat-Shock Protein Inhibitor of Hsp90 and Hsc70 that Manifests Isoform Selectivity for Hsp90α in Prostate Cancer Cells

PubMed Central

Liu, Weiya; Vielhauer, George A.; Zhao, Huiping; Ghosh, Suman; Brown, Douglas; Lee, Eugene

2015-01-01

The 90-kDa heat-shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Inhibiting Hsp90 consequently is an attractive strategy for cancer therapy as the concomitant degradation of multiple oncoproteins may lead to effective antineoplastic agents. Here we report a novel C-terminal Hsp90 inhibitor, designated KU675, that exhibits potent antiproliferative and cytotoxic activity along with client protein degradation without induction of the heat-shock response in both androgen-dependent and -independent prostate cancer cell lines. In addition, KU675 demonstrates direct inhibition of Hsp90 complexes as measured by the inhibition of luciferase refolding in prostate cancer cells. In direct binding studies, the internal fluorescence signal of KU675 was used to determine the binding affinity of KU675 to recombinant Hsp90α, Hsp90β, and Hsc70 proteins. The binding affinity (Kd) for Hsp90α was determined to be 191 μM, whereas the Kd for Hsp90β was 726 μM, demonstrating a preference for Hsp90α. Western blot experiments with four different prostate cancer cell lines treated with KU675 supported this selectivity by inducing the degradation of Hsp90α-dependent client proteins. KU675 also displayed binding to Hsc70 with a Kd value at 76.3 μM, which was supported in cellular by lower levels of Hsc70-specific client proteins on Western blot analyses. Overall, these findings suggest that KU675 is an Hsp90 C-terminal inhibitor, as well as a dual inhibitor of Hsc70, and may have potential use for the treatment of cancer. PMID:25939977

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

PubMed Central

Uchida, Akira; Murugesapillai, Divakaran; Kastner, Markus; Wang, Yao; Lodeiro, Maria F; Prabhakar, Shaan; Oliver, Guinevere V; Arnold, Jamie J; Maher, L James; Williams, Mark C; Cameron, Craig E

2017-01-01

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications. DOI: http://dx.doi.org/10.7554/eLife.27283.001 PMID:28745586

The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

PubMed Central

Schneider, H C; Westermann, B; Neupert, W; Brunner, M

1996-01-01

Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle. Images PMID:8918457

Molecular cloning, characterization and expression analysis of HSP60, HSP70 and HSP90 in the golden apple snail, Pomacea canaliculata.

PubMed

Xu, Yipeng; Zheng, Guowan; Dong, Shengzhang; Liu, Guangfu; Yu, Xiaoping

2014-12-01

The golden apple snail, Pomacea canaliculata, has strong tolerance to high temperature, facilitating its invasion in East and Southeast Asia. In the present study, three cDNAs encoding heat shock proteins (PocaHSP60, PocaHSP70, PocaHSP90) in P. canaliculata were cloned and characterized. The PocaHSP60 cDNA was 2447 bp, containing an ORF encoding a polypeptide of 574 amino acids. The PocaHSP70 cDNA was 2644 bp, containing an ORF encoding a polypeptide of 643 amino acids. The PocaHSP90 cDNA was 2546 bp, containing an ORF encoding a polypeptide of 726 amino acids. Genomic DNA analysis showed that PocaHSP60 had 11 introns in the coding region and PocaHSP90 had 7 introns but PocaHSP70 had no one. The expression changes of these three PocaHSPs in the gill, digestive gland, kidney and foot muscle of P. canaliculata exposed to high and low temperature were investigated. The results of quantitative PCR and western blotting showed that the expression level of PocaHSP90 was much higher than PocaHSP60 and PocaHSP70 at room temperature, and PocaHSP70 expression level was the lowest among them. Afterheat shock, PocaHSP70 expression increased rapidly, much more significantly than PocaHSP90 expression, and the effect of heat shock on the expression of PocaHSP70 and PocaHSP90 in the different tissues of P. canaliculata was not the same. Unlike PocaHSP70 and PocaHSP90, PocaHSP60 expression seemed not to be affected by heat shock, because its expression was moderately induced only in the foot muscle. However, cool shock had little effect on the expression change of above three PocaHSPs. These results indicated that HSPs might be related to the thermal resistance of P. canaliculata.

Heat shock protein 90{beta}: A novel mediator of vitamin D action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angelo, Giana; Mineral Bioavailability Laboratory, 711 Washington Street, Boston, MA 02111; Lamon-Fava, Stefania

2008-03-14

We investigated the role of Heat shock protein 90 (Hsp90) in vitamin D action in Caco-2 cells using geldanamycin (GA) to block Hsp90 function and RNA interference to reduce Hsp90{beta} expression. When cells were exposed to GA, vitamin D-mediated gene expression and transcriptional activity were inhibited by 69% and 54%, respectively. Gel shift analysis indicated that GA reduced vitamin D-mediated DNA binding activity of the vitamin D receptor (VDR). We tested the specific role of Hsp90{beta} by knocking down its expression with stably expressed short hairpin RNA. Vitamin D-induced gene expression and transcriptional activity were reduced by 90% and 80%,more » respectively, in Hsp90{beta}-deficient cells. Nuclear protein for VDR and RXR{alpha}, its heterodimer partner, were not reduced in Hsp90{beta}-deficient cells. These findings indicate that Hsp90{beta} is needed for optimal vitamin D responsiveness in the enterocyte and demonstrate a specific role for Hsp90{beta} in VDR signaling.« less

Temperature effects on gene expression and morphological development of European eel, Anguilla anguilla larvae

PubMed Central

Mazurais, David; Servili, Arianna; Zambonino-Infante, Jose-Luis; Miest, Joanna J.; Sørensen, Sune R.; Tomkiewicz, Jonna; Butts, Ian A. E.

2017-01-01

Temperature is important for optimization of rearing conditions in aquaculture, especially during the critical early life history stages of fish. Here, we experimentally investigated the impact of temperature (16, 18, 20, 22 and 24°C) on thermally induced phenotypic variability, from larval hatch to first-feeding, and the linked expression of targeted genes [heat shock proteins (hsp), growth hormone (gh) and insulin-like growth factors (igf)] associated to larval performance of European eel, Anguilla anguilla. Temperature effects on larval morphology and gene expression were investigated throughout early larval development (in real time from 0 to 18 days post hatch) and at specific developmental stages (hatch, jaw/teeth formation, and first-feeding). Results showed that hatch success, yolk utilization efficiency, survival, deformities, yolk utilization, and growth rates were all significantly affected by temperature. In real time, increasing temperature from 16 to 22°C accelerated larval development, while larval gene expression patterns (hsp70, hsp90, gh and igf-1) were delayed at cold temperatures (16°C) or accelerated at warm temperatures (20–22°C). All targeted genes (hsp70, hsp90, gh, igf-1, igf-2a, igf-2b) were differentially expressed during larval development. Moreover, expression of gh was highest at 16°C during the jaw/teeth formation, and the first-feeding developmental stages, while expression of hsp90 was highest at 22°C, suggesting thermal stress. Furthermore, 24°C was shown to be deleterious (resulting in 100% mortality), while 16°C and 22°C (~50 and 90% deformities respectively) represent the lower and upper thermal tolerance limits. In conclusion, the high survival, lowest incidence of deformities at hatch, high yolk utilization efficiency, high gh and low hsp expression, suggest 18°C as the optimal temperature for offspring of European eel. Furthermore, our results suggest that the still enigmatic early life history stages of European eel may inhabit the deeper layer of the Sargasso Sea and indicate vulnerability of this critically endangered species to increasing ocean temperature. PMID:28806748

Cold-Induced Accumulation of hsp90 Transcripts in Brassica napus.

PubMed Central

Krishna, P.; Sacco, M.; Cherutti, J. F.; Hill, S.

1995-01-01

Characterization of the expression of hsp90 genes of Brassica napus by northern blot analysis and immunoblotting showed that the hsp90 mRNA and protein are present in all B. napus tissues examined, albeit at different levels. High levels of hsp90 mRNA and protein were found in young and rapidly dividing tissues such as shoot apices and flower buds, suggesting that hsp90 may have an important role in plant growth and development. A significant increase in hsp90 mRNA levels was detected in seedlings exposed to 5[deg]C. The transcript levels reached a maximum within 1 d of cold treatment and remained elevated for the entire duration of cold treatment. The levels of hsp90 mRNA rapidly decreased to the level found in control plants upon return to 20[deg]C. The cold-induced accumulation of hsp90 mRNA closely resembles the expression of two previously identified cold-regulated genes of B. napus. We have also confirmed cold regulation of hsp90 mRNA in spinach (Spinacea oleracea). Our results suggest a role for hsp90 in adaptation to cold temperature stress. PMID:12228411

Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: Transcriptional induction in response to thermal stress in hemocyte primary culture.

PubMed

Farcy, Emilie; Serpentini, Antoine; Fiévet, Bruno; Lebel, Jean-Marc

2007-04-01

Heat-shock proteins are a multigene family of proteins whose expression is induced by a variety of stress factors. This work reports the cloning and sequencing of HSP70 and HSP90 cDNAs in the gastropod Haliotis tuberculata. The deduced amino acid sequences of both HSP70 and HSP90 from H. tuberculata shared a high degree of homology with their homologues in other species, including typical eukaryotic HSP70 and HSP90 signature sequences. We examined their transcription expression pattern in abalone hemocytes exposed to thermal stress. Real-time PCR analysis indicated that both HSP70 and HSP90 mRNA were expressed in control animals but rapidly increased after heat-shock.

The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth

PubMed Central

Sun, Hongchao; Zhuo, Xunhui; Zhao, Xianfeng; Yang, Yi; Chen, Xueqiu; Yao, Chaoqun; Du, Aifang

2017-01-01

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects almost all warm-blooded vertebrates. Heat shock proteins (HSP) regulate key signal transduction events in many organisms, and heat shock protein 90 (Hsp90) plays an important role in growth, development, and virulence in several parasitic protozoa. Here, we discovered increased transcription of the Hsp90 gene under conditions for bradyzoite differentiation, i.e. alkaline and heat shock conditions in vitro, suggesting that Hsp90 may be connected with bradyzoite development in T. gondii. A knockout of the TgHsp90 strain (ΔHsp90) and a complementation strain were constructed. The TgHsp90 knockout cells were found to be defective in host-cell invasion, were not able to proliferate in vitro in Vero cells, and did not show long-time survival in mice in vivo. These inabilities of the knockout parasites were restored upon complementation of TgHsp90. These data unequivocally show that TgHsp90 contributes to bradyzoite development, and to invasion and replication of T. gondii in host cells. PMID:28627357

1,4-Naphthoquinone activates the HSP90/HSF1 pathway through the S-arylation of HSP90 in A431 cells: Negative regulation of the redox signal transduction pathway by persulfides/polysulfides.

PubMed

Abiko, Yumi; Sha, Liang; Shinkai, Yasuhiro; Unoki, Takamitsu; Luong, Nho Cong; Tsuchiya, Yukihiro; Watanabe, Yasuo; Hirose, Reiko; Akaike, Takaaki; Kumagai, Yoshito

2017-03-01

The current consensus is that environmental electrophiles activate redox signal transduction pathways through covalent modification of sensor proteins with reactive thiol groups at low concentrations, while they cause cell damage at higher concentrations. We previously exposed human carcinoma A431 cells to the atmospheric electrophile 1,4-naphthoquinone (1,4-NQ) and found that heat shock protein 90 (HSP90), a negative regulator of heat shock factor 1 (HSF1), was a target of 1,4-NQ. In the study presented here, we determined whether 1,4-NQ activates HSF1. We also examined whether such redox signaling could be regulated by nucleophilic sulfur species. Exposure of A431 cells to 1,4-NQ covalently modified cellular HSP90, resulting in repression of the association between HSF1 with HSP90, thereby enhancing HSF1 translocation into the nuclei. Liquid chromatography-tandem mass spectrometry analysis with recombinant HSP90 revealed that the modifications site were Cys412 and Cys564. We found that HSF1 activation mediated by 1,4-NQ upregulated downstream genes, such as HSPA6. HSF1 knockdown accelerated 1,4-NQ-mediated cytotoxicity in the cells. While simultaneous treatment with reactive persulfide and polysulfide, Na 2 S 2 and Na 2 S 4 , blocked 1,4-NQ-dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) enhanced these phenomena. 1,4-NQ-thiol adduct and 1,4-NQ-S-1,4-NQ adduct were produced during the enzymatic reaction of recombinant CSE in the presence of 1,4-NQ. The results suggest that activation of the HSP90-HSF1 signal transduction pathway mediated by 1,4-NQ protects cells against 1,4-NQ and that per/polysulfides can diminish the reactivity of 1,4-NQ by forming sulfur adducts. Copyright © 2017 Elsevier Inc. All rights reserved.

Increased extracellular heat shock protein 90α in severe sepsis and SIRS associated with multiple organ failure and related to acute inflammatory-metabolic stress response in children.

PubMed

Fitrolaki, Michaela-Diana; Dimitriou, Helen; Venihaki, Maria; Katrinaki, Marianna; Ilia, Stavroula; Briassoulis, George

2016-08-01

Mammalian heat-shock-protein (HSP) 90α rapidly responses to environmental insults. We examined the hypothesis that not only serum HSP72 but also HSP90α is increased in the systemic inflammatory response syndrome (SIRS), severe-sepsis (SS), and/or sepsis (S) compared to healthy children (H); we assessed HSP90α relation to (a) multiple organ system failure (MOSF) and (b) inflammatory-metabolic response and severity of illness.A total of 65 children with S, SS, or SIRS and 25 H were included. ELISA was used to evaluate extracellular HSP90α and HSP72, chemiluminescence interleukins (ILs), flow-cytometry neutrophil-CD64 (nCD64)-expression.HSP90α, along with HSP72, were dramatically increased among MOSF patients. Patients in septic groups and SIRS had elevated HSP90α compared to H (P < 0.01). HSP90α was independently related to predicted death rate and severity of illness; positively to HSP72, nCD64, ILs, length of stay, days on ventilator, and fever; negatively to HDL and LDL (P < 0.05). The HSP72 was increased in SS/S and related negatively to HDL and LDL (P < 0.05).Serum HSP90α is markedly elevated in children with severe sepsis and is associated with MOSF. Better than the HSP72, also increased in SS, SIRS, and MOSF, HSP90α is related to the inflammatory stress, fever, outcome endpoints, and predicted mortality and inversely related to the low-LDL/low-HDL stress metabolic pattern.

Increased extracellular heat shock protein 90α in severe sepsis and SIRS associated with multiple organ failure and related to acute inflammatory-metabolic stress response in children

PubMed Central

Fitrolaki, Michaela-Diana; Dimitriou, Helen; Venihaki, Maria; Katrinaki, Marianna; Ilia, Stavroula; Briassoulis, George

2016-01-01

Abstract Mammalian heat-shock-protein (HSP) 90α rapidly responses to environmental insults. We examined the hypothesis that not only serum HSP72 but also HSP90α is increased in the systemic inflammatory response syndrome (SIRS), severe-sepsis (SS), and/or sepsis (S) compared to healthy children (H); we assessed HSP90α relation to (a) multiple organ system failure (MOSF) and (b) inflammatory-metabolic response and severity of illness. A total of 65 children with S, SS, or SIRS and 25 H were included. ELISA was used to evaluate extracellular HSP90α and HSP72, chemiluminescence interleukins (ILs), flow-cytometry neutrophil-CD64 (nCD64)-expression. HSP90α, along with HSP72, were dramatically increased among MOSF patients. Patients in septic groups and SIRS had elevated HSP90α compared to H (P < 0.01). HSP90α was independently related to predicted death rate and severity of illness; positively to HSP72, nCD64, ILs, length of stay, days on ventilator, and fever; negatively to HDL and LDL (P < 0.05). The HSP72 was increased in SS/S and related negatively to HDL and LDL (P < 0.05). Serum HSP90α is markedly elevated in children with severe sepsis and is associated with MOSF. Better than the HSP72, also increased in SS, SIRS, and MOSF, HSP90α is related to the inflammatory stress, fever, outcome endpoints, and predicted mortality and inversely related to the low-LDL/low-HDL stress metabolic pattern. PMID:27583886

Neurite outgrowth mediated by the heat shock protein Hsp90α: a novel target for the antipsychotic drug aripiprazole

PubMed Central

Ishima, T; Iyo, M; Hashimoto, K

2012-01-01

Aripiprazole is an atypical antipsychotic drug approved for the treatment of psychiatric disorders such as schizophrenia, bipolar disorder, major depressive disorder and autism. The drug shows partial agonistic activity at dopamine D2 receptors and 5-hydroxytryptamine (5-HT) 5-HT1A receptors, and antagonistic activity at 5-HT2A receptors. However, the precise mechanistic pathways remain unclear. In this study, we examined the effects of aripiprazole on neurite outgrowth. Aripiprazole significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, in a concentration-dependent manner. The 5-HT1A receptor antagonist WAY-100635, but not the dopamine D2 receptor antagonist sulpiride, blocked the effects of aripiprazole, although, only partially. Specific inhibitors of inositol 1,4,5-triphosphate (IP3) receptors and BAPTA-AM, a chelator of intracellular Ca2+, blocked the effects of aripiprazole. Moreover, specific inhibitors of several common signaling pathways phospholipase C-γ (PLC-γ), phosphatidylinositol-3 kinase (PI3K), mammalian target of rapamycin, p38 MAPK, c-Jun N-terminal kinase, Akt, Ras, Raf, ERK, MAPK) also blocked the effects of aripiprazole. Using proteomic analysis, we found that aripiprazole significantly increased levels of the heat shock protein Hsp90α in cultured cells. The effects of aripiprazole on NGF-induced neurite outgrowth were significantly attenuated by treatment with Hsp90α RNA interference, but not by the negative control of Hsp90α. These findings suggest that both 5-HT1A receptor activation and Ca2+ signaling via IP3 receptors, as well as their downstream cellular signaling pathways play a role in the promotion of aripiprazole-induced neurite outgrowth. Furthermore, aripiprazole-induced increases in Hsp90α protein expression may form part of the therapeutic mechanism for this drug. PMID:23047241

Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone sis1

DOE PAGES

Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; ...

2015-02-13

Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activitymore » with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor proteins. Finally, these interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.« less

Roles of Intramolecular and Intermolecular Interactions in Functional Regulation of the Hsp70 J-protein Co-Chaperone Sis1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy

2015-04-01

Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at heir C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activitymore » with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively.« less

Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1.

PubMed

Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A

2015-04-10

Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways, Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70∆EEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interactions between the J-domain and glycine-rich region control co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. However, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

Roles of Intramolecular and Intermolecular Interactions in Functional Regulation of the Hsp70 J-protein Co-chaperone Sis1

PubMed Central

Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy; Ciesielski, Szymon J.; Baranowski, Maciej; Zhou, Min; Joachimiak, Andrzej; Craig, Elizabeth A.

2015-01-01

Unlike other Hsp70 molecular chaperones, those of the eukaryotic cytosol have four residues, EEVD, at their C-termini. EEVD(Hsp70) binds adaptor proteins of the Hsp90 chaperone system and mitochondrial membrane preprotein receptors, thereby facilitating processing of Hsp70-bound clients through protein folding and translocation pathways. Among J-protein co-chaperones functioning in these pathways Sis1 is unique, as it also binds the EEVD(Hsp70) motif. However, little is known about the role of the Sis1:EEVD(Hsp70) interaction. We found that deletion of EEVD(Hsp70) abolished the ability of Sis1, but not the ubiquitous J-protein Ydj1, to partner with Hsp70 in in vitro protein refolding. Sis1 co-chaperone activity with Hsp70ΔEEVD was restored upon substitution of a glutamic acid of the J-domain. Structural analysis revealed that this key glutamic acid, which is not present in Ydj1, forms a salt bridge with an arginine of the immediately adjacent glycine-rich region. Thus, restoration of Sis1 in vitro activity suggests that intramolecular interaction(s) between the J-domain and glycine-rich region controls co-chaperone activity, which is optimal only when Sis1 interacts with the EEVD(Hsp70) motif. Yet, we found that disruption of the Sis1:EEVD(Hsp70) interaction enhances the ability of Sis1 to substitute for Ydj1 in vivo. Our results are consistent with the idea that interaction of Sis1 with EEVD(Hsp70) minimizes transfer of Sis1-bound clients to Hsp70s that are primed for client transfer to folding and translocation pathways by their preassociation with EEVD-binding adaptor proteins. These interactions may be one means by which cells triage Ydj1- and Sis1-bound clients to productive and quality control pathways, respectively. PMID:25687964

Immunogenic apoptosis in human acute myeloid leukemia (AML): primary human AML cells expose calreticulin and release heat shock protein (HSP) 70 and HSP90 during apoptosis.

PubMed

Fredly, Hanne; Ersvær, Elisabeth; Gjertsen, Bjørn-Tore; Bruserud, Oystein

2011-06-01

Several previous studies have demonstrated that both conventional cytotoxic drugs as well as targeted therapeutics can induce apoptosis in primary human acute myelogenous leukemia (AML) cells. However, the apoptotic phenotype of dying AML cells has been less extensively characterized. Even though specific antileukemic immune reactivity is important in AML, especially for allotransplanted patients, it has not been investigated whether dying primary human AML cells show phenotypic characteristics consistent with immunogenic apoptosis [calreticulin exposure, heat shock protein (HSP) release]. We therefore investigated whether in vitro cultured primary human acute myeloid leukemia (AML) cells show calreticulin exposure and HSP70/HSP90 release during spontaneous (stress-induced) apoptosis when cultured in medium alone and when cultured in the presence of antileukemic drugs. Both surface exposure of calreticulin and release of HSP70 and HSP90 was detected but showed a wide variation between patients. This variation was also maintained when the AML cells were cultured in the presence of cytotoxic drugs (cytarabine, daunorubicin, mitomycin), all-trans retinoic acid (ATRA) and valproic acid. Finally, AML cells collected during in vivo ATRA therapy showed increased calreticulin exposure during spontaneous in vitro apoptosis, suggesting that in vivo pharmacotherapy can modulate the apoptotic phenotype. To conclude, apoptotic AML cells can show phenotypic characteristics consistent with immunogenic apoptosis, but there is a wide variation between patients and the level of calreticulin exposure/HSP release seems to depend on individual patient characteristics rather than the apoptosis-inducing agent.

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

Discovery of new molecular entities able to strongly interfere with Hsp90 C-terminal domain.

PubMed

Terracciano, Stefania; Russo, Alessandra; Chini, Maria G; Vaccaro, Maria C; Potenza, Marianna; Vassallo, Antonio; Riccio, Raffaele; Bifulco, Giuseppe; Bruno, Ines

2018-01-26

Heat shock protein 90 (Hsp90) is an ATP dependent molecular chaperone deeply involved in the complex network of cellular signaling governing some key functions, such as cell proliferation and survival, invasion and angiogenesis. Over the past years the N-terminal protein domain has been fully investigated as attractive strategy against cancer, but despite the many efforts lavished in the field, none of the N-terminal binders (termed "classical inhibitors"), currently in clinical trials, have yet successfully reached the market, because of the detrimental heat shock response (HSR) that showed to induce; thus, recently, the selective inhibition of Hsp90 C-terminal domain has powerfully emerged as a more promising alternative strategy for anti-cancer therapy, not eliciting this cell rescue cascade. However, the structural complexity of the target protein and, mostly, the lack of a co-crystal structure of C-terminal domain-ligand, essential to drive the identification of new hits, represent the largest hurdles in the development of new selective C-terminal inhibitors. Continuing our investigations on the identification of new anticancer drug candidates, by using an orthogonal screening approach, here we describe two new potent C-terminal inhibitors able to induce cancer cell death and a considerable down-regulation of Hsp90 client oncoproteins, without triggering the undesired heat shock response.

The Molecular Chaperone HSP90 Promotes Notch Signaling in the Germline of Caenorhabditis elegans

PubMed Central

Lissemore, James L.; Connors, Elyse; Liu, Ying; Qiao, Li; Yang, Bing; Edgley, Mark L.; Flibotte, Stephane; Taylor, Jon; Au, Vinci; Moerman, Donald G.; Maine, Eleanor M.

2018-01-01

In a genetic screen to identify genes that promote GLP-1/Notch signaling in Caenorhabditis elegans germline stem cells, we found a single mutation, om40, defining a gene called ego-3. ego-3(om40) causes several defects in the soma and the germline, including paralysis during larval development, sterility, delayed proliferation of germline stem cells, and ectopic germline stem cell proliferation. Whole genome sequencing identified om40 as an allele of hsp-90, previously known as daf-21, which encodes the C. elegans ortholog of the cytosolic form of HSP90. This protein is a molecular chaperone with a central position in the protein homeostasis network, which is responsible for proper folding, structural maintenance, and degradation of proteins. In addition to its essential role in cellular function, HSP90 plays an important role in stem cell maintenance and renewal. Complementation analysis using a deletion allele of hsp-90 confirmed that ego-3 is the same gene. hsp-90(om40) is an I→N conservative missense mutation of a highly conserved residue in the middle domain of HSP-90. RNA interference-mediated knockdown of hsp-90 expression partially phenocopied hsp-90(om40), confirming the loss-of-function nature of hsp-90(om40). Furthermore, reduced HSP-90 activity enhanced the effect of reduced function of both the GLP-1 receptor and the downstream LAG-1 transcription factor. Taken together, our results provide the first experimental evidence of an essential role for HSP90 in Notch signaling in development. PMID:29507057

Hsp90 interaction with Cdc2 and Plo1 kinases contributes to actomyosin ring condensation in fission yeast.

PubMed

Santino, Andrea; Tallada, Victor A; Jimenez, Juan; Garzón, Andrés

2012-08-01

In Schizosaccharomyces pombe, cytokinesis occurs by ordered recruitment of actomyosin components at the division site, followed by lateral condensation to produce a ring-like structure early in anaphase, which eventually matures and contracts at the end of mitosis. We found that in temperature-sensitive hsp90-w1 mutant cells, encoding an Hsp90 mutant protein, ring components were recruited to form a cortical network at the division site, but this network failed to condense into a compact ring, suggesting a role for Hsp90 in this particular step. hsp90-w1 mutant shows strong genetic interaction with specific mutant alleles of the fission yeast cdc2, such as cdc2-33. Interestingly, actomyosin ring defects in hsp90-w1 cdc2-33 mutant cells resembled that of hsp90-w1 single mutant at restrictive temperature. Noteworthy, similar genetic interaction was found with a mutant allele of polo-like kinase, plo1-ts4, suggesting that Hsp90 collaborates with Cdc2 and Plo1 cell cycle kinases to condense medial ring components. In vitro analyses suggested that Cdc2 and Plo1 physically interact with Hsp90. Association of Cdc2 to Hsp90 was ATP independent, while Plo1 binds to this chaperone in an ATP-dependent manner, indicating that these two kinases interact with different Hsp90 complexes. Overall, our analyses of hsp90-w1 reveal a possible role for this chaperone in medial ring condensation in association with Cdc2 and Plo1 kinases.

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

The Molecular Chaperone HSP90 Promotes Notch Signaling in the Germline of Caenorhabditis elegans.

PubMed

Lissemore, James L; Connors, Elyse; Liu, Ying; Qiao, Li; Yang, Bing; Edgley, Mark L; Flibotte, Stephane; Taylor, Jon; Au, Vinci; Moerman, Donald G; Maine, Eleanor M

2018-05-04

In a genetic screen to identify genes that promote GLP-1/Notch signaling in Caenorhabditis elegans germline stem cells, we found a single mutation, om40 , defining a gene called ego-3. ego-3(om40) causes several defects in the soma and the germline, including paralysis during larval development, sterility, delayed proliferation of germline stem cells, and ectopic germline stem cell proliferation. Whole genome sequencing identified om40 as an allele of hsp-90 , previously known as daf-21 , which encodes the C. elegans ortholog of the cytosolic form of HSP90. This protein is a molecular chaperone with a central position in the protein homeostasis network, which is responsible for proper folding, structural maintenance, and degradation of proteins. In addition to its essential role in cellular function, HSP90 plays an important role in stem cell maintenance and renewal. Complementation analysis using a deletion allele of hsp-90 confirmed that ego-3 is the same gene. hsp-90(om40) is an I→N conservative missense mutation of a highly conserved residue in the middle domain of HSP-90 RNA interference-mediated knockdown of hsp-90 expression partially phenocopied hsp-90(om40) , confirming the loss-of-function nature of hsp-90(om40) Furthermore, reduced HSP-90 activity enhanced the effect of reduced function of both the GLP-1 receptor and the downstream LAG-1 transcription factor. Taken together, our results provide the first experimental evidence of an essential role for HSP90 in Notch signaling in development. Copyright © 2018 Lissemore et al.

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

2015-12-08

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.

ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1.

PubMed

Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

2010-02-12

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1*

PubMed Central

Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

2010-01-01

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Δhep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer. PMID:20007714

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

PubMed

Chung, Chaeuk; Yoo, Geon; Kim, Tackhoon; Lee, Dahye; Lee, Choong-Sik; Cha, Hye Rim; Park, Yeon Hee; Moon, Jae Young; Jung, Sung Soo; Kim, Ju Ock; Lee, Jae Cheol; Kim, Sun Young; Park, Hee Sun; Park, Myoungrin; Park, Dong Il; Lim, Dae-Sik; Jang, Kang Won; Lee, Jeong Eun

2016-10-14

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein sigma1.

PubMed

Gilmore, R; Coffey, M C; Lee, P W

1998-06-12

The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.

KU675, a Concomitant Heat-Shock Protein Inhibitor of Hsp90 and Hsc70 that Manifests Isoform Selectivity for Hsp90α in Prostate Cancer Cells.

PubMed

Liu, Weiya; Vielhauer, George A; Holzbeierlein, Jeffrey M; Zhao, Huiping; Ghosh, Suman; Brown, Douglas; Lee, Eugene; Blagg, Brian S J

2015-07-01

The 90-kDa heat-shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Inhibiting Hsp90 consequently is an attractive strategy for cancer therapy as the concomitant degradation of multiple oncoproteins may lead to effective antineoplastic agents. Here we report a novel C-terminal Hsp90 inhibitor, designated KU675, that exhibits potent antiproliferative and cytotoxic activity along with client protein degradation without induction of the heat-shock response in both androgen-dependent and -independent prostate cancer cell lines. In addition, KU675 demonstrates direct inhibition of Hsp90 complexes as measured by the inhibition of luciferase refolding in prostate cancer cells. In direct binding studies, the internal fluorescence signal of KU675 was used to determine the binding affinity of KU675 to recombinant Hsp90α, Hsp90β, and Hsc70 proteins. The binding affinity (Kd) for Hsp90α was determined to be 191 μM, whereas the Kd for Hsp90β was 726 μM, demonstrating a preference for Hsp90α. Western blot experiments with four different prostate cancer cell lines treated with KU675 supported this selectivity by inducing the degradation of Hsp90α -: dependent client proteins. KU675 also displayed binding to Hsc70 with a Kd value at 76.3 μM, which was supported in cellular by lower levels of Hsc70-specific client proteins on Western blot analyses. Overall, these findings suggest that KU675 is an Hsp90 C-terminal inhibitor, as well as a dual inhibitor of Hsc70, and may have potential use for the treatment of cancer. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

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.

The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization

PubMed Central

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. PMID:26562659

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

PubMed

Karayazi Atici, Ödül; Urbanska, Anna; Gopinathan, Sesha Gopal; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

2018-02-01

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents. Copyright © 2018 Endocrine Society.

Herpes Simplex Virus 1 Inhibits TANK-Binding Kinase 1 through Formation of the Us11-Hsp90 Complex.

PubMed

Liu, Xing; Main, David; Ma, Yijie; He, Bin

2018-05-09

The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA dependent protein kinase PKR, 2',5'-oligoadenylate synthetase, RIG-I and MDA-5. However, its precise role is incompletely defined. By screening human cDNA library, we show that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes the access of TBK1 to Hsp90 and IFN promoter activation. Consistently, upon HSV infection the Us11 protein suppresses the expression of IFN-β, RANTES, and interferon stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome dependent pathway. Accordingly, Us11 expression facilitates HSV growth. Conversely, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection. IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrate that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions. Copyright © 2018 American Society for Microbiology.

The Prognostic Significance of Hsp70/Hsp90 Expression in Breast Cancer: A Systematic Review and Meta-analysis.

PubMed

Dimas, Dionysios Th; Perlepe, Christina D; Sergentanis, Theodoros N; Misitzis, Ioannis; Kontzoglou, Konstantinos; Patsouris, Efstratios; Kouraklis, Gregory; Psaltopoulou, Theodora; Nonni, Afroditi

2018-03-01

Studies have focused on heat shock protein (Hsp) inhibitors as potential treatment agents in breast cancer, with controversial results. Adopting a pathophysiological perspective, this systematic review aims to synthesize the evidence examining the association between Hsp70/Hsp90 expression and breast cancer prognosis, as well as prognosis-related clinicopathological indices. Secondarily, changes in Hsp70/Hsp90 expression in the continuum of breast neoplasia were assessed. Hsp70/Hsp90 expression was approached globally, quantified by means of immunohistochemistry, western blot or PCR. This study was performed in accordance with the PRISMA guidelines. Relevant studies were sought in PubMed, up to December 31, 2015. A total of 23 eligible studies were identified (7,288 breast cancer cases). High Hsp90 expre s sion was associated with worse overall survival (pooled RR=1.48, 95%CI=1.21-1.82) and marginally with worse disease-free survival. High Hsp70 expression also correlated with worse disease-free survival (pooled RR=1.77, 95%CI=1.71-2.82). Hsp70 intense expression correlated with ER positivity (pooled OR=3.51, 95%CI=1.31-9.40) and PR positivity (pooled OR=2.48, 95%CI=1.39-4.44). No significant associations were noted between Hsp70/Hsp90 expression and clinicopathological variables including histological grade, tumor size, nodal metastasis or patient age at diagnosis. No clear pattern emerged for Hsp70/Hsp90 expression along the breast neoplasia continuum. This systematic review and meta-analysis highlights the prognostic role of Hsp90 and Hsp70 expression in breast cancer. Further high-quality studies, with detailed reporting are needed to provide epidemiological evidence complementing the findings of ongoing clinical trials on Hsp inhibitors. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Molecular cloning and sequence analysis of heat shock proteins 70 (HSP70) and 90 (HSP90) and their expression analysis when exposed to benzo(a)pyrene in the clam Ruditapes philippinarum.

PubMed

Liu, Tong; Pan, Luqing; Cai, Yuefeng; Miao, Jingjing

2015-01-25

HSP70 and HSP90 are the most important heat shock proteins (HSPs), which play the key roles in the cell as molecular chaperones and may involve in metabolic detoxification. The present research has obtained full-length cDNAs of genes HSP70 and HSP90 from the clam Ruditapes philippinarum and studied the transcriptional responses of the two genes when exposed to benzo(a)pyrene (BaP). The full-length RpHSP70 cDNA was 2336bp containing a 5' untranslated region (UTR) of 51bp, a 3' UTR of 335bp and an open reading frame (ORF) of 1950bp encoding 650 amino acid residues. The full-length RpHSP90 cDNA was 2839bp containing a 107-bp 5' UTR, a 554-bp 3' UTR and a 2178-bp ORF encoding 726 amino acid residues. The deduced amino acid sequences of RpHSP70 and RpHSP90 shared the highest identity with the sequences of Paphia undulata, and the phylogenetic trees showed that the evolutions of RpHSP70 and RpHSP90 were almost in accord with the evolution of species. The RpHSP70 and RpHSP90 mRNA expressions were detected in all tested tissues in the adult clams (digestive gland, gill, adductor muscle and mantle) and the highest mRNA expression level was observed in the digestive gland compared to other tissues. Quantitative real-time RT-PCR analysis revealed that mRNA expression levels of the clam RpHSP70, RpHSP90 and other xenobiotic metabolizing enzymes (XMEs) (AhR, DD, GST, GPx) in the digestive gland of R. philippinarum were induced by benzo(a)pyrene (BaP) and the absolute expression levels of these genes showed a temporal and dose-dependent response. The results suggested that RpHSP70 and RpHSP90 were involved in the metabolic detoxification of BaP in the clam R. philippinarum. Copyright © 2014 Elsevier B.V. All rights reserved.

The Hsp90-Sti1 interaction is critical for Leishmania donovani proliferation in both life cycle stages.

PubMed

Hombach, Antje; Ommen, Gabi; Chrobak, Mareike; Clos, Joachim

2013-04-01

The heat shock protein 90 plays a pivotal role in the life cycle control of Leishmania donovani promoting the fast-growing insect stage of this parasite. Equally important for insect stage growth is the co-chaperone Sti1. We show that replacement of Sti1 is only feasible in the presence of additional Sti1 transgenes indicating an essential role. To better understand the impact of Sti1 and its interaction with Hsp90, we performed a mutational analysis of Hsp90. We established that a single amino acid exchange in the Leishmania Hsp90 renders that protein resistant to the inhibitor radicicol (RAD), yet does not interfere with its functionality. Based on this RAD-resistant Hsp90, we established a combined chemical knockout/gene complementation (CKC) approach. We can show that Hsp90 function is required in both insect and mammalian life stages and that the Sti1-binding motif of Hsp90 is crucial for proliferation of insect and mammalian stages of the parasite. The Sti1-binding motif in Leishmania Hsp90 is suboptimal - optimizing the motif increased initial intracellular proliferation underscoring the importance of the Hsp90-Sti1 interaction for this important parasitic protozoan. The CKC strategy we developed will allow the future analysis of more Hsp90 domains and motifs in parasite viability and infectivity. © 2012 Blackwell Publishing Ltd.

TaRAR1 and TaSGT1 associate with TaHsp90 to function in bread wheat (Triticum aestivum L.) seedling growth and stripe rust resistance.

PubMed

Wang, Guan-Feng; Fan, Renchun; Wang, Xianping; Wang, Daowen; Zhang, Xiangqi

2015-04-01

RAR1 and SGT1 are important co-chaperones of Hsp90. We previously showed that TaHsp90.1 is required for wheat seedling growth, and that TaHsp90.2 and TaHsp90.3 are essential for resistance (R) gene mediated resistance to stripe rust fungus. Here, we report the characterization of TaRAR1 and TaSGT1 genes in bread wheat. TaRAR1 and TaSGT1 each had three homoeologs, which were located on wheat groups 2 and 3 chromosomes, respectively. Strong inhibition of seedling growth was observed after silencing TaSGT1 but not TaRAR1. In contrast, decreasing the expression of TaRAR1 or TaSGT1 could all compromise R gene mediated resistance to stripe rust fungus infection. Protein-protein interactions were found among TaRAR1, TaSGT1 and TaHsp90. The N-terminus of TaHsp90, the CHORD-I and CHORD-II domains of TaRAR1 and the CS domain of TaSGT1 may be instrumental for the interactions among the three proteins. Based on this work and our previous study on TaHsp90, we speculate that the TaSGT1-TaHsp90.1 interaction is important for maintaining bread wheat seedling growth. The TaRAR1-TaSGT1-TaHsp90.2 and TaRAR1-TaSGT1-TaHsp90.3 interactions are involved in controlling the resistance to stripe rust disease. The new information obtained here should aid further functional investigations of TaRAR1-TaSGT1-TaHsp90 complexes in regulating bread wheat growth and disease resistance.

E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78

PubMed Central

Ajiro, Masahiko

2015-01-01

ABSTRACT Transcripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy. PMID:25691589

Molecular characterization of three Hsp90 from Pieris and expression patterns in response to cold and thermal stress in summer and winter diapause of Pieris melete.

PubMed

Wu, Yue-Kun; Zou, Chao; Fu, Dao-Meng; Zhang, Wan-Na; Xiao, Hai-Jun

2018-04-01

Heat shock proteins (Hsps) have been linked to stresses and winter diapause in insects, but whether they are components of summer diapause is still unknown. In this study, complementary DNAs of Hsp90 from Pieris melete, Pieris rapae and Pieris canidia named PmHsp90, PrHsp90 and PcHsp90, respectively, were cloned and sequenced. The deduced amino acid sequence consisted of 718 amino acid residues with a putative molecular mass of 82.6, 82.6 and 82.7 kDa, respectively. The amino acid sequences contained all of the five conserved signature motifs in the Hsp90 family and a bHLH protein folding activity region. The differential expression pattern of PmHsp90 in response to summer diapause and winter diapause, which are related to heat/cold stress, was investigated. Cold stress induced Hsp90 up-regulation in summer and winter diapause pupae, but not in non-diapause individuals. Heat shock up-regulated PmHsp90 gradually with an increase in temperature in summer diapause, and PmHsp90 was rapidly up-regulated in winter diapause. After 30 min heat shock at 39°C, substantial up-regulation of PmHsp90 transcript levels were observed both in summer and winter diapause. However, in non-diapause a relatively stable expression was found under different durations of 39°C heat shock. Compared to the optimal treatment of 18°C for diapause development, a high temperature acclimation of 31°C induced PmHsp90 up-regulation in summer diapause, whereas a low temperature acclimation of 4°C induced up-regulation in winter diapause. The current results indicate that Hsp90 may play an important role in response to heat/cold stress both in summer and winter diapause. © 2016 Institute of Zoology, Chinese Academy of Sciences.

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.

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

High-Throughput Gene Expression Profiles to Define Drug Similarity and Predict Compound Activity.

PubMed

De Wolf, Hans; Cougnaud, Laure; Van Hoorde, Kirsten; De Bondt, An; Wegner, Joerg K; Ceulemans, Hugo; Göhlmann, Hinrich

2018-04-01

By adding biological information, beyond the chemical properties and desired effect of a compound, uncharted compound areas and connections can be explored. In this study, we add transcriptional information for 31K compounds of Janssen's primary screening deck, using the HT L1000 platform and assess (a) the transcriptional connection score for generating compound similarities, (b) machine learning algorithms for generating target activity predictions, and (c) the scaffold hopping potential of the resulting hits. We demonstrate that the transcriptional connection score is best computed from the significant genes only and should be interpreted within its confidence interval for which we provide the stats. These guidelines help to reduce noise, increase reproducibility, and enable the separation of specific and promiscuous compounds. The added value of machine learning is demonstrated for the NR3C1 and HSP90 targets. Support Vector Machine models yielded balanced accuracy values ≥80% when the expression values from DDIT4 & SERPINE1 and TMEM97 & SPR were used to predict the NR3C1 and HSP90 activity, respectively. Combining both models resulted in 22 new and confirmed HSP90-independent NR3C1 inhibitors, providing two scaffolds (i.e., pyrimidine and pyrazolo-pyrimidine), which could potentially be of interest in the treatment of depression (i.e., inhibiting the glucocorticoid receptor (i.e., NR3C1), while leaving its chaperone, HSP90, unaffected). As such, the initial hit rate increased by a factor 300, as less, but more specific chemistry could be screened, based on the upfront computed activity predictions.

Heat Treatment Improves Glucose Tolerance and Prevents Skeletal Muscle Insulin Resistance in Rats Fed a High-Fat Diet

PubMed Central

Gupte, Anisha A.; Bomhoff, Gregory L.; Swerdlow, Russell H.; Geiger, Paige C.

2009-01-01

OBJECTIVE—Heat treatment and overexpression of heat shock protein 72 (HSP72) have been shown to protect against high-fat diet–induced insulin resistance, but little is known about the underlying mechanism or the target tissue of HSP action. The purpose of this study is to determine whether in vivo heat treatment can prevent skeletal muscle insulin resistance. RESEARCH DESIGN AND METHODS—Male Wistar rats were fed a high-fat diet (60% calories from fat) for 12 weeks and received a lower-body heat treatment (41°C for 20 min) once per week. RESULTS—Our results show that heat treatment shifts the metabolic characteristics of rats on a high-fat diet toward those on a standard diet. Heat treatment improved glucose tolerance, restored insulin-stimulated glucose transport, and increased insulin signaling in soleus and extensor digitorum longus (EDL) muscles from rats fed a high-fat diet. Heat treatment resulted in decreased activation of Jun NH2-terminal kinase (JNK) and inhibitor of κB kinase (IKK-β), stress kinases implicated in insulin resistance, and upregulation of HSP72 and HSP25, proteins previously shown to inhibit JNK and IKK-β activation, respectively. Mitochondrial citrate synthase and cytochrome oxidase activity decreased slightly with the high-fat diet, but heat treatment restored these activities. Data from L6 cells suggest that one bout of heat treatment increases mitochondrial oxygen consumption and fatty acid oxidation. CONCLUSIONS—Our results indicate that heat treatment protects skeletal muscle from high-fat diet–induced insulin resistance and provide strong evidence that HSP induction in skeletal muscle could be a potential therapeutic treatment for obesity-induced insulin resistance. PMID:19073766

Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Yoon Sik, E-mail: yumshak@naver.com; Seo, Hyun Wook, E-mail: suruk@naver.com; Jung, Guhung, E-mail: drjung@snu.ac.kr

2015-02-13

Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study thatmore » Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.« less

Molecular characterization and expression analysis of a heat shock protein 90 gene from disk abalone (Haliotis discus).

PubMed

Wang, Ning; Whang, Ilson; Lee, Jae-Seong; Lee, Jehee

2011-06-01

Heat shock protein 90s (hsp90s) are chaperones that contribute to the proper folding of cellular proteins and help animals cope with the cellular protein damages in stress conditions. In this study, an hsp90 gene was isolated from disc abalone (Haliotis discus). The complete nucleotide sequence of the hsp90 gene contains an open reading frame of 2,184 base pairs, encoding an 84 kDa protein. Disk abalone hsp90 shares high sequence similarity with other hsp90 family proteins. Although the phylogenetic analysis did not classify it into the hsp90α group, the inductivity of this gene was confirmed by heat shock and lipopolysaccharide (LPS) challenge test. Disk abalone hsp90 gene displayed a rapid and reversible induction response to both an exposure of typical heat shock and the LPS challenge. Once given the sublethal heat shock treatment, the transcription of disk abalone hsp90 gene was significantly up-regulated. With a recovery of 12 h, the transcription of disk abalone hsp90 gene gradually attenuated to the control level. These observations reflected the feedback regulation of abalone heat shock responses faithfully. In response to LPS challenge, the transcription of disk abalone hsp90 gene was significantly increased within 2 h and it approached maximum induction at 4 h later and recovered finally the reference level in 24 h. Take all together, the cloning and expression analysis of disk abalone hsp90 gene provided useful molecular information of abalone responses in stress conditions and potential ways to monitor the chronic stressors in abalone culture environments and diagnose the animal health status.

HSP27 Inhibits Homocysteine-Induced Endothelial Apoptosis by Modulation of ROS Production and Mitochondrial Caspase-Dependent Apoptotic Pathway.

PubMed

Tian, Xin; Zhao, Lei; Song, Xianjing; Yan, Youyou; Liu, Ning; Li, Tianyi; Yan, Bingdi; Liu, Bin

2016-01-01

Objectives. Elevated plasma homocysteine (Hcy) could lead to endothelial dysfunction and is viewed as an independent risk factor for atherosclerosis. Heat shock protein 27 (HSP27), a small heat shock protein, is reported to exert protective effect against atherosclerosis. This study aims to investigate the protective effect of HSP27 against Hcy-induced endothelial cell apoptosis in human umbilical vein endothelial cells (HUVECs) and to determine the underlying mechanisms. Methods. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) of normal or HSP27-overexpressing HUVECs in the presence of Hcy were analyzed by flow cytometry. The mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Results. We found that Hcy could induce cell apoptosis with corresponding decrease of nitric oxide (NO) level, increase of endothelin-1 (ET-1), intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) levels, elevation of ROS, and dissipation of MMP. In addition, HSP27 could protect the cell against Hcy-induced apoptosis and inhibit the effect of Hcy on HUVECs. Furthermore, HSP27 could increase the ratio of Bcl-2/Bax and inhibit caspase-3 activity. Conclusions. Therefore, we concluded that HSP27 played a protective role against Hcy-induced endothelial apoptosis through modulation of ROS production and the mitochondrial caspase-dependent apoptotic pathway.

A Novel Hsp90 Inhibitor Activates Compensatory Heat Shock Protein Responses and Autophagy and Alleviates Mutant A53T α-Synuclein Toxicity

PubMed Central

Xiong, Rui; Zhou, Wenbo; Siegel, David; Kitson, Russell R. A.; Freed, Curt R.; Moody, Christopher J.

2015-01-01

A potential cause of neurodegenerative diseases, including Parkinson’s disease (PD), is protein misfolding and aggregation that in turn leads to neurotoxicity. Targeting Hsp90 is an attractive strategy to halt neurodegenerative diseases, and benzoquinone ansamycin (BQA) Hsp90 inhibitors such as geldanamycin (GA) and 17-(allylamino)-17-demethoxygeldanamycin have been shown to be beneficial in mutant A53T α-synuclein PD models. However, current BQA inhibitors result in off-target toxicities via redox cycling and/or arylation of nucleophiles at the C19 position. We developed novel 19-substituted BQA (19BQA) as a means to prevent arylation. In this study, our data demonstrated that 19-phenyl-GA, a lead 19BQA in the GA series, was redox stable and exhibited little toxicity relative to its parent quinone GA in human dopaminergic SH-SY5Y cells as examined by oxygen consumption, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and apoptosis assays. Meanwhile, 19-phenyl-GA retained the ability to induce autophagy and potentially protective heat shock proteins (HSPs) such as Hsp70 and Hsp27. We found that transduction of A53T, but not wild type (WT) α-synuclein, induced toxicity in SH-SY5Y cells. 19-Phenyl-GA decreased oligomer formation and toxicity of A53T α-synuclein in transduced cells. Mechanistic studies indicated that mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase signaling was activated by A53T but not WT α-synuclein, and 19-phenyl-GA decreased mTOR activation that may be associated with A53T α-synuclein toxicity. In summary, our results indicate that 19BQAs such as 19-phenyl-GA may provide a means to modulate protein-handling systems including HSPs and autophagy, thereby reducing the aggregation and toxicity of proteins such as mutant A53T α-synuclein. PMID:26405178

Heat shock protein 10 (Hsp10) in immune-related diseases: one coin, two sides

PubMed Central

Jia, Haibo; Halilou, Amadou I.; Hu, Liang; Cai, Wenqian; Liu, Jing; Huang, Bo

2011-01-01

Heat shock protein 10 (Hsp10) in eukaryotes, originally identified as a mitochondrial chaperone, now is also known to be present in cytosol, cell surface, extracellular space and peripheral blood. Functionally besides participating in mitochondrial protein folding in association with Hsp60, Hsp10 appears to be related to pregnancy, cancer and autoimmune inhibition. Hsp10 can be released to peripheral blood at very early time point of pregnancy and given another name called early pregnancy factor (EPF), which seems to play a critical role in developing a pregnant niche. In malignant disorders, Hsp10 is usually abnormally expressed in the cytosol of malignant cells and further released to extracellular space, resulting in tumor-promoting effect from various aspects. Furthermore, distinct from other heat shock protein members, whose soluble form is recognized as danger signal by immune cells and triggers immune responses, Hsp10 after release, however, is designed to be an inhibitory signal by limiting immune response. This review discusses how Hsp10 participates in various physiological and pathological processes from basic protein molecule folding to pregnancy, cancer and autoimmune diseases, and emphasizes how important the location is for the function exertion of a molecule. PMID:21969171

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

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.

Evidence of a Cell Surface Role for Hsp90 Complex Proteins Mediating Neuroblast Migration in the Subventricular Zone.

PubMed

Miyakoshi, Leo M; Marques-Coelho, Diego; De Souza, Luiz E R; Lima, Flavia R S; Martins, Vilma R; Zanata, Silvio M; Hedin-Pereira, Cecilia

2017-01-01

In most mammalian brains, the subventricular zone (SVZ) is a germinative layer that maintains neurogenic activity throughout adulthood. Neuronal precursors arising from this region migrate through the rostral migratory stream (RMS) and reach the olfactory bulbs where they differentiate and integrate into the local circuitry. Recently, studies have shown that heat shock proteins have an important role in cancer cell migration and blocking Hsp90 function was shown to hinder cell migration in the developing cerebellum. In this work, we hypothesize that chaperone complexes may have an important function regulating migration of neuronal precursors from the subventricular zone. Proteins from the Hsp90 complex are present in the postnatal SVZ as well as in the RMS. Using an in vitro SVZ explant model, we have demonstrated the expression of Hsp90 and Hop/STI1 by migrating neuroblasts. Treatment with antibodies against Hsp90 and co-chaperone Hop/STI1, as well as Hsp90 and Hsp70 inhibitors hinder neuroblast chain migration. Time-lapse videomicroscopy analysis revealed that cell motility and average migratory speed was decreased after exposure to both antibodies and inhibitors. Antibodies recognizing Hsp90, Hsp70, and Hop/STI1 were found bound to the membranes of cells from primary SVZ cultures and biotinylation assays demonstrated that Hsp70 and Hop/STI1 could be found on the external leaflet of neuroblast membranes. The latter could also be detected in conditioned medium samples obtained from cultivated SVZ cells. Our results suggest that chaperones Hsp90, Hsp70, and co-chaperone Hop/STI1, components of the Hsp90 complex, regulate SVZ neuroblast migration in a concerted manner through an extracellular mechanism.

Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma.

PubMed

Calero, R; Morchon, E; Martinez-Argudo, I; Serrano, R

2017-10-10

Drug resistance by MAPK signaling recovery or activation of alternative signaling pathways, such as PI3K/AKT/mTOR, is an important factor that limits the long-term efficacy of targeted therapies in melanoma patients. In the present study, we investigated the phospho-proteomic profile of RTKs and its correlation with downstream signaling pathways in human melanoma. We found that tyrosine kinase receptors expression correlated with the expression of pivotal downstream components of the RAS/RAF/MAPK and PI3K/AKT/mTOR pathways in melanoma cell lines and tumors. We also found high expression of HSP90 and the PI3K/AKT/mTOR pathway proteins, 4EBP1 and AKT compared with healthy tissue and this correlated with poor overall survival of melanoma patients. The combination of the HSP90 inhibitor 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 showed a synergistic activity decreasing melanoma cell growth, inducing apoptosis and targeting simultaneously the MAPK and PI3K/AKT/mTOR pathways. These results demonstrate that the combination of HSP90 and PI3K/mTOR inhibitors could be an effective therapeutic strategy that target the main survival pathways in melanoma and must be considered to overcome resistance to BRAF inhibitors in melanoma patients. Copyright © 2017 Elsevier B.V. All rights reserved.

HSP90 empowers evolution of resistance to hormonal therapy in human breast cancer models.

PubMed

Whitesell, Luke; Santagata, Sandro; Mendillo, Marc L; Lin, Nancy U; Proia, David A; Lindquist, Susan

2014-12-23

The efficacy of hormonal therapies for advanced estrogen receptor-positive breast cancers is limited by the nearly inevitable development of acquired resistance. Efforts to block the emergence of resistance have met with limited success, largely because the mechanisms underlying it are so varied and complex. Here, we investigate a new strategy aimed at the very processes by which cancers evolve resistance. From yeast to vertebrates, heat shock protein 90 (HSP90) plays a unique role among molecular chaperones by promoting the evolution of heritable new traits. It does so by regulating the folding of a diverse portfolio of metastable client proteins, many of which mediate adaptive responses that allow organisms to adapt and thrive in the face of diverse challenges, including those posed by drugs. Guided by our previous work in pathogenic fungi, in which very modest HSP90 inhibition impairs resistance to mechanistically diverse antifungals, we examined the effect of similarly modest HSP90 inhibition on the emergence of resistance to antiestrogens in breast cancer models. Even though this degree of inhibition fell below the threshold for proteotoxic activation of the heat-shock response and had no overt anticancer activity on its own, it dramatically impaired the emergence of resistance to hormone antagonists both in cell culture and in mice. Our findings strongly support the clinical testing of combined hormone antagonist-low-level HSP90 inhibitor regimens in the treatment of metastatic estrogen receptor-positive breast cancer. At a broader level, they also provide promising proof of principle for a generalizable strategy to combat the pervasive problem of rapidly emerging resistance to molecularly targeted therapeutics.

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia-lymphoma cells.

PubMed

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-12-01

Adult T-cell leukemia-lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia–lymphoma cells

PubMed Central

Taniguchi, Hiroaki; Hasegawa, Hiroo; Sasaki, Daisuke; Ando, Koji; Sawayama, Yasushi; Imanishi, Daisuke; Taguchi, Jun; Imaizumi, Yoshitaka; Hata, Tomoko; Tsukasaki, Kunihiro; Uno, Naoki; Morinaga, Yoshitomo; Yanagihara, Katsunori; Miyazaki, Yasushi

2014-01-01

Adult T-cell leukemia–lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. PMID:25263741

Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.

PubMed

Montgomery, Ellyn R; Temple, Brenda R S; Peters, Kimberly A; Tolbert, Caitlin E; Booker, Brandon K; Martin, Joseph W; Hamilton, Tyler P; Tagliatela, Alicia C; Smolski, William C; Rogers, Stephen L; Jones, Alan M; Meigs, Thomas E

2014-04-01

The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12 and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Gα12 determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in Gα12 and Gα13. Mutation of these residues in Gα12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13 exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12 than Gα13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12 determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12 and Gα13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

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

PubMed Central

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

2015-01-01

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

Hsp90 binds microtubules and is involved in the reorganization of the microtubular network in angiosperms.

PubMed

Krtková, Jana; Zimmermann, Aleksandra; Schwarzerová, Kateřina; Nick, Peter

2012-09-15

Microtubules (MTs) are essential for many processes in plant cells. MT-associated proteins (MAPs) influence MT polymerization dynamics and enable them to perform their functions. The molecular chaperone Hsp90 has been shown to associate with MTs in animal and plant cells. However, the role of Hsp90-MT binding in plants has not yet been investigated. Here, we show that Hsp90 associates with cortical MTs in tobacco cells and decorates MTs in the phragmoplast. Further, we show that tobacco Hsp90_MT binds directly to polymerized MTs in vitro. The inhibition of Hsp90 by geldanamycin (GDA) severely impairs MT re-assembly after cold-induced de-polymerization. Our results indicate that the plant Hsp90 interaction with MTs plays a key role in cellular events, where MT re-organization is needed. Copyright © 2012 Elsevier GmbH. All rights reserved.

The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone.

PubMed

Marcu, M G; Chadli, A; Bouhouche, I; Catelli, M; Neckers, L M

2000-11-24

Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185(ErbB2). In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the co-chaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.

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.

DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging.

PubMed

Park, Sung-Jun; Gavrilova, Oksana; Brown, Alexandra L; Soto, Jamie E; Bremner, Shannon; Kim, Jeonghan; Xu, Xihui; Yang, Shutong; Um, Jee-Hyun; Koch, Lauren G; Britton, Steven L; Lieber, Richard L; Philp, Andrew; Baar, Keith; Kohama, Steven G; Abel, E Dale; Kim, Myung K; Chung, Jay H

2017-05-02

Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases, including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function, and decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity. Published by Elsevier Inc.

Novel Entropically Driven Conformation-specific Interactions with Tomm34 Protein Modulate Hsp70 Protein Folding and ATPase Activities*

PubMed Central

Durech, Michal; Trcka, Filip; Man, Petr; Blackburn, Elizabeth A.; Hernychova, Lenka; Dvorakova, Petra; Coufalova, Dominika; Kavan, Daniel; Vojtesek, Borivoj; Muller, Petr

2016-01-01

Co-chaperones containing tetratricopeptide repeat (TPR) domains enable cooperation between Hsp70 and Hsp90 to maintain cellular proteostasis. Although the details of the molecular interactions between some TPR domains and heat shock proteins are known, we describe a novel mechanism by which Tomm34 interacts with and coordinates Hsp70 activities. In contrast to the previously defined Hsp70/Hsp90-organizing protein (Hop), Tomm34 interaction is dependent on the Hsp70 chaperone cycle. Tomm34 binds Hsp70 in a complex process; anchorage of the Hsp70 C terminus by the TPR1 domain is accompanied by additional contacts formed exclusively in the ATP-bound state of Hsp70 resulting in a high affinity entropically driven interaction. Tomm34 induces structural changes in determinants within the Hsp70-lid subdomain and modulates Hsp70/Hsp40-mediated refolding and Hsp40-stimulated Hsp70 ATPase activity. Because Tomm34 recruits Hsp90 through its TPR2 domain, we propose a model in which Tomm34 enables Hsp70/Hsp90 scaffolding and influences the Hsp70 chaperone cycle, providing an additional role for co-chaperones that contain multiple TPR domains in regulating protein homeostasis. PMID:26944342

The regulation mechanisms of AhR by molecular chaperone complex.

PubMed

Kudo, Ikuru; Hosaka, Miki; Haga, Asami; Tsuji, Noriko; Nagata, Yuhtaroh; Okada, Hirotaka; Fukuda, Kana; Kakizaki, Yuka; Okamoto, Tomoya; Grave, Ewa; Itoh, Hideaki

2018-03-01

The AhR, so called the dioxin receptor, is a member of the nuclear receptor superfamily. The ligand-free AhR forms a cytosolic protein complex with the molecular chaperone HSP90, co-chaperone p23, and XAP2 in the cytoplasm. Following ligand binding like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR translocates into the nucleus. Although it has been reported that HSP90 regulates the translocation of the AhR to the nucleus, the precise activation mechanisms of the AhR have not yet been fully understood. AhR consists of the N-terminal bHLH domain containing NLS and NES, the middle PAS domain and the C-terminal transactivation domain. The PAS domain is familiar as a ligand and HSP90 binding domain. In this study, we focused on the bHLH domain that was thought to be a HSP90 binding domain. We investigated the binding properties of bHLH to HSP90. We analyzed the direct interaction of bHLH with HSP90, p23 and XAP2 using purified proteins. We found that not only the PAS domain but also the bHLH domain bound to HSP90. The bHLH domain forms complex with HSP90, p23 and XAP2. We also determined the bHLH binding domain was HSP90 N-domain. The bHLH domain makes a complex with HSP90, p23 and XAP2 via the HSP90 N-domain. Although the NLS is closed in the absence of a ligand, the structure of AhR will be changed in the presence of a ligand, which leads to NLS open, result in the nuclear translocation of AhR.

Plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity.

PubMed

Joseph, Anna-Maria; Hood, David A

2012-03-01

We investigated the assembly of the TOM complex within skeletal muscle under conditions of chronic contractile activity-induced mitochondrial biogenesis. Tom40 import into mitochondria was increased by chronic contractile activity, as was its time-dependent assembly into the TOM complex. These changes coincided with contractile activity-induced augmentations in the expression of key protein import machinery components Tim17, Tim23, and Tom22, as well as the cytosolic chaperone Hsp90. These data indicate the adaptability of the TOM protein import complex and suggest a regulatory role for the assembly of this complex in exercise-induced mitochondrial biogenesis. Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.

Gene expression of Hsp70, Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis.

PubMed

Zhu, Yongfei; Ren, Chuanlu; Wan, Xuying; Zhu, Yuping; Zhu, Jiangbo; Zhou, Hongyuan; Zhang, Tianbao

2013-11-01

Most previous studies focused on a small number of heat shock proteins (Hsps) and their relationships with embryogenesis, and the actual roles of these Hsps in normal and abnormal embryonic development remain unclear. It was found in the present systemic study that except for Grp170, whose expression was not detectable at GD18, all 19 Hsps of Hsp70, Hsp90 and Hsp110 families were expressed in the normal development of embryonic palate tissue in mice, but their expression patterns varied with different Hsps, presenting as a correlation with the developmental phases. In the treatment group by all-trans retinoic acid (atRA), the messenger RNA (mRNA) abundance of HspA1A, HspA1L, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14, Hsp90AA1, Hsp90AB1, Grp94, Trap1, Hsp105, Hsp110 and Grp170 was higher in the palates at GD11 (the beginning of palate development), the mRNA abundance of HspA1A, HspA12A and HspA12B was higher at GD18 (before birth) and an mRNA expression peak of HspA1L, HspA8, HspA9, Hsp90AA1, Grp94, Hsp110 and Grp170 was observed at GD17. The mRNA abundance of most genes in atRA-induced cleft palates of the treatment group was different from that of the control group. Grp78, HspA14 and Hsp105 were closely associated with the normal palate development and cleft palate in mouse embryo, possibly as palate development-related genes. Except Grp170, the other genes may be closely associated with the development of mouse palates through participating in the stress response process and/or the antiapoptosis process.

Uncaria rhynchophylla Ameliorates Parkinson's Disease by Inhibiting HSP90 Expression: Insights from Quantitative Proteomics.

PubMed

Lan, Yu-Long; Zhou, Jun-Jun; Liu, Jing; Huo, Xiao-Kui; Wang, Ya-Li; Liang, Jia-Hao; Zhao, Jian-Chao; Sun, Cheng-Peng; Yu, Zhen-Long; Fang, Lin-Lin; Tian, Xiang-Ge; Feng, Lei; Ning, Jing; Zhang, Bao-Jing; Wang, Chao; Zhao, Xin-Yu; Ma, Xiao-Chi

2018-06-21

Uncaria rhynchophylla, known as "Gou-teng", is a traditional Chinese medicine (TCM) used to extinguish wind, clear heat, arrest convulsions, and pacify the liver. Although U. rhynchophylla has a long history of being often used to treat central nervous system (CNS) diseases, its efficacy and potential mechanism are still uncertain. This study investigated neuroprotective effect and the underlying mechanism of U. rhynchophylla extract (URE) in MPP+-induced SH-SY5Y cells and MPTP-induced mice. MPP+-induced SH-SY5Y cells and MPTP-induced mice were used to established Parkinson's disease (PD) models. Quantitative proteomics and bioinformatics were used to uncover proteomics changes of URE. Western blotting was used to validate main differentially expressed proteins and test HSP90 client proteins (apoptosis-related, autophagy-related, MAPKs, PI3K, and AKT proteins). Flow cytometry and JC-1 staining assay were further used to confirm the effect of URE on MPP+-induced apoptosis in SH-SY5Y cells. Gait analysis was used to detect the behavioral changes in MPTP-induced mice. The levels of dopamine (DA) and their metabolites were examined in striatum (STR) by HPLC-EC. The positive expression of tyrosine hydroxylase (TH) was detected by immunohischemical staining and Western blotting. URE dose-dependently increased the cell viability in MPP+-induced SH-SY5Y cells. Quantitative proteomics and bioinformatics results confirmed that HSP90 was an important differentially expressed protein of URE. URE inhibited the expression of HSP90, which further reversed MPP+-induced cell apoptosis and autophagy by increasing the expressions of Bcl-2, Cyclin D1, p-ERK, p-PI3K p85, PI3K p110α, p-AKT, and LC3-I and decreasing cleaved caspase 3, Bax, p-JNK, p-p38, and LC3-II. URE also markedly decreased the apoptotic ratio and elevated mitochondrial transmembrane potential (DΨm). Furthermore, URE treatment ameliorated behavioral impairments, increased the contents of DA and its metabolites and elevated the positive expressions of TH in SN and STR as well as the TH protein. URE possessed the neuroprotective effect in vivo and in vitro, regulated MAPK and PI3K-AKT signal pathways, and inhibited the expression of HSP90. U. rhynchophylla has potentials as therapeutic agent in PD treatment. © 2018 The Author(s). Published by S. Karger AG, Basel.

Natural Product Inspired Hsp90 N-Terminal Inhibitors for the Treatment of Cancer: From Bench to Bedside

PubMed Central

Blagg, Brian S. J.

2015-01-01

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. PMID:26010985

The evolutionary capacitor HSP90 buffers the regulatory effects of mammalian endogenous retroviruses.

PubMed

Hummel, Barbara; Hansen, Erik C; Yoveva, Aneliya; Aprile-Garcia, Fernando; Hussong, Rebecca; Sawarkar, Ritwick

2017-03-01

Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.

Induction of hsp70, hsp90, and catalase activity in planarian Dugesia japonica exposed to cadmium.

PubMed

Zhang, Xiufang; Mo, Yehua; Zhou, Luming; Wang, Yinan; Wang, Zhongchen; Zhao, Bosheng

2016-08-01

The hsp70 and hsp90 expression patterns and catalase (CAT) activity in the freshwater planaria Dugesia japonica exposed to cadmium (Cd) under laboratory conditions were investigated. Planaria were exposed to a range of Cd concentrations (0-150 μg Cd/L) for 24 h. The expression levels of hsp70 and hsp90 were determined by relative quantitative real-time polymerase chain reaction. Within the overall dose range in the experiment, the expression level of hsp70 and the activity of CAT in D. japonica were altered significantly. Hsp70 was induced in D. japonica upon Cd exposure concentrations as low as 9.375 μg Cd/L. No significant effect on the expression level of hsp90 was observed. Our findings demonstrated that stress gene hsp70, but not hsp90, was responsive to Cd contamination in D. japonica CAT activity was significantly induced at concentrations of 18.75, 37.5, and 75 μg Cd/L after 24-h exposure. We recommend that the use of hsp70 as a biomarker should be complemented by evidence of changes in other parameters, such as CAT activity, in D. japonica. © The Author(s) 2014.

17-ABAG, a novel geldanamycin derivative, inhibits LNCaP-cell proliferation through heat shock protein 90 inhibition

PubMed Central

LIN, ZHIYUAN; PENG, RUIXIAN; LI, ZHENYU; WANG, YANG; LU, CHUNHUA; SHEN, YUEMAO; WANG, JIFENG; SHI, GUOWEI

2015-01-01

Prostate cancer is one of the most common cancer types worldwide. In 2014, there were an estimated 233,000 new cases and 29,480 mortalities in the United States. Androgen deprivation therapy, also called androgen suppression therapy, targets androgen signaling and remains the standard treatment for patients with advanced prostate cancer; however, responses to treatment are not durable and most patients advance to castrate-resistant prostate cancer. Therefore, novel therapeutic strategies to treat prostate cancer are urgently required. Heat shock protein 90 (Hsp90) is a chaperone protein that has been shown to regulate the progression of tumor cells. Numerous Hsp90 inhibitors show anti-tumor activity and several of them have entered clinical trials. Geldanamycin (GA) was identified as the first Hsp90 inhibitor, but shows hepatotoxicity at its effective concentrations, limiting its clinical use. In previous studies by our group, the GA derivative 17-ABAG was designed and synthesized. The present study showed that 17-ABAG inhibits the proliferation and induces apoptosis of LNCaP, an androgen-dependent prostate cancer cell line, in vitro through a classic apoptotic pathway. 17-ABAG also downregulated the Hsp90 client protein and inhibited androgen receptor nuclear localization in LNCaP cells. In addition, 17-ABAG suppressed the growth of LNCaP xenograft tumors without any obvious side-effects. The present study demonstrated that 17-ABAG is a promising anti-tumor agent and warrants further validation in prospective studies. PMID:26059743

Acclimation of killifish to thermal extremes of hot spring: Transcription of gonadal and liver heat shock genes.

PubMed

Akbarzadeh, Arash; Leder, Erica H

2016-01-01

In this study, we explored the hypothesis that killifish acclimate to thermal extremes through regulation of genes involved in stress and metabolism. We examined the liver and gonadal transcription of heat shock proteins (hsp70, hsp90a, hsp90b), glucokinase (gck), and high mobility group b1 (hmgb1) protein in wild killifish species from hot springs and rivers using quantitative real-time PCR. Moreover, we exposed a river killifish species to a long-term thermal regime of hot spring (37-40°C) and examined the liver transcription of the heat shock genes. Our results showed that hot spring killifish showed a significant, strong upregulation of liver hsp90a. Moreover, the testicular transcript levels of hsp90a, hsp90b, and hsp70 were higher in hot spring killifish than the river ones. The results of the common garden experiments showed that the transcripts of hsp70, hsp90b, and hmgb1 were mildly induced (> twofold) at the time when temperature reached to 37-40°C, while the transcripts of hsp90a were strongly induced (17-fold increase). The level of hsp90a was dramatically more upregulated when fish were maintained in thermal extreme (42-fold change higher than in ambient temperature). Moreover, a significant downregulation of gck transcripts was observed at the time when temperature was raised to 37-40°C (80-fold decrease) and during exposure to long-term thermal extreme (56-fold decrease). It can be concluded that the regulation of heat shock genes particularly hsp90a might be a key factor of the acclimation of fish to high temperature environments like hot springs. Copyright © 2015 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bi, Rui; Bao, Chunrong; Jiang, Lianyong

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 PAHmore » 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.« less

Protective Role of Hsp27 Protein Against Gamma Radiation-Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aloy, Marie-Therese; Hospices Civils de Lyon, Service de Radiotherapie, Centre Hospitalier Lyon-Sud, Pierre-Benite; Hadchity, Elie

Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively ormore » in response to {gamma}-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.« less

Applying chaperones to protein-misfolding disorders: molecular chaperones against α-synuclein in Parkinson's disease.

PubMed

Chaari, Ali; Hoarau-Véchot, Jessica; Ladjimi, Moncef

2013-09-01

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of a protein called α-synuclein (α-syn) into inclusions known as lewy bodies (LB) within neurons. This accumulation is also due to insufficient formation and activity of dopamine produced in certain neurons within the substantia nigra. Lewy bodies are the pathological hallmark of the idiopathic disorder and the cascade that allows α-synuclein to misfold, aggregate and form these inclusions has been the subject of intensive research. Targeting these early steps of oligomerization is one of the main therapeutic approaches in order to develop neurodegenerative-modifying agents. Because the folding and refolding of alpha synuclein is the key point of this cascade, we are interested in this review to summarize the role of some molecular chaperones proteins such as Hsp70, Hsp90 and small heat shock proteins (sHsp) and Hsp 104. Hsp70 and its co-chaperone, Hsp70 and small heat shock proteins can prevent neurodegeneration by preventing α-syn misfolding, oligomerization and aggregation in vitro and in Parkinson disease animal models. Hsp104 is able to resolve disordered protein aggregates and cross beta amyloid conformers. Together, these chaperones have a complementary effect and can be a target for therapeutic intervention in PD. Published by Elsevier B.V.

Pertuzumab Increases 17-AAG-Induced Degradation of ErbB2, and This Effect Is Further Increased by Combining Pertuzumab with Trastuzumab

PubMed Central

Hughes, Juliana Bentes; Rødland, Marianne Skeie; Hasmann, Max; Madshus, Inger Helene; Stang, Espen

2012-01-01

ErbB2 is an important oncogenic protein involved in carcinogenesis of, among others, breast, gastric, and ovarian carcinoma. Over-expression of ErbB2 is found in almost 20% of breast cancers, and this results in proliferative and anti-apoptotic signalling. ErbB2 is therefore an important treatment target. Antibodies recognizing full-length ErbB2 are clinically established, and drugs targeting the ErbB2 stabilizing heat shock protein 90 (Hsp90) are under clinical evaluation. We have investigated effects of the ErbB2-binding antibodies trastuzumab and pertuzumab alone and in combination, as well as the effect of the antibodies in combination with the Hsp90 inhibitor 17-AAG. Our results confirm the notion that combination of different ErbB2-binding antibodies more efficiently down-regulates ErbB2 than does one antibody in isolation. Additionally, our data demonstrate that ErbB2 is most efficiently down-regulated upon incubation with anti-ErbB2 antibodies in combination with Hsp90 inhibitors. The combination of anti-ErbB2 antibodies, and especially the combination of antibodies with 17-AAG, did also increase the inhibition of Akt activation of either agent, which could suggest an anti-proliferative effect. In such case, combining these agents could be beneficial in treatment of tumors not responding to trastuzumab only. PMID:24281706

Characterize and Gene Expression of Heat Shock Protein 90 in Marine Crab Charybdis japonica following Bisphenol A and 4-Nonylphenol Exposures

PubMed Central

Park, Kiyun

2014-01-01

Objectives Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of protein. In this study, an HSP90 gene was isolated from Asian paddle crab, Charybdis japonica, as a bio-indicator to monitor the marine ecosystem. Methods This work reports the responses of C. japonica HSP90 mRNA expression to cellular stress by endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 4-nonylphenol (NP) using real-time. reverse transcription polymerase chain reaction. Results The deduced amino acid sequence of HSP90 from C. japonica shared a high degree of homology with their homologues in other species. In a phylogenetic analysis, C. japonica HSP90 is evolutionally related with an ortholog of the other crustacean species. The expression of HSP90 gene was almost distributed in all the examined tissues of the C. japonica crab but expression levels varied among the different body parts of the crabs. We examined HSP90 mRNA expression pattern in C. japonica crabs exposed to EDCs for various exposure times. The expression of HSP90 transcripts was significantly increased in C. japonica crabs exposed to BPA and NP at different concentrations for 12, 24, 48 and 96 hours. The mRNA expression of HSP90 gene was significantly induced in a concentration- and time-dependent manner after BPA or NP exposures for 96 hours. Conclusions Taken together, expression analysis of Asian paddle crab HSP90 gene provided useful molecular information about crab responses in stress conditions and potential ways to monitor the EDCs stressors in marine environments. PMID:24955332

Characterize and Gene Expression of Heat Shock Protein 90 in Marine Crab Charybdis japonica following Bisphenol A and 4-Nonylphenol Exposures.

PubMed

Park, Kiyun; Kwak, Ihn-Sil

2014-01-01

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone important in the maturation of a broad spectrum of protein. In this study, an HSP90 gene was isolated from Asian paddle crab, Charybdis japonica, as a bio-indicator to monitor the marine ecosystem. This work reports the responses of C. japonica HSP90 mRNA expression to cellular stress by endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 4-nonylphenol (NP) using real-time. reverse transcription polymerase chain reaction. The deduced amino acid sequence of HSP90 from C. japonica shared a high degree of homology with their homologues in other species. In a phylogenetic analysis, C. japonica HSP90 is evolutionally related with an ortholog of the other crustacean species. The expression of HSP90 gene was almost distributed in all the examined tissues of the C. japonica crab but expression levels varied among the different body parts of the crabs. We examined HSP90 mRNA expression pattern in C. japonica crabs exposed to EDCs for various exposure times. The expression of HSP90 transcripts was significantly increased in C. japonica crabs exposed to BPA and NP at different concentrations for 12, 24, 48 and 96 hours. The mRNA expression of HSP90 gene was significantly induced in a concentration- and time-dependent manner after BPA or NP exposures for 96 hours. Taken together, expression analysis of Asian paddle crab HSP90 gene provided useful molecular information about crab responses in stress conditions and potential ways to monitor the EDCs stressors in marine environments.

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

PubMed Central

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

2011-01-01

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

Role of Heat Shock Protein 90 and Endothelial Nitric Oxide Synthase during Early Anesthetic and Ischemic Preconditioning

PubMed Central

Amour, Julien; Brzezinska, Anna K.; Weihrauch, Dorothee; Billstrom, Amie R.; Zielonka, Jacek; Krolikowski, John G.; Bienengraeber, Martin W.; Warltier, David C.; Pratt, Philip F.; Kersten, Judy R.

2009-01-01

Background Nitric oxide is known to be essential for early anesthetic (APC) and ischemic (IPC) preconditioning of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, we tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. Methods Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning with 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pre-treatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or NG-nitro-L-arginine methylester, a non-specific NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or NG-nitro-L-arginine methylester. Interactions between Hsp90 and eNOS, and eNOS activation were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. Results APC and IPC decreased infarct size (50% and 59%, respectively) and this action was abolished by Hsp90 inhibitors. NG-nitro-L-arginine methylester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells, concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes and eNOS was below the level of detection. Conclusion The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signalling during APC. PMID:19194158

Targeting Hodgkin and Reed–Sternberg Cells with an Inhibitor of Heat-Shock Protein 90: Molecular Pathways of Response and Potential Mechanisms of Resistance

PubMed Central

Corrêa, Stephany; Du Rocher, Bárbara; Krsticevic, Flavia; Arce, Debora; Sternberg, Cinthya; Abdelhay, Eliana

2018-01-01

Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed–Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol’s toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research. PMID:29534015

Targeting Hodgkin and Reed-Sternberg Cells with an Inhibitor of Heat-Shock Protein 90: Molecular Pathways of Response and Potential Mechanisms of Resistance.

PubMed

Segges, Priscilla; Corrêa, Stephany; Du Rocher, Bárbara; Vera-Lozada, Gabriela; Krsticevic, Flavia; Arce, Debora; Sternberg, Cinthya; Abdelhay, Eliana; Hassan, Rocio

2018-03-13

Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed-Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol's toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research.

Pattern of heat shock factor and heat shock protein expression in lymphocytes of bipolar patients: increased HSP70-glucocorticoid receptor heterocomplex.

PubMed

Bei, E S; Salpeas, V; Alevizos, B; Anagnostara, C; Pappa, D; Moutsatsou, P

2013-11-01

Bipolar disorder (BD), a stress-related disease, is characterized by altered glucocorticoid receptor (GR) signalling. Stress response includes activation of heat shock factor (HSF) and subsequent heat shock protein (HSP) synthesis which regulate GR folding and function. The objective of this study was to investigate the possible role of HSFs, HSPs and their interaction with GR in BD. We applied immunoprecipitation, SDS-PAGE/Western blot analysis and electrophoretic mobility shift assay (EMSA) in lymphocytes (whole cell or nuclear extracts) from BD patients and healthy subjects and determined the HSPs (HSP90 and HSP70), the heterocomplexes HSP90-GR and HSP70-GR, the HSFs (HSF1 and HSF4) as well as the HSF-DNA binding. The HSP70-GR heterocomplex was elevated (p < 0.05) in BD patients vs healthy subjects, and nuclear HSP70 was reduced (p ≤ 0.01) in bipolar manic patients. Protein levels of HSF1, HSF4, HSP90, HSP90-GR heterocomplex, and HSF-DNA binding remained unaltered in BD patients vs healthy subjects. The corresponding effect sizes (ES) indicated a large ES for HSP70-GR, HSP70, HSF-DNA binding and HSF4, and a medium ES for HSP90, HSF1 and HSP90-GR between healthy subjects and bipolar patients. Significant correlations among HSFs, HSPs, GR and HSP70-GR heterocomplex were observed in healthy subjects, which were abrogated in bipolar patients. The higher interaction between GR and HSP70 and the disturbances in the relations among heat shock response parameters and GR as observed in our BD patients may provide novel insights into the contribution of these factors in BD aetiopathogenesis. Copyright © 2013. Published by Elsevier Ltd.

Overexpression and localization of heat shock proteins mRNA in pancreatic carcinoma.

PubMed

Ogata, M; Naito, Z; Tanaka, S; Moriyama, Y; Asano, G

2000-06-01

In the present study we examined the localization and overexpression of heat shock proteins (hsps), mainly hsp90, in pancreatic carcinoma tissue compared with control tissue (including chronic pancreatitis and normal pancreas tissue), with the aid of immunohistochemical staining, in situ hybridization and reverse transcriptase polymerase chain reaction. Hsp90 alpha mRNA was overexpressed more highly in pancreatic carcinoma than in the control tissue. The proliferating-cell-nuclear-antigen labeling index was also high in pancreatic carcinoma tissue compared with the other tissue. These findings suggest that the overexpression of hsp90 alpha mRNA in carcinomas may be correlated with cell proliferation. However, hsp90 beta was constitutively overexpressed almost equally in all groups of pancreatic tissue including pancreatic carcinoma, chronic pancreatitis and normal pancreas tissue. Immunohistochemical staining demonstrated a differentiation in the expression of hsp90 between histological types of pancreatic carcinoma. These findings suggest that hsp90 alpha is involved in carcinogenesis and that hsp90 beta is correlated to structural conformation. Hsp90 alpha and hsp90 beta seem to perform different functions in tissue containing malignant cells. P53, MDM2 and WAF1, that were cell-cycle-related oncogene product were more strongly expressed in the nuclei of the cancer cells of the cancer tissue. Especially, MDM2 was more strongly expressed in mucinous carcinoma and the mucin secreting tissues surrounding pancreatic carcinoma tissue. The expression of MDM2 protein might also be correlated to secretion systems during structural conformation and be correlated to hsp90 beta.

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling.

PubMed

Chen, Christine Y; Balch, William E

2006-08-01

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.

A Novel Class of Small Molecule Inhibitors of Hsp90

PubMed Central

Yi, Fang; Regan, Lynne

2012-01-01

Unregulated cellular proliferation, caused by mutation or dysregulation of growth-promoting proteins, is an underlying cause of cancer. Many such growth-promoting proteins exhibit an increased dependence on the activity of the chaperone heat-shock protein 90 (Hsp90) for correct folding and maturation in the cell. One can therefore envision that inhibition of Hsp90 would be an effective and broadly applicable strategy for the development of anticancer agents. Hsp90 functions in multichaperone complexes driven by the binding and hydrolysis of ATP. Encouraging results have been obtained by inhibiting Hsp90 with 17-AAG, an active-site binding ATP analog. Here we present the results of a different approach to inhibiting Hsp90 by disrupting its interaction with a cochaperone named Hsp organizing protein (HOP). We have used an AlphaScreen technology based high-throughput in vitro screen to identify compounds that inhibit this interaction. In addition, we demonstrate that these compounds are active in vivo. Treatment of human breast cancer cell lines BT474 and SKBR3 with these compounds decreases the levels of the Hsp90-dependent client protein HER2, with associated cell death. PMID:18785742

The threshold induction temperature of the 90-kDa heat shock protein is subject to acclimatization in eurythermal goby fishes (genus Gillichthys).

PubMed Central

Dietz, T J; Somero, G N

1992-01-01

Two extremely eurythermal goby fishes, Gillichthys mirabilis and Gillichthys seta, which encounter habitat temperature variations of approximately 30 degrees C, showed seasonal acclimatization of endogenous levels and of onset temperatures for enhanced synthesis of a 90-kDa-class heat shock protein (HSP90). Summer-acclimatized fishes had higher levels of HSP90 in brain tissue than winter-acclimatized specimens, as shown by Western blot analysis. For winter-acclimatized fishes, increased synthesis of HSP90 was observed when the temperature was raised from a control temperature (18 degrees C) to 28 degrees C. For summer-acclimatized fish, no significantly increased synthesis of HSP90 occurred until the experimental temperature was raised to 32 degrees C. These data suggest that the threshold temperature at which enhanced expression of HSP-encoding genes occurs is not hard-wired genetically but may be subject to acclimatization. A causal relationship between seasonal changes in steady-state levels of HSP90 and the threshold temperature for enhanced HSP90 synthesis is discussed in terms of existing models for the regulation of HSP gene expression. Images PMID:1565632

[Determination of mRNA-transcripts and heat shock proteins HSP70 and HSP90 in retina of the adult Spanish Ribbed Newt Pleurodeles waltl].

PubMed

Avdonin, P P; Markitantova, Yu V; Poplinskaya, V A; Grigoryan, E N

2013-01-01

Expression of genes and heat shock proteins in normal intact retina of the Spanish Ribbed Newt Pleurodeles waltl was studied using polymerase chain reaction, Western blot hybridization, and immunohistochemistry. It was shown that the proteins HSP70 and HSP90, as well as their encoding transcripts of relevant genes, are constitutively expressed in eye tissues. These proteins were distributed differentially, and they were characterized by expression of different levels in the retina: HSP70 dominated in the external retina, while HSP90 dominated in the internal one, in particular, in Muller glial cells and the optic nerve. Transcripts and heat shock proteins HSP70 and HSP90 were also found in the retinal pigment epithelium and eye growth zone.

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

PubMed Central

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

2014-01-01

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

Inhibition of HSP90α protects cultured neurons from oxygen-glucose deprivation induced necroptosis by decreasing RIP3 expression.

PubMed

Wang, Zhen; Guo, Li-Min; Wang, Yong; Zhou, Hong-Kang; Wang, Shu-Chao; Chen, Dan; Huang, Ju-Fang; Xiong, Kun

2018-06-01

Heat shock protein 90α (HSP90α) maintains cell stabilization and regulates cell death, respectively. Recent studies have shown that HSP90α is involved in receptor interacting protein 3 (RIP3)-mediated necroptosis in HT29 cells. It is known that oxygen and glucose deprivation (OGD) can induce necroptosis, which is regulated by RIP3 in neurons. However, it is still unclear whether HSP90α participates in the process of OGD-induced necroptosis in cultured neurons via the regulation of RIP3. Our study found that necroptosis occurs in primary cultured cortical neurons and PC-12 cells following exposure to OGD insult. Additionally, the expression of RIP3/p-RIP3, MLKL/p-MLKL, and the RIP1/RIP3 complex (necrosome) significantly increased following OGD, as measured through immunofluorescence (IF) staining, Western blotting (WB), and immunoprecipitation (IP) assay. Additionally, data from computer simulations and IP assays showed that HSP90α interacts with RIP3. In addition, HSP90α was overexpressed following OGD in cultured neurons, as measured through WB and IF staining. Inhibition of HSP90α in cultured neurons, using the specific inhibitor, geldanamycin (GA), and siRNA/shRNA of HSP90α, protected cultured neurons from necrosis. Our study showed that the inhibitor of HSP90α, GA, rescued cultured neurons not only by decreasing the expression of total RIP3/MLKL, but also by decreasing the expression of p-RIP3/p-MLKL and the RIP1/RIP3 necrosome. In this study, we reveal that inhibition of HSP90α protects primary cultured cortical neurons and PC-12 cells from OGD-induced necroptosis through the modulation of RIP3 expression. © 2017 Wiley Periodicals, Inc.

Apoptosis in response to heat stress is positively associated with heat-shock protein 90 expression in chicken myocardial cells in vitro.

PubMed

Zhang, Xiao-Hui; Wu, Hong; Tang, Shu; Li, Qiao-Ning; Xu, Jiao; Zhang, Miao; Su, Ya-Nan; Yin, Bin; Zhao, Qi-Ling; Kemper, Nicole; Hartung, Joerg; Bao, En-Dong

2017-06-30

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken ( Gallus gallus ) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.

miR-223/Hsp70/JNK/JUN/miR-223 feedback loop modulates the chemoresistance of osteosarcoma to cisplatin.

PubMed

Tang, Qi; Yuan, Qi; Li, Hui; Wang, Wanchun; Xie, Guangrong; Zhu, Kewei; Li, Ding

2018-03-11

Osteosarcoma (OS) is a primary bone malignancy with a five-year survival rate of 60%; the chemoresistance of OS still remains a huge challenge. Heat shock protein 70 (Hsp70), a member of HSP family, is overexpressed in OS cell lines and involved in the resistance of OS cell lines. In addition, miRNAs have been involved in the carcinogenesis and chemoresistance of OS; of them, miR-223 has been reported to be underexpressed and serve as a tumor suppressor in OS through targeting Hsp90B1, also a member of HSP family. Herein, online tools predicted that Hsp70 might be a direct target of miR-223. In the present study, miR-223 expression was down-regulated in OS tissues and cell lines; miR-223 overexpression enhanced the cellular effects of cisplatin (CDDP) on OS cell lines. Through binding to the HSPA1A 3'UTR, miR-223 could regulate Hsp70 protein levels and downstream JNK/JUN signaling pathway, thus modulating OS cell apoptosis through Hsp70 under CDDP stress. Finally, JUN, a downstream transcription factor of JNK signaling, could bind to the promoter region of miR-223 to promote its transcription. In summary, miR-223, Hsp70 and downstream JNK/JUN formed a feedback loop to modulate the chemoresistance of OS to CDDP. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Resolving Hot Spots in the C-Terminal Dimerization Domain that Determine the Stability of the Molecular Chaperone Hsp90

PubMed Central

Reimann, Sven; Smits, Sander H. J.; Schmitt, Lutz; Groth, Georg; Gohlke, Holger

2014-01-01

Human heat shock protein of 90 kDa (hHsp90) is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD) should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScorePPI. Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization. PMID:24760083

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

PubMed

Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

2016-01-01

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

Identification of highly effective target genes for RNAi-mediated control of emerald ash borer, Agrilus planipennis.

PubMed

Rodrigues, Thais B; Duan, Jian J; Palli, Subba R; Rieske, Lynne K

2018-03-22

Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), caused up to 90% mortality of larvae and adults. In EAB eggs, larvae, and adults, the hsp is expressed at higher levels when compared to that of shi. Ingestion of dsHSP and dsSHI caused mortality in both neonate larvae and adults. Administration of a mixture of both dsRNAs worked better than either dsRNA by itself. In contrast, injection of EAB.dsHSP and EAB.dsSHI did not cause mortality in T. castaneum. Thus, the two genes identified cause high mortality in the EAB with no apparent phenotype effects in a non-target organism, the red flour beetle, and could be used in RNAi-mediated control of this invasive pest.

Heat shock response and metabolic stress in the tropical estuarine copepod Pseudodiaptomus annandalei converge at its upper thermal optimum.

PubMed

Low, Joyce S Y; Chew, Li Lee; Ng, Ching Ching; Goh, Hao Chin; Lehette, Pascal; Chong, Ving Ching

2018-05-01

Heat shock response (HSR), in terms of transcription regulation of two heat shock proteins genes hsp70 and hsp90), was analysed in a widespread tropical copepod Pseudodiaptomus annandalei. The mRNA transcripts of both genes were quantified after copepods at a salinity of 20 underwent an acclimation process involving an initial acclimation temperature of 29 °C, followed by gradual thermal ramping to the target exposure temperature range of 24-36 °C. The respective cellular HSR and organismal metabolism, measured by respiratory activity at exposure temperatures, were compared. The fold change in mRNA expression for both hsp70 and hsp90 (8-9 fold) peaks at 32 °C, which is very close to 32.4 °C, the upper thermal optimum for respiration in the species. Unexpectedly, the modelled HSR curves peak at only 3 °C (hsp90) and 3.5 °C (hsp70) above the mean water temperature (29.32 °C) of the copepod in the field. We propose that copepods in tropical waters adopt a preparative HSR strategy, early at the upper limit of its thermal optimum, due to the narrow thermal range of its habitat thus precluding substantial energy demand at higher temperatures. However, the model suggests that the species could survive to at least 36 °C with short acclimation time. Nevertheless, the significant overlap between its thermal range of hsp synthesis and the narrow temperature range of its habitat also suggests that any unprecedented rise in sea temperature would have a detrimental effect on the species. Copyright © 2018 Elsevier Ltd. All rights reserved.

Anti-hypoxia effect of adenovirus-mediated expression of heat shock protein 70 (HSP70) on primary cultured neurons.

PubMed

Hu, Dan; Chen, Fuqiang; Guan, Chun; Yang, Fangfang; Qu, Yan

2013-09-01

Heat shock protein 70 (HSP70) has attracted great attention recently in hypoxia injury because of its close link to the recovery after hypoxic-ischemic damage in organs. However, the cellular mechanism underlying its protective roles remains unclear. In this study, we developed a recombinant adenovirus containing HSP70-GFP (vAd-HSP70-GFP) and studied the effect of virus-mediated expression of exogenous HSP70 gene on neurons in response to hypoxia-reoxygenation injury. Virus-mediated expression of HSP70 was detected as early as 24 hr and lasted until 10 days after infection. Neurons with 48 hr vAd-HSP70-GFP infection were exposed to 0, 0.5, 1, 2, 3, or 4 hr hypoxia followed by 1 hr reoxygenation. The mRNA and protein levels of HSP70 in neurons exposed to different lengths of hypoxia were compared by using RT-PCR and Western blotting (WB). The 1-hr hypoxia exposure showed the most significant increases in the HSP70 mRNA and protein level compared with other exposure durations. MTT assay showed that HSP70 overexpression significantly increased the neuronal viability, accompanied by decreased lactate dehydrogenase (LDH) activity in the culture medium after hypoxia-reoxygenation. Neurons with vAd-HSP70-GFP exhibited increased levels of mitochondrial cytochrome C (Cyt-C) and decreased levels of cytoplasmic Cyt-C compared with vAd-GFP-infected cells. These results suggest a neuroprotective role of exogenous HSP70 against hypoxia-reoxygenation injury, possibly via preventing initiation of mitochondrial apoptosis. Copyright © 2013 Wiley Periodicals, Inc.

Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles (Trachemys scripta) using an external RNA control for normalization.

PubMed

Stecyk, Jonathan A W; Couturier, Christine S; Fagernes, Cathrine E; Ellefsen, Stian; Nilsson, Göran E

2012-03-01

The mRNA expression of heat-shock protein 90 (HSP90) and heat-shock cognate 70 (HSC70) was examined in cardiac chambers and telencephalon of warm- (21°C) and cold-acclimated (5°C) turtles (Trachemys scripta) exposed to normoxia, prolonged anoxia or anoxia followed by reoxygenation. Additionally, the suitability of total RNA as well as mRNA from β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclophilin A (PPIA) for normalizing gene expression data was assessed, as compared to the use of an external RNA control. Measurements of HSP90 and HSC70 mRNA expression revealed that anoxia and reoxygenation have tissue- and gene-specific effects. By and large, the alterations support previous investigations on HSP protein abundance in the anoxic turtle heart and brain, as well as the hypothesized roles of HSP90 and HSC70 during stress and non-stress conditions. However, more prominent was a substantially increased HSP90 and HSC70 mRNA expression in the cardiac chambers with cold acclimation. The finding provides support for the notion that cold temperature induces a number of adaptations in tissues of anoxia-tolerant vertebrates that precondition them for winter anoxia. β-actin, GAPDH and PPIA mRNA expression and total RNA also varied with oxygenation state and acclimation temperature in a tissue- and gene-specific manner, as well as among tissue types, thus disqualifying them as suitable for real-time RT-PCR normalization. Thus, the present data highlights the advantages of normalizing real-time RT-PCR data to an external RNA control, an approach that also allows inter-tissue and potentially inter-species comparisons of target gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Somatostatin increases glucocorticoid binding and signaling in macrophages by blocking the calpain-specific cleavage of Hsp 90.

PubMed

Bellocq, A; Doublier, S; Suberville, S; Perez, J; Escoubet, B; Fouqueray, B; Puyol, D R; Baud, L

1999-12-24

Somatostatin has direct anti-inflammatory actions and participates in the anti-inflammatory actions of glucocorticoids, but the mechanisms underlying this regulation remain poorly understood. The objective of this study was to evaluate whether somatostatin increases glucocorticoid responsiveness by up-regulating glucocorticoid receptor (GR) expression and signaling. Somatostatin promoted a time- and dose-dependent increase in [(3)H]dexamethasone binding to RAW 264.7 macrophages. Cell exposure to 10 nM somatostatin for 18 h promoted a 2-fold increase in the number of GR sites per cell without significant modification of the affinity. Analysis of GR heterocomplex components demonstrated that somatostatin increased the level of heat shock protein (Hsp) 90, whereas the level of GR remained almost unchanged. The increase in Hsp 90 was associated with a decrease in the cleavage of its carboxyl-terminal domain. Evidence for the involvement of calpain inhibition in this process was obtained by the demonstration that 1) somatostatin induced a dose-dependent decrease in calpain activity and 2) calpain inhibitors, calpain inhibitor I and calpeptin, both abolished the cleavage of Hsp 90 and induced a dose-dependent increase in [(3)H]dexamethasone binding. Increases in glucocorticoid binding after somatostatin treatment were associated with similar increases in the ability of GR to transactivate a minimal promoter containing two glucocorticoid response elements (GRE) and to interfere with the activation of nuclear factor-kappaB (NF-kappaB). Thus, the present findings indicate that somatostatin increases glucocorticoid binding and signaling by limiting the calpain-specific cleavage of GR-associated Hsp 90. This mechanism may represent a novel target for intervention to increase glucocorticoid responsiveness.

A Phase 1 study of PF-04929113 (SNX-5422), an orally bioavailable heat shock protein 90 inhibitor in patients with refractory solid tumor malignancies and lymphomas

PubMed Central

Rajan, Arun; Kelly, Ronan J.; Trepel, Jane B.; Kim, Yeong Sang; Alarcon, Sylvia V.; Kummar, Shivaani; Gutierrez, Martin; Crandon, Sonja; Zein, Wadih M.; Jain, Lokesh; Mannargudi, Baskar; Figg, William D.; Houk, Brett E.; Shnaidman, Michael; Brega, Nicoletta; Giaccone, Giuseppe

2011-01-01

Purpose To determine the maximum tolerated dose (MTD), toxicities, and pharmacokinetic-pharmacodynamic profile of the heat shock protein 90 (Hsp90) inhibitor PF-04929113 (SNX-5422) in patients with advanced solid tumors and lymphomas. Methods This was a single institution, phase I, dose-escalation study of PF-04929113 dosed twice-weekly. Endpoints included determination of dose-limiting toxicities (DLT), MTD, the safety profile of PF-04929113, pharmacodynamic assessment of PF-04929113 on Hsp70 induction, pharmacokinetic (PK) analysis of PF 04928473 (SNX-2112) and its prodrug PF-04929113 and assessment of response. Results Thirty three patients with advanced malignancies were treated. Dose escalation was continued up to 177 mg/m2 administered orally twice a week. One DLT (non-septic arthritis) was noted. No grade 4 adverse events (AEs) were seen; grade 3 AEs included diarrhea (9%), non-septic arthritis (3%), AST elevation (3%) and thrombocytopenia (3%). No objective responses were seen in 32 evaluable patients. Fifteen patients (47%) had stable disease; 17 patients (53%) had progressive disease. PK data revealed rapid absorption, hepatic and extra-hepatic clearance, extensive tissue binding and almost linear pharmacokinetics of the active drug PF 04928473. PD studies confirmed inhibition of Hsp90 and a linear correlation between PK parameters and Hsp70 induction. Conclusions PF-04929113 administered orally twice weekly is well tolerated and inhibits its intended target Hsp90. No objective responses were seen but long lasting stabilizations were obtained. Although no clinically significant drug-related ocular toxicity was seen in this study the development of PF-04929113 has been discontinued due to ocular toxicity seen in animal models and in a separate phase I study. PMID:21908572

The Hsp90 co-chaperone p23 of Toxoplasma gondii: Identification, functional analysis and dynamic interactome determination

PubMed Central

Echeverria, Pablo C.; Figueras, Maria J.; Vogler, Malvina; Kriehuber, Thomas; de Miguel, Natalia; Deng, Bin; Dalmasso, Maria C.; Matthews, Dwight E.; Matrajt, Mariana; Haslbeck, Martin; Buchner, Johannes; Angel, Sergio O.

2010-01-01

Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. Recently a link between the T. gondii Hsp90 chaperone machinery and parasite development was observed. Here, the T. gondii Hsp90 co-chaperones p23 and Hip were identified mining the Toxoplasma- database (www.toxodb.org). Their identity was confirmed by domain structure and blast analysis. Additionally, analysis of the secondary structure and studies on the chaperone function of the purified protein verified the p23 identity. Studies of co-immunoprecipitation (co-IP) identified two different types of complexes, one comprising at least Hip-Hsp70-Hsp90 and another containing at least p23-Hsp90. Indirect immunofluorescence assays showed that Hip is localized in the cytoplasm in tachyzoites and as well in bradyzoites. For p23 in contrast, a solely cytoplasmic localization was only observed in the tachyzoite stage whereas nuclear and cytosolic distribution and colocalization with Hsp90 was observed in bradyzoites. These results indicate that the T. gondii Hsp90-heterocomplex cycle is similar to the one proposed for higher eukaryotes, further highlighting the implication of the Hsp90/p23 in parasite development. Furthermore, co-IP experiments of tachyzoite/bradyzoite lysates with anti-p23 antiserum and identification of the complexed proteins together with the use of the curated interaction data available from different source (orthologs and Plasmodium databases) allowed us to construct an interaction network (interactome) covering the dynamics of the Hsp90 chaperone machinery. PMID:20403389

The change in heat shock protein expression in avermectin induced neurotoxicity of the pigeon (Columba livia) both in vivo and in vitro.

PubMed

Li, Ming; Wang, Xian-Song; Xu, Feng-Ping; Liu, Shuang; Xu, Shi-Wen; Li, Shu

2014-12-01

The expression of heat shock proteins (Hsps) commonly increases to provide neuroprotection when brain tissues are under stress conditions. Residues of avermectins (AVMs) have neurotoxic effects on a number of non-target organisms. The aim of this study was to investigate the effects of AVM exposure on the expression levels of Hsp 60, Hsp 70 and Hsp 90 for pigeon (Columba livia) neurons both in vivo and in vitro. The results showed that in general, the mRNA and protein levels of Hsps were increased in treated groups relative to control groups after AVM exposure for 30d, 60d and 90d in the cerebrum, cerebellum and optic lobe in vivo. However, AVM exposure had no significant effects on the transcription expression of Hsps for 90d in the optic lobe and decreased the translation expression of Hsps significantly for 90d in the optic lobe. In vitro, the LC50 of avermectin for King pigeon neurons is between 15μgL(-1) and 20μgL(-1). Following AVM (2.5-20μgL(-1)) exposure, the mRNA expression of the 3 Hsps was up-regulated to different degrees. Compared with the control groups, a significant decrease, a remarkable increase and a non-significant change was found in the protein expression of Hsp 60, Hsp 70 and Hsp 90 separately following AVM (2.5-20μgL(-1)) exposure. Based on these results, we conclude that AVM exposure can induce a protective stress response in pigeons by means of promoting the mRNA and protein expression of Hsps under in vivo and in vitro conditions, thus easing the neurotoxic effects of AVM to some extent. Copyright © 2014 Elsevier Inc. All rights reserved.

Heat shock protein 90 functions to stabilize and activate the testis-specific serine/threonine kinases, a family of kinases essential for male fertility.

PubMed

Jha, Kula N; Coleman, Alyssa R; Wong, Lily; Salicioni, Ana M; Howcroft, Elizabeth; Johnson, Gibbes R

2013-06-07

Spermiogenesis is characterized by a profound morphological differentiation of the haploid spermatid into spermatozoa. The testis-specific serine/threonine kinases (TSSKs) comprise a family of post-meiotic kinases expressed in spermatids, are critical to spermiogenesis, and are required for male fertility in mammals. To explore the role of heat shock protein 90 (HSP90) in regulation of TSSKs, the stability and catalytic activity of epitope-tagged murine TSSKs were assessed in 293T and COS-7 cells. TSSK1, -2, -4, and -6 (small serine/threonine kinase) were all found to associate with HSP90, and pharmacological inhibition of HSP90 function using the highly specific drugs 17-AAG, SNX-5422, or NVP-AUY922 reduced TSSK protein levels in cells. The attenuation of HSP90 function abolished the catalytic activities of TSSK4 and -6 but did not significantly alter the specific activities of TSSK1 and -2. Inhibition of HSP90 resulted in increased TSSK ubiquitination and proteasomal degradation, indicating that HSP90 acts to control ubiquitin-mediated catabolism of the TSSKs. To study HSP90 and TSSKs in germ cells, a mouse primary spermatid culture model was developed and characterized. Using specific antibodies against murine TSSK2 and -6, it was demonstrated that HSP90 inhibition resulted in a marked decrease of the endogenous kinases in spermatids. Together, our findings demonstrate that HSP90 plays a broad and critical role in stabilization and activation of the TSSK family of protein kinases.

Virtual prototyping study shows increased ATPase activity of Hsp90 to be the key determinant of cancer phenotype.

PubMed

Vali, Shireen; Pallavi, Rani; Kapoor, Shweta; Tatu, Utpal

2010-03-01

Hsp90 is an ATP-dependent molecular chaperone that regulates key signaling proteins and thereby impacts cell growth and development. Chaperone cycle of Hsp90 is regulated by ATP binding and hydrolysis through its intrinsic ATPase activities, which is in turn modulated by interaction with its co-chaperones. Hsp90 ATPase activity varies in different organisms and is known to be increased in tumor cells. In this study we have quantitatively analyzed the impact of increasing Hsp90 ATPase activity on the activities of its clients through a virtual prototyping technology, which comprises a dynamic model of Hsp90 interaction with clients involved in proliferation pathways. Our studies highlight the importance of increased ATPase activity of Hsp90 in cancer cells as the key modulator for increased proliferation and survival. A tenfold increase in ATPase activity of Hsp90 often seen in cancer cells increases the levels of active client proteins such as Akt-1, Raf-1 and Cyclin D1 amongst others to about 12-, 8- and 186-folds respectively. Additionally we studied the effect of a competitive inhibitor of Hsp90 activity on the reduction in the client protein levels. Virtual prototyping experiments corroborate with findings that the drug has almost 10- to 100-fold higher affinity as indicated by a lower IC(50) value (30-100 nM) in tumor cells with higher ATPase activity. The results also indicate a 15- to 25-fold higher efficacy of the inhibitor in reducing client levels in tumor cells. This analysis provides mechanistic insights into the links between increased Hsp90 ATPase activity, tumor phenotype and the hypersensitivity of tumor Hsp90 to inhibition by ATP analogs. The online version of this article (doi:10.1007/s11693-009-9046-3) contains supplementary material, which is available to authorized users.

GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases.

PubMed

Roberts, Jane L; Tavallai, Mehrad; Nourbakhsh, Aida; Fidanza, Abigail; Cruz-Luna, Tanya; Smith, Elizabeth; Siembida, Paul; Plamondon, Pascale; Cycon, Kelly A; Doern, Christopher D; Booth, Laurence; Dent, Paul

2015-10-01

Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged 'rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the 'Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. 'Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of 'Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections. © 2015 Wiley Periodicals, Inc.

The stress protein level under clinorotation in context of the seedling developmental program and the stress response

NASA Astrophysics Data System (ADS)

Kozeko, Lyudmyla; Kordyum, Elizabeth

2006-09-01

Heat-shock proteins (HSP70 and HSP90) are present in plant cells under the normal growth conditions. At the same time, a variety of environmental disruptions results in their rapid synthesis as a substantial part of adaptation. HSP amounts can be indicative of a cellular stress level. Altered gravity (clinorotation) is unnatural for plants, so it may be a kind of stress. The aim of this study was to analyze the influence of horizontal clinorotation on the HSP70 and HSP90 level during seedling development. Pea (Pisum sativum L.) seedlings grown for 3 days from seed imbibitions in stationary control and under slow clinorotation (2 rpm) are used for this investigation. Western blot analysis indicated that HSP70 and HSP90 were abundant in the embryos of dry seeds and their amount decreased significantly during seed germination. But under horizontal clinorotation, their level in seedlings remained higher compared to the control. Furthermore, a comparison of the influence of horizontal and vertical clinorotation on the HSP level was carried out. On the ELISA data, HSP70 and HSP90 amounts in the 3-day old seedlings were higher after horizontal clinorotation than after vertical. The obtained data show an increased HSP70 and HSP90 level in pea seedlings under clinorotation. Both, rotation and change in the cell position relatively to a gravity vector affect the HSP level.

Novel arrangement and comparative analysis of hsp90 family genes in three thermotolerant species of Stratiomyidae (Diptera).

PubMed

Astakhova, L N; Zatsepina, O G; Przhiboro, A A; Evgen'ev, M B; Garbuz, D G

2013-06-01

The heat shock proteins belonging to the Hsp90 family (Hsp83 in Diptera) play a crucial role in the protection of cells due to their chaperoning functions. We sequenced hsp90 genes from three species of the family Stratiomyidae (Diptera) living in thermally different habitats and characterized by extraordinarily high thermotolerance. The sequence variation and structure of the hsp90 family genes were compared with previously described features of hsp70 copies isolated from the same species. Two functional hsp83 genes were found in the species studied, that are arranged in tandem orientation at least in one of them. This organization was not previously described. Stratiomyidae hsp83 genes share a high level of identity with hsp83 of Drosophila, and the deduced protein possesses five conserved amino acid sequence motifs characteristic of the Hsp90 family as well as the C-terminus MEEVD sequence characteristic of the cytosolic isoform. A comparison of the hsp83 promoters of two Stratiomyidae species from thermally contrasting habitats demonstrated that while both species contain canonical heat shock elements in the same position, only one of the species contains functional GAF-binding elements. Our data indicate that in the same species, hsp83 family genes show a higher evolution rate than the hsp70 family. © 2013 Royal Entomological Society.

Impact of heat-shock protein 90 on cancer metastasis

PubMed Central

Tsutsumi, Shinji; Beebe, Kristin; Neckers, Len

2009-01-01

Cancer metastasis is the result of complex processes, including alteration of cell adhesion/motility in the microenvironment and neoangiogenesis, that are necessary to support cancer growth in tissues distant from the primary tumor. The molecular chaperone heat-shock protein 90 (Hsp90), also termed the ‘cancer chaperone’, plays a crucial role in maintaining the stability and activity of numerous signaling proteins involved in these processes. Small-molecule Hsp90 inhibitors display anticancer activity both in vitro and in vivo, and multiple Phase II and Phase III clinical trials of several structurally distinct Hsp90 inhibitors are currently underway. In this review, we will highlight the importance of Hsp90 in cancer metastasis and the therapeutic potential of Hsp90 inhibitors as antimetastasis drugs. PMID:19519207

Elucidation of the Hsp90 C-terminal Inhibitor Binding Site

PubMed Central

Matts, Robert L.; Dixit, Anshuman; Peterson, Laura B.; Sun, Liang; Voruganti, Sudhakar; Kalyanaraman, Palgunan; Hartson, Steve D.; Verkhivker, Gennady M.; Blagg, Brian S. J.

2011-01-01

The Hsp90 chaperone machine is required for the folding, activation and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains, however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein. PMID:21548602

Spermatogenesis arrest caused by conditional deletion of Hsp90α in adult mice

PubMed Central

Kajiwara, Chiaki; Kondo, Shiho; Uda, Shizuha; Dai, Lei; Ichiyanagi, Tomoko; Chiba, Tomoki; Ishido, Satoshi; Koji, Takehiko; Udono, Heiichiro

2012-01-01

Summary It is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90α KO mice. We had generated Hsp90α KO mice independently and confirmed this phenotype. However, the important question of whether Hsp90α is required to maintain spermatogenesis in adult mice in which testicular maturation is already completed could not be addressed using these conventional KO mice. To answer this question, we generated a tamoxifen-inducible deletion mutant of Hsp90α and found that conditional deletion of Hsp90α in adult mice caused even more severe apoptosis in germ cells beyond the pachytene stage, leading to complete arrest of spermatogenesis and testicular atrophy. Importantly, immunohistochemical analysis revealed that AR expression in WT testis was more evident in spermatogonia than in spermatocytes, whereas its expression was aberrant and ectopic in Hsp90α KO testis, raising the possibility that an AR abnormality in primordial germ cells is involved in spermatogenesis arrest in the Hsp90α KO mice. Our results suggest that the AR, specifically chaperoned by Hsp90α in spermatogonia, is critical for maintenance of established spermatogenesis and for survival of spermatocytes in adult testis, in addition to setting the first wave of spermatogenesis before puberty. PMID:23213375

Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9

PubMed Central

Schmitz-Abe, Klaus; Ciesielski, Szymon J.; Schmidt, Paul J.; Campagna, Dean R.; Rahimov, Fedik; Schilke, Brenda A.; Cuijpers, Marloes; Rieneck, Klaus; Lausen, Birgitte; Linenberger, Michael L.; Sendamarai, Anoop K.; Guo, Chaoshe; Hofmann, Inga; Newburger, Peter E.; Matthews, Dana; Shimamura, Akiko; Snijders, Pieter J. L. M.; Towne, Meghan C.; Niemeyer, Charlotte M.; Watson, Henry G.; Dziegiel, Morten H.; Heeney, Matthew M.; May, Alison; Bottomley, Sylvia S.; Swinkels, Dorine W.; Markianos, Kyriacos; Craig, Elizabeth A.

2015-01-01

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance. PMID:26491070

FT-IR Study Reveals Intrinsically Disordered Nature of Heat Shock Protein 90

NASA Astrophysics Data System (ADS)

Xie, Aihua; Neto, David; Balch, Maurie; Hendriks, Johnny; Causey, Oliver; Deng, Junpeng; Matts, Robert

Heat shock protein 90 (Hsp90) is a highly conserved chaperone protein that enables the proper folding of a large number of structurally diverse proteins (a.k.a., clients) in the crowded cytosolic environment and plays a key role in regulating the heat shock response. A long standing open question is how Hsp90 accommodates the structural diversity of a large cohort of client proteins? We report ATR FTIR study on structural properties of Hsp90 C-terminal domain (CTD) and their temperature dependences. Effects of temperature on Hsp90 structure are dissected into the C-terminal domain (CTD) and the N-terminal/middle domain (NTMD). One of our major findings reveals that within a narrow temperature window across the physiological temperatures (35 to 45 C), Hsp90CTD exhibits significant increases in protein aggregation and increases in unordered structures. Despite the intrinsically disordered nature of Hsp90CTD, it retains a protected hydrophobic core at 40 C. Implications of these results will be discussed in the light of the structural dynamics and client diversity of Hsp90. AX is grateful for Grant supports from OCAST HR10-078 and NSF MRI DBI1338097.

Steroid resistance in COPD is associated with impaired molecular chaperone Hsp90 expression by pro-inflammatory lymphocytes.

PubMed

Hodge, Greg; Roscioli, Eugene; Jersmann, Hubertus; Tran, Hai B; Holmes, Mark; Reynolds, Paul N; Hodge, Sandra

2016-10-21

Corticosteroid resistance is a major barrier to effective treatment of COPD. We have shown that the resistance is associated with decreased expression of glucocorticoid receptor (GCR) by senescent CD28nullCD8+ pro-inflammatory lymphocytes in peripheral blood of COPD patients. GCR must be bound to molecular chaperones heat shock proteins (Hsp) 70 and Hsp90 to acquire a high-affinity steroid binding conformation, and traffic to the nucleus. We hypothesized a loss of Hsp70/90 from these lymphocytes may further contribute to steroid resistance in COPD. Blood was collected from COPD (n = 10) and aged-matched controls (n = 10). To assess response to steroids, cytotoxic mediators, intracellular pro-inflammatory cytokines, CD28, GCR, Hsp70 and Hsp90 were determined in T and NKT-like cells in the presence of ± 10 μM prednisolone and 2.5 ng/mL cyclosporine A (binds to GCR-Hsp70/90 complex) using flow cytometry, western blot and fluorescence microscopy. A loss of expression of Hsp90 and GCR from CD28null CD8+ T and NKT-like cells in COPD was noted (Hsp70 unchanged). Loss of Hsp90 expression correlated with the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -0.763, p = 0.007 for T-cell IFNγ). Up-regulation of Hsp90 and associated decrease in pro-inflammatory cytokine production was found in CD28nullCD8+ T and NKT-like cells in the presence of 10 μM prednisolone and 2.5 ng/mL cyclosporine A. Loss of Hsp90 from cytotoxic/pro-inflammatory CD28nullCD8+ T and NKT-like cells could contribute to steroid resistance in COPD. Combination prednisolone and low-dose cyclosporine A therapy inhibits these pro-inflammatory cells and may reduce systemic inflammation in COPD.

Dual interaction of scaffold protein Tim44 of mitochondrial import motor with channel-forming translocase subunit Tim23

PubMed Central

Ting, See-Yeun; Yan, Nicholas L; Schilke, Brenda A; Craig, Elizabeth A

2017-01-01

Proteins destined for the mitochondrial matrix are targeted to the inner membrane Tim17/23 translocon by their presequences. Inward movement is driven by the matrix-localized, Hsp70-based motor. The scaffold Tim44, interacting with the matrix face of the translocon, recruits other motor subunits and binds incoming presequence. The basis of these interactions and their functional relationships remains unclear. Using site-specific in vivo crosslinking and genetic approaches in Saccharomyces cerevisiae, we found that both domains of Tim44 interact with the major matrix-exposed loop of Tim23, with the C-terminal domain (CTD) binding Tim17 as well. Results of in vitro experiments showed that the N-terminal domain (NTD) is intrinsically disordered and binds presequence near a region important for interaction with Hsp70 and Tim23. Our data suggest a model in which the CTD serves primarily to anchor Tim44 to the translocon, whereas the NTD is a dynamic arm, interacting with multiple components to drive efficient translocation. DOI: http://dx.doi.org/10.7554/eLife.23609.001 PMID:28440746

Multifaceted Intervention by the Hsp90 Inhibitor Ganetespib (STA-9090) in Cancer Cells with Activated JAK/STAT Signaling

PubMed Central

Proia, David A.; Foley, Kevin P.; Korbut, Tim; Sang, Jim; Smith, Don; Bates, Richard C.; Liu, Yuan; Rosenberg, Alex F.; Zhou, Dan; Koya, Keizo; Barsoum, James; Blackman, Ronald K.

2011-01-01

There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2V617F mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors. PMID:21533169

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

PubMed Central

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

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors.

PubMed

Austin, Carol; Pettit, Simon N; Magnolo, Sharon K; Sanvoisin, Jonathan; Chen, Wenjie; Wood, Stephen P; Freeman, Lauren D; Pengelly, Reuben J; Hughes, Dallas E

2012-08-01

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.

Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation.

PubMed

Münch, Christian; Harper, J Wade

2016-06-30

The mitochondrial matrix is unique in that it must integrate the folding and assembly of proteins derived from the nuclear and mitochondrial genomes. In Caenorhabditis elegans, the mitochondrial unfolded protein response (UPRmt) senses matrix protein misfolding and induces a program of nuclear gene expression, including mitochondrial chaperonins, to promote mitochondrial proteostasis. While misfolded mitochondrial-matrix-localized ornithine transcarbamylase induces chaperonin expression, our understanding of mammalian UPRmt is rudimentary, reflecting a lack of acute triggers for UPRmt activation. This limitation has prevented analysis of the cellular responses to matrix protein misfolding and the effects of UPRmt on mitochondrial translation to control protein folding loads. Here we combine pharmacological inhibitors of matrix-localized HSP90/TRAP1 (ref. 8) or LON protease, which promote chaperonin expression, with global transcriptional and proteomic analysis to reveal an extensive and acute response of human cells to UPRmt. This response encompasses widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. Functional studies revealed rapid but reversible translation inhibition in mitochondria occurring concurrently with defects in pre-RNA processing caused by transcriptional repression and LON-dependent turnover of the mitochondrial pre-RNA processing nuclease MRPP3 (ref. 10). This study reveals that acute mitochondrial protein folding stress activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition, and provides a framework for further dissection of mammalian UPRmt.

Serine/Threonine Kinase Unc-51-like Kinase-1 (Ulk1) Phosphorylates the Co-chaperone Cell Division Cycle Protein 37 (Cdc37) and Thereby Disrupts the Stability of Cdc37 Client Proteins.

PubMed

Li, Ran; Yuan, Fengjie; Fu, Wan; Zhang, Luyao; Zhang, Nan; Wang, Yanan; Ma, Ke; Li, Xue; Wang, Lina; Zhu, Wei-Guo; Zhao, Ying

2017-02-17

The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (Cdc37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in Cdc37 compromised the recruitment of client kinases to a complex comprising Cdc37 and heat shock protein 90 (Hsp90) but only modestly affected Cdc37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of Cdc37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-Cdc37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Systematic Protocol for the Characterization of Hsp90 Modulators

PubMed Central

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.

2015-01-01

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

Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts

PubMed Central

Chan, Wen-Hsiung

2007-01-01

The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras→ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 μM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras→ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo. PMID:17331071

Evaluation of host Hsp(s) as potential biomarkers for the diagnosis of tuberculous meningitis.

PubMed

Shekhawat, Seema D; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

2016-01-01

Diagnosis of tuberculosis meningitis (TBM) remains challenging in tuberculosis (TB) endemic countries. The need for TB biomarkers arises, in part, from the difficulty of accurately diagnosing TBM with the available methods. To explore the potential of host Hsps (Hsp 25, Hsp 60, Hsp 70 and Hsp 90) as an alternative marker in TBM diagnosis, we evaluated cerebrospinal fluid (CSF) sample of TBM (n=49), Pyogenic Meningitis (PM) (n=20), Viral Meningitis (VM) (n=09), Fungal Meningitis (FM) (n=04) and non infectious control (n=79) patients using indirect ELISA. Out of four Hsps, Hsp 70 and Hsp 90 yields 89% & 88% sensitivity and 82% & 89% specificity, respectively. The positive (PPV) and negative (NPV) predictive values yielded in TBM group for Hsp 70 was 86.27% (73.74-94.27) and 93.51% (85.48-97.83), respectively. For Hsp 90 the obtained PPV was 89.36% (76.88-96.41) and NPV was 91.36% (82.99-96.44). In 86% of TBM patients all the four Hsps were found to be positive and none of the patient was found to be negative for all Hsps in the same group. The data presented in the study indicate that host Hsp 70 and Hsp 90 shows good sensitivity and specificity and have potential in the diagnosis of TBM disease. The combined use of all Hsps (Hsp 25, Hsp 60, Hsp 70 and Hsp 90) effectively distinguishes patients with TBM from other disease controls. Copyright © 2015 Elsevier B.V. All rights reserved.

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. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90.

PubMed

Oroz, Javier; Kim, Jin Hae; Chang, Bliss J; Zweckstetter, Markus

2017-04-01

The critical toxic species in over 40 human diseases are misfolded proteins. Their interaction with molecular chaperones such as Hsp90, which preferentially interacts with metastable proteins, is essential for the blocking of disease progression. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the three-dimensional structure of the misfolded cytotoxic monomer of the amyloidogenic human protein transthyretin, which is characterized by the release of the C-terminal β-strand and perturbations of the A-B loop. The misfolded transthyretin monomer, but not the wild-type protein, binds to human Hsp90. In the bound state, the Hsp90 dimer predominantly populates an open conformation, and transthyretin retains its globular structure. The interaction surface for the transthyretin monomer comprises the N-terminal and middle domains of Hsp90 and overlaps with that of the Alzheimer's-disease-related protein tau. Taken together, the data suggest that Hsp90 uses a mechanism for the recognition of aggregation-prone proteins that is largely distinct from those of other Hsp90 clients.

The crystal structure of the Hsp90 co-chaperone Cpr7 from Saccharomyces cerevisiae.

PubMed

Qiu, Yu; Ge, Qiangqiang; Wang, Mingxing; Lv, Hui; Ebrahimi, Mohammad; Niu, Liwen; Teng, Maikun; Li, Xu

2017-03-01

The versatility of Hsp90 can be attributed to the variety of co-chaperone proteins that modulate the role of Hsp90 in many cellular processes. As a co-chaperone of Hsp90, Cpr7 is essential for accelerating the cell growth in an Hsp90-containing trimeric complex. Here, we report the crystal structure of Cpr7 at a resolution of 1.8Å. It consists of an N-terminal PPI domain and a C-terminal TPR domain, and exhibits a U-shape conformation. Our studies revealed the aggregation state of Cpr7 in solution and the interaction properties between Cpr7 and the MEEVD sequence from the C-terminus of Hsp90. In addition, the structure and sequence analysis between Cpr7 and homologues revealed the structure basis both for the function differences between Cpr6 and Cpr7 and the functional complements between Cns1 and Cpr7. Our studies facilitate the understanding of Cpr7 and provide decent insights into the molecular mechanisms of the Hsp90 co-chaperone pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Little effect of HSP90 inhibition on the quantitative wing traits variation in Drosophila melanogaster.

PubMed

Takahashi, Kazuo H

2017-02-01

Drosophila wings have been a model system to study the effect of HSP90 on quantitative trait variation. The effect of HSP90 inhibition on environmental buffering of wing morphology varies among studies while the genetic buffering effect of it was examined in only one study and was not detected. Variable results so far might show that the genetic background influences the environmental and genetic buffering effect of HSP90. In the previous studies, the number of the genetic backgrounds used is limited. To examine the effect of HSP90 inhibition with a larger number of genetic backgrounds than the previous studies, 20 wild-type strains of Drosophila melanogaster were used in this study. Here I investigated the effect of HSP90 inhibition on the environmental buffering of wing shape and size by assessing within-individual and among-individual variations, and as a result, I found little or very weak effects on environmental and genetic buffering. The current results suggest that the role of HSP90 as a global regulator of environmental and genetic buffering is limited at least in quantitative traits.

Maximizing the Therapeutic Potential of Hsp90 Inhibitors

PubMed Central

Butler, Lisa M.; Ferraldeschi, Roberta; Armstrong, Heather K.; Centenera, Margaret M.; Workman, Paul

2015-01-01

Hsp90 is required for maintaining the stability and activity of a diverse group of client proteins, including protein kinases, transcription factors and steroid hormone receptors involved in cell signaling, proliferation, survival, oncogenesis and cancer progression. Inhibition of Hsp90 alters the Hsp90-client protein complex, leading to reduced activity, misfolding, ubiquitination and, ultimately, proteasomal degradation of client proteins. Hsp90 inhibitors have demonstrated significant antitumor activity in a wide variety of preclinical models with evidence of selectivity for cancer versus normal cells. In the clinic however, the efficacy of this class of therapeutic agents has been relatively limited to date, with promising responses mainly observed in breast and lung cancer, but no major activity seen in other tumor types. In addition, adverse events and some significant toxicities have been documented. Key to improving these clinical outcomes is a better understanding of the cellular consequences of inhibiting Hsp90 that may underlie treatment response or resistance. This review considers the recent progress that has been made in the study of Hsp90 and its inhibitors, and highlights new opportunities to maximize their therapeutic potential. PMID:26219697

Sulforaphane inhibits pancreatic cancer through disrupting Hsp90-p50(Cdc37) complex and direct interactions with amino acids residues of Hsp90.

PubMed

Li, Yanyan; Karagöz, G Elif; Seo, Young Ho; Zhang, Tao; Jiang, Yiqun; Yu, Yanke; Duarte, Afonso M S; Schwartz, Steven J; Boelens, Rolf; Carroll, Kate; Rüdiger, Stefan G D; Sun, Duxin

2012-12-01

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl) butane)], an isothiocyanate derived from cruciferous vegetables, has been shown to possess potent chemopreventive activity. We analyzed the effect of sulforaphane on the proliferation of pancreatic cancer cells. Sulforaphane inhibited pancreatic cancer cell growth in vitro with IC(50)s of around 10-15 μM and induced apoptosis. In pancreatic cancer xenograft mouse model, administration of sulforaphane showed remarkable inhibition of tumor growth without apparent toxicity noticed. We found that sulforaphane induced the degradation of heat shock protein 90 (Hsp90) client proteins and blocked the interaction of Hsp90 with its cochaperone p50(Cdc37) in pancreatic cancer cells. Using nuclear magnetic resonance spectroscopy (NMR) with an isoleucine-specific labeling strategy, we overcame the protein size limit of conventional NMR and studied the interaction of sulforaphane with full-length Hsp90 dimer (170 kDa) in solution. NMR revealed multiple chemical shifts in sheet 2 and the adjacent loop in Hsp90 N-terminal domain after incubation of Hsp90 with sulforaphane. Liquid chromatography coupled to mass spectrometry further mapped a short peptide in this region that was tagged with sulforaphane. These data suggest a new mechanism of sulforaphane that disrupts protein-protein interaction in Hsp90 complex for its chemopreventive activity. Copyright © 2012 Elsevier Inc. All rights reserved.

A C-terminal HSP90 inhibitor restores glucocorticoid sensitivity and relieves a mouse allograft model of Cushing disease.

PubMed

Riebold, Mathias; Kozany, Christian; Freiburger, Lee; Sattler, Michael; Buchfelder, Michael; Hausch, Felix; Stalla, Günter K; Paez-Pereda, Marcelo

2015-03-01

One function of the glucocorticoid receptor (GR) in corticotroph cells is to suppress the transcription of the gene encoding proopiomelanocortin (POMC), the precursor of the stress hormone adrenocorticotropin (ACTH). Cushing disease is a neuroendocrine condition caused by partially glucocorticoid-resistant corticotroph adenomas that excessively secrete ACTH, which leads to hypercortisolism. Mutations that impair GR function explain glucocorticoid resistance only in sporadic cases. However, the proper folding of GR depends on direct interactions with the chaperone heat shock protein 90 (HSP90, refs. 7,8). We show here that corticotroph adenomas overexpress HSP90 compared to the normal pituitary. N- and C-terminal HSP90 inhibitors act at different steps of the HSP90 catalytic cycle to regulate corticotroph cell proliferation and GR transcriptional activity. C-terminal inhibitors cause the release of mature GR from HSP90, which promotes its exit from the chaperone cycle and potentiates its transcriptional activity in a corticotroph cell line and in primary cultures of human corticotroph adenomas. In an allograft mouse model, the C-terminal HSP90 inhibitor silibinin showed anti-tumorigenic effects, partially reverted hormonal alterations, and alleviated symptoms of Cushing disease. These results suggest that the pathogenesis of Cushing disease caused by overexpression of heat shock proteins and consequently misregulated GR sensitivity may be overcome pharmacologically with an appropriate HSP90 inhibitor.

Sulforaphane inhibits pancreatic cancer through disrupting Hsp90-p50Cdc37 complex and direct interactions with amino acids residues of Hsp90

PubMed Central

Li, Yanyan; Karagöz, G. Elif; Seo, Young Ho; Zhang, Tao; Jiang, Yiqun; Yu, Yanke; Duarte, Afonso M.S.; Schwartz, Steven J.; Boelens, Rolf; Carroll, Kate; Rüdiger, Stefan G. D.; Sun, Duxin

2011-01-01

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl) butane)], an isothiocyanate derived from cruciferous vegetables, has been shown to possess potent chemopreventive activity. We analyzed the effect of sulforaphane on the proliferation of pancreatic cancer cells. Sulforaphane inhibited pancreatic cancer cell growth in vitro with the IC50's around 10-15 μM and induced apoptosis. In pancreatic cancer xenograft mouse model, administration of sulforaphane showed remarkable inhibition of tumor growth without apparent toxicity noticed. We found that sulforaphane induced the degradation of heat shock protein 90 (Hsp90) client proteins and blocked the interaction of Hsp90 with its cochaperone p50Cdc37 in pancreatic cancer cells. Using Nuclear Magnetic Resonance Spectroscopy (NMR) with an isoleucine-specific labeling strategy, we overcame the protein size limit of conventional NMR and studied the interaction of sulforaphane with full-length Hsp90 dimer (170 kDa) in solution. NMR revealed multiple chemical shifts in sheet 2 and the adjacent loop in Hsp90 N-terminal domain after incubation of Hsp90 with sulforaphane. Liquid Chromatography coupled to Mass Spectrometry (LC-MS) further mapped a short peptide in this region that was tagged with sulforaphane. These data suggest a new mechanism of sulforaphane that disrupts protein-protein interaction in Hsp90 complex for its chemopreventive activity. PMID:22444872

Surface heat shock protein 90 serves as a potential receptor for calcium oxalate crystal on apical membrane of renal tubular epithelial cells.

PubMed

Fong-Ngern, Kedsarin; Sueksakit, Kanyarat; Thongboonkerd, Visith

2016-07-01

Adhesion of calcium oxalate monohydrate (COM) crystals on renal tubular epithelial cells is a crucial step in kidney stone formation. Finding potential crystal receptors on the apical membrane of the cells may lead to a novel approach to prevent kidney stone disease. Our previous study identified a large number of crystal-binding proteins on the apical membrane of MDCK cells. However, their functional role as potential crystal receptors had not been validated. The present study aimed to address the potential role of heat shock protein 90 (HSP90) as a COM crystal receptor. The apical membrane was isolated from polarized MDCK cells by the peeling method and recovered proteins were incubated with COM crystals. Western blot analysis confirmed the presence of HSP90 in the apical membrane and the crystal-bound fraction. Immunofluorescence staining without permeabilization and laser-scanning confocal microscopy confirmed the surface HSP90 expression on the apical membrane of the intact cells. Crystal adhesion assay showed that blocking surface HSP90 by specific anti-HSP90 antibody and knockdown of HSP90 by small interfering RNA (siRNA) dramatically reduced crystal binding on the apical surface of MDCK cells (by approximately 1/2 and 2/3, respectively). Additionally, crystal internalization assay revealed the presence of HSP90 on the membrane of endocytic vesicle containing the internalized COM crystal. Moreover, pretreatment of MDCK cells with anti-HSP90 antibody significantly reduced crystal internalization (by approximately 1/3). Taken together, our data indicate that HSP90 serves as a potential receptor for COM crystals on the apical membrane of renal tubular epithelial cells and is involved in endocytosis/internalization of the crystals into the cells.

A Mechanistic and Structural Analysis of the Inhibition of the 90-kDa Heat Shock Protein by the Benzoquinone and Hydroquinone AnsamycinsS⃞

PubMed Central

Reigan, Philip; Siegel, David; Guo, Wenchang

2011-01-01

The benzoquinone ansamycins inhibit the ATPase activity of the 90-kDa heat shock protein (Hsp90), disrupting the function of numerous client proteins involved in oncogenesis. In this study, we examine the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the metabolism of trans- and cis-amide isomers of the benzoquinone ansamycins and their mechanism of Hsp90 inhibition. Inhibition of purified human Hsp90 by a series of benzoquinone ansamycins was examined in the presence and absence of NQO1, and their relative rate of NQO1-mediated reduction was determined. Computational-based molecular docking simulations indicated that the trans- but not the cis-amide isomers of the benzoquinone ansamycins could be accommodated by the NQO1 active site, and the ranking order of binding energies correlated with the relative reduction rate using purified human NQO1. The trans-cis isomerization of the benzoquinone ansamycins in Hsp90 inhibition has been disputed in recent reports. Previous computational studies have used the closed or cocrystallized Hsp90 structures in an attempt to explore this isomerization step; however, we have successfully docked both the trans- and cis-amide isomers of the benzoquinone ansamycins into the open Hsp90 structure. The results of these studies indicate that both trans- and cis-amide isomers of the hydroquinone ansamycins exhibited increased binding affinity for Hsp90 relative to their parent quinones. Our data support a mechanism in which trans- rather than cis-amide forms of benzoquinone ansamycins are metabolized by NQO1 to hydroquinone ansamycins and that Hsp90-mediated trans-cis isomerization via tautomerization plays an important role in subsequent Hsp90 inhibition. PMID:21285336

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

PubMed

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

2014-10-17

Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Assessment of heat shock protein (HSP60, HSP72, HSP90, and HSC70) expression in cultured limbal stem cells following air lifting

PubMed Central

Mohammadi, Parvaneh; Daryadel, Arezoo; Baharvand, Hossein

2010-01-01

Objectives The aim of this study is to create an ex vivo model to examine the expression of major heat-shock protein (HSP) families; HSP60, HSP72, and HSP90, and heat-shock cognate 70 (HCS70) at the mRNA and protein level in differentiating corneal cells from limbal stem cells (LSC) following air exposure. Methods Limbal biopsies taken from cadaveric normal human limbus were cultivated as explants on human amniotic membrane (HAM) and plastic dish (PD). Corneal differentiation was induced by air lifting for 16 days. The expression of putative LSC markers (P63 and ATP-binding cassette G2 [ABCG2]), corneal markers (keratin 3 [K3/12] and connexin 43 [CX43]), and HSP60, HSP72, HSP90, and HSC70 were tested by RT–PCR, immunofluorescence, and flow cytometry pre- and post-air exposure. Fresh limbal and corneal tissues were used as control groups. Results Air lifting induced corneal differentiation with a decrease in the number of P63+ cells and an increase in the number of K3+/CX43+ cells, which characterized transient amplifying cells (TACs). Moreover, denuded HAM provided a superior niche for LSC proliferation and phenotype maintenance in vitro. Additionally, we have evidence that expressions of HSC70 as well as HSP72 were enhanced through corneal differentiation and HSP90 post-air lifting in vitro and in vivo. HSP60, however, was not detected in either LSC or corneal cells, in vivo and in vitro. Conclusions These results suggest that corneal differentiation following air exposure may regulate HSP72 and HSC70 expression. In addition, HSP72 and HSP90 may protect LSC and corneal cells against oxidative stress. PMID:20806039

Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction.

PubMed

Dhamad, Ahmed E; Zhou, Zhenqi; Zhou, Jianhong; Du, Yuchun

2016-01-01

Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90β) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2-3 times more abundant than Hsp90β in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17β-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET.

PubMed

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-05-29

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.

Expression Patterns of Three Genes Under Short and Long Term Cold Exposure in Thitarodes pui (Lepidoptera: Hepialidae), A Host of Ophiocordyceps sinensis.

PubMed

Min, Q; Cheng, S Y; Xi, J F; Ma, J; Xin, T R; Xia, B; Zou, Z W

　　BACKGROUND: Thitarodes larvae are the host of the caterpillar fungus Ophiocordyceps sinensis. Low temperature is the main environmental limitation for larvae growth. To better understand the cold adaption process in T. pui larvae, the expression patterns of trehalose-6-phosphate synthase (TpTPS), heat shock protein 70 (TpHSP70), and heat shock protein 90 (TpHSP90) were investigated upon short and long-term exposure to 0°C. The 6th instar T. pui larvae were collected in July 2013. TpTPS was firstly sequenced and expression patterns of TpTPS, TpHSP70 and TpHSP90 were investigated using quantitative PCR. Full-length cDNA of TpTPS was 3,012 bp, with an open reading frame of 2,472 bp and an encoding protein of 823 amino acids. TpTPS up-regulation was induced by cold exposure. TpHSP70 expression is altered by cold exposure, but remained low. TpHSP90 expression was obviously up regulated in long-term cold stimulation. All three genes (TpTPS, TpHSP70 and TpHSP90) have likely contributed to cold tolerance in T. pui larvae, TpTPS and TpHSP90 potentially being more important.

Characterization of novel heat-responsive transcription factor (TaHSFA6e) gene involved in regulation of heat shock proteins (HSPs) - A key member of heat stress-tolerance network of wheat.

PubMed

Kumar, Ranjeet R; Goswami, Suneha; Singh, Khushboo; Dubey, Kavita; Rai, Gyanendra K; Singh, Bhupinder; Singh, Shivdhar; Grover, Monendra; Mishra, Dwijesh; Kumar, Sanjeev; Bakshi, Suman; Rai, Anil; Pathak, Himanshu; Chinnusamy, Viswanathan; Praveen, Shelly

2018-08-10

Heat stress has an adverse effect on the quality and quantity of agriculturally important crops, especially wheat. The tolerance mechanism has not been explored much in wheat and very few genes/ TFs responsive to heat stress is available on public domain. Here, we identified, cloned and characterized a putative TaHSFA6e TF gene of 1.3 kb from wheat cv. HD2985. We observed an ORF of 368 aa with Hsf DNA binding signature domain in the amino acid sequence. Single copy number of TaHSFA6e was observed integrated in the genome of wheat. Expression analysis of TaHSFA6e under differential HS showed maximum transcripts in wheat cv. Halna (thermotolerant) in response to 38 °C for 2 h during pollination and grain-filling stages, as compared to PBW343, HD2329 and HD2985. Putative target genes of TaHSFA6e (HSP17, HSP70 and HSP90) showed upregulation in response to differential HS (30 & 38 °C, 2 h) during pollination and grain-filling stages. Small HSP17 was observed most triggered in Halna under HS. We observed increase in the catalase, guaiacol peroxidase, total antioxidant capacity (TAC), and decrease in the lipid peroxidation in thermotolerant cvs. (Halna, HD2985), as compared to thermosusceptible (PBW343, HD2329) under differential HS. Multiple stresses (heat - 38 °C, 2 h, and drought - 100 mL of 20% polyethylene Glycol 6000) during seedling stage of wheat showed positive correlation between the expression of TaHSFA6e, putative targets (HSP70, HSP90, HSP17) and TAC. Halna (thermotolerant) performed better, as compared to other contrasting cvs. TaHSFA6e TF can be used as promising candidate gene for manipulating the heat stress-tolerance network. Copyright © 2018 Elsevier B.V. All rights reserved.

Alteration of Fatty-Acid-Metabolizing Enzymes Affects Mitochondrial Form and Function in Hereditary Spastic Paraplegia

PubMed Central

Tesson, Christelle; Nawara, Magdalena; Salih, Mustafa A.M.; Rossignol, Rodrigue; Zaki, Maha S.; Al Balwi, Mohammed; Schule, Rebecca; Mignot, Cyril; Obre, Emilie; Bouhouche, Ahmed; Santorelli, Filippo M.; Durand, Christelle M.; Oteyza, Andrés Caballero; El-Hachimi, Khalid H.; Al Drees, Abdulmajeed; Bouslam, Naima; Lamari, Foudil; Elmalik, Salah A.; Kabiraj, Mohammad M.; Seidahmed, Mohammed Z.; Esteves, Typhaine; Gaussen, Marion; Monin, Marie-Lorraine; Gyapay, Gabor; Lechner, Doris; Gonzalez, Michael; Depienne, Christel; Mochel, Fanny; Lavie, Julie; Schols, Ludger; Lacombe, Didier; Yahyaoui, Mohamed; Al Abdulkareem, Ibrahim; Zuchner, Stephan; Yamashita, Atsushi; Benomar, Ali; Goizet, Cyril; Durr, Alexandra; Gleeson, Joseph G.; Darios, Frederic; Brice, Alexis; Stevanin, Giovanni

2012-01-01

Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function. PMID:23176821

Mutation of Hip’s Carboxy-Terminal Region Inhibits a Transitional Stage of Progesterone Receptor Assembly

PubMed Central

Prapapanich, Viravan; Chen, Shiying; Smith, David F.

1998-01-01

Steroid receptor complexes are assembled through an ordered, multistep pathway involving multiple components of the cytoplasmic chaperone machinery. Two of these components are Hsp70-binding proteins, Hip and Hop, that have some limited homology in their C-terminal regions, outside the sequences mapped for Hsp70 binding. Within this region of Hip is a DPEV sequence that occurs twice; in Hop, one DPEV sequence plus a partial second sequence occurs. In an effort to better understand Hip function as it relates to assembly of progesterone receptor complexes, the DPEV region of Hip was targeted for mutations. Each DPEV sequence was mutated to an APAV sequence, singly or in combination. The combined mutation, APAV2, was further combined with a deletion of Hip’s tetratricopeptide repeat region that is required for Hsp70 binding or with a deletion of Hip’s GGMP repeat. An additional mutant was prepared by truncation of Hip’s DPEV-containing C terminus. By comparing interactions of various Hip forms with Hsp70, it was determined that mutation of the DPEV sequences created a dominant inhibitory form of Hip. The mutant Hip-Hsp70 complex was not prevented from interacting with progesterone receptor, but the mutant caused a dose-dependent inhibition of receptor assembly with Hsp90. The behavior of the Hip mutant is consistent with a model in which Hip and Hop are required to facilitate the transition from an early receptor complex with Hsp70 into later complexes containing Hsp90. PMID:9447991

The role of heat shock protein 70 (Hsp70) in radiation-induced immunomodulation.

PubMed

Multhoff, Gabriele; Pockley, Alan G; Schmid, Thomas E; Schilling, Daniela

2015-11-28

Despite enormous progress in radiation technologies (high precision image-guided irradiation, proton irradiation, heavy ion irradiation) and radiotherapeutic concepts (hypofractionated irradiation schemes), the clinical outcome of radiotherapy in locally advanced and metastasized tumors and in hypoxic tumors which are radiation-resistant remains unsatisfactory. Given their key influence on a number of biological and immunological parameters, this article considers the influence of irradiation-induced stress proteins on radiation-induced immunomodulation. Depending on its location, the major stress-inducible Heat shock protein 70 (Hsp70) has been found to fulfill multiple roles. On the one hand, increased intracellular Hsp70 levels have been found to play a key role in the recovery from stress such as radio(chemo)therapy, and on the other hand extracellular Hsp70 proteins are potent stimulators of the innate immune system and mediators of anti-tumor immunity. Furthermore, if loaded with tumor-derived peptides, members of the Heat Shock Protein 70 (HSP70) and 90 (HSP90) families can stimulate the adaptive immune system via antigen cross-presentation. An irradiation-induced enhancement of the selective expression of a membrane form of Hsp70 on the surface of tumor cells which can act as a recognition structure for activated NK cells might have significant clinical relevance, in that the outcome of irradiation therapy for advanced tumors could be improved by combining it with cell-based and other immunotherapies that target this membrane form of Hsp70. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Identification of a heat shock protein 90 gene involved in resistance to temperature stress in two wing-morphs of Nilaparvata lugens (Stål).

PubMed

Lu, Kai; Chen, Xia; Liu, Wenting; Zhou, Qiang

2016-07-01

The brown planthopper, Nilaparvata lugens, is one of the most destructive pests damaging rice in Asia and exhibits wing dimorphism, with brachypters possessing severely reduced wings and macropters bearing fully developed wings. Previous studies have shown that macropters are more heat resistant than brachypters. To understand the molecular mechanism underlying the differential thermotolerance abilities of these two morphs, a full-length Hsp gene, NlHsp90 was cloned from N. lugen. Our results showed that the relative expression levels of NlHsp90 in N. lugens females increased with the rise of temperature. Interestingly, NlHsp90 in macropters females could be induced at lower temperature (32°C) than that in brachypters (34°C), and the NlHsp90 mRNA levels in macropters were significantly higher than those in brachypters from 34 to 40°C. In addition, the maximum expression levels of NlHsp90 were achieved much earlier in macropters, and NlHsp90 mRNA levels in macropters were significantly higher than those in brachypters from 1 to 6h of recovery after temperature stress. Furthermore, knockdown of NlHsp90 by dsRNA injection reduced survival in both morphs with a greater reduction in the macropters relative to that of the brachyters. These results indicated that NlHsp90 plays an important role for thermotolerance in N. lugens, and there is difference on induction between two morphs. Copyright © 2016 Elsevier Inc. All rights reserved.

A Non-ATP Competitive Inhibitor of BCR-ABL for the Therapy of Imatinib-Resistant Cmls

DTIC Science & Technology

2008-05-01

HSP90 members (e.g. HSP70 and HSP27 ) help maintain the macromolecular complex that assembled the Bcr-Abl/Jak2 signaling Network. Our published papers 4...kinase, Akt and GSK3 beta. Our recent findings suggest that the HSP90/HSP70/ HSP27 chaperone proteins are also part of this Network, and may play a

Effectively delivering a unique hsp90 inhibitor using star polymers.

PubMed

Kim, Seong Jong; Ramsey, Deborah M; Boyer, Cyrille; Davis, Thomas P; McAlpine, Shelli R

2013-07-25

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 comprised of 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 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.

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

Hsp90 dependence of a kinase is determined by its conformational landscape

PubMed Central

Luo, Qi; Boczek, Edgar E.; Wang, Qi; Buchner, Johannes; Kaila, Ville R. I.

2017-01-01

Heat shock protein 90 (Hsp90) is an abundant molecular chaperone, involved in the folding and activation of 60% of the human kinome. The oncogenic tyrosine kinase v-Src is one of the most stringent client proteins of Hsp90, whereas its almost identical homolog c-Src is only weakly affected by the chaperone. Here, we perform atomistic molecular simulations and in vitro kinase assays to explore the mechanistic differences in the activation of v-Src and c-Src. While activation in c-Src is strictly controlled by ATP-binding and phosphorylation, we find that activating conformational transitions are spontaneously sampled in Hsp90-dependent Src mutants. Phosphorylation results in an enrichment of the active conformation and in an increased affinity for Hsp90. Thus, the conformational landscape of the mutated kinase is reshaped by a broken “control switch”, resulting in perturbations of long-range electrostatics, higher activity and increased Hsp90-dependence. PMID:28290541

Effect of thermal stress on HSP90 expression of Bali cattle in Barru district, South Sulawesi

NASA Astrophysics Data System (ADS)

Aritonang, S. B.; Yuniati, R.; Abinawanto, Imron, M.; Bowolaksono, A.

2017-07-01

Heat shock protein 90-kDa is induced stress protein that expressed in response to stress and play crucial roles in environmental stress tolerance and adaptation. This study aimed to determine effect of environmental heat stress on the HSP90 expression of Bali cattle. Heat stress was measured by temperature humidity index in the morning and evening across 5-days on August 2016. The blood samples of Bali cattle were taken from venous jungularis. HSP90 was derived from RNA isolation of whole blood then was followed reverse transcription two steps. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to analyze the transcript variants of HSP90, followed by comparative ΔΔCt to determine HSP90 expression. The results of temperature and humidity index (THI) measurement indicated THI on afternoon was higher than in the morning. The difference in environmental conditions in the morning and afternoon effected changes on rectal temperature but neither did on Hsp90 expression.

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.

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. © 2014 FEBS.

Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

PubMed Central

Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

2014-01-01

In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

HSP90 gene expression induced by aspirin is associated with damage remission in a chicken myocardial cell culture exposed to heat stress.

PubMed

Zhang, X; Qian, Z; Zhu, H; Tang, S; Wu, D; Zhang, M; Kemper, N; Hartung, J; Bao, E

2016-08-01

To understand the potential protection of heat shock protein 90 (HSP90) induced by aspirin against heat stress damage in chicken myocardial cells, enzyme activities related to stress damage, cytopathological changes, the expression and distribution of HSP90, and HSP90 mRNA levels in the myocardial cells exposed to heat stress (42°C) for different durations with or without aspirin administration (1 mg/ml, 2 h prior) in vitro were investigated. Significant increase of enzyme levels in the supernatant of heat-stressed myocardial cells and cellular lesions characterised by acute degeneration, karyopyknosis and karyorrhexis were observed, compared to non-treated cells. However, the lesions of cells treated with aspirin were milder, characterised by earlier recovery of enzyme levels to the control levels and no obvious heat stress-related cellular necrosis. Stronger positive signals in the cytoplasm and longer retention of HSP90 signal in nuclei were observed in aspirin-treated myocardial cells than those of only heat-stressed cells. HSP90 level in the aspirin-treated myocardial cells was 11.1-fold higher than that in non-treated cells, and remained at a high level at the early stage of heat stress, whereas it was just 4.1-fold higher in only heat-stressed cells and returned rapidly to a low level. Overexpression of HSP90 mRNA in aspirin-treated cells was observed throughout the experiment, whereas HSP90 mRNA decreased significantly only in heat-stressed cells. The early higher HSP90 expression induced by aspirin during heat stress was accompanied by decreased heat stress damage, suggesting that aspirin might play an important role in preventing myocardial cells from heat stress damage in vitro.

Heat Shock Proteins Promote Cancer: It's a Protection Racket.

PubMed

Calderwood, Stuart K; Gong, Jianlin

2016-04-01

Heat shock proteins (HSP) are expressed at high levels in cancer and form a fostering environment that is essential for tumor development. Here, we review the recent data in this area, concentrating mainly on Hsp27, Hsp70, and Hsp90. The overriding role of HSPs in cancer is to stabilize the active functions of overexpressed and mutated cancer genes. Thus, elevated HSPs are required for many of the traits that underlie the morbidity of cancer, including increased growth, survival, and formation of secondary cancers. In addition, HSPs participate in the evolution of cancer treatment resistance. HSPs are also released from cancer cells and influence malignant properties by receptor-mediated signaling. Current data strongly support efforts to target HSPs in cancer treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Overexpression of Three Heat Shock Proteins Protects Monochamus alternatus (Coleoptera: Cerambycidae) From Thermal Stress

PubMed Central

Cai, Ziling; Chen, Jingxiang; Cheng, Jie

2017-01-01

Abstract Ambient temperature is an important factor limiting the abundance and distribution of insects, and heat shock protein (Hsp) gene expression is sensitive to extremes of cold and heat. In order to explore the role of Hsps during thermal stress and development in Monochamus alternatus Hope (Coleoptera: Cerambycidae), we cloned and characterized full-length Hsp genes, including MaHsp60, MaHsp70, and MaHsp90. M. alternatus were exposed to different temperatures (−15, −5, 5, 15, 25, 35, and 40℃) for 1 h and was allowed to recover at 25℃ for 1 h. Following the treatments, we investigated the expression of the Hsps by quantitative real-time polymerase chain reaction. In third instar larvae, MaHsp60, MaHsp70, and MaHsp90 expression was upregulated in response to cold and heat, but the three Hsps were especially sensitive to heat, specifically at 35℃ and 40℃. After heating M. alternatus to 35℃, the expression of MaHsp60, MaHsp70, and MaHsp90 was higher than at 5℃ and 25℃ in nearly all developmental stages. MaHsp60, MaHsp70, and MaHsp90 expression was highest in later pupal, early adult, and early adult stages, respectively. These results suggest that compared with normal ambient temperatures, thermal stress could induce high expression of the three Hsps.

Caffeine Induces the Stress Response and Up-Regulates Heat Shock Proteins in Caenorhabditis elegans.

PubMed

Al-Amin, Mohammad; Kawasaki, Ichiro; Gong, Joomi; Shim, Yhong-Hee

2016-02-01

Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner. C. elegans has been used as an animal model to investigate the effects of caffeine on development. Caffeine treatment at a high dose (30 mM) showed detrimental effects and caused early larval arrest. We performed a comparative proteomic analysis to investigate the mode of action of high-dose caffeine treatment in C. elegans and found that the stress response proteins, heat shock protein (HSP)-4 (endoplasmic reticulum [ER] chaperone), HSP-6 (mitochondrial chaperone), and HSP-16 (cytosolic chaperone), were induced and their expression was regulated at the transcriptional level. These findings suggest that high-dose caffeine intake causes a strong stress response and activates all three stress-response pathways in the worms, including the ER-, mitochondrial-, and cytosolic pathways. RNA interference of each hsp gene or in triple combination retarded growth. In addition, caffeine treatment stimulated a food-avoidance behavior (aversion phenotype), which was enhanced by RNAi depletion of the hsp-4 gene. Therefore, up-regulation of hsp genes after caffeine treatment appeared to be the major responses to alleviate stress and protect against developmental arrest.

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases

PubMed Central

Bloyet, Louis-Marie; Welsch, Jérémy; Enchery, François; Mathieu, Cyrille; de Breyne, Sylvain

2016-01-01

ABSTRACT Nonsegmented negative-stranded RNA viruses, or members of the order Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have coevolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes the L protein through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-dimethylaminoethylamino-17-demethoxygeldanamycin as specific inhibitors of cellular heat shock protein 90 (HSP90), we found that efficient chaperoning of L by HSP90 requires P in the measles, Nipah, and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L, as evidenced by a luciferase reporter insert fused within measles virus L. HSP90 acts as a true chaperon; its activity is transient and dispensable for the activity of measles and Nipah virus polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order. IMPORTANCE Viruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus, successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, while P protein binds to the N terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded, and functional L proteins. Once formed, the mature P+L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved in several members of the Mononegavirales order, including the Nipah and vesicular stomatitis viruses. PMID:27170753

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases.

PubMed

Bloyet, Louis-Marie; Welsch, Jérémy; Enchery, François; Mathieu, Cyrille; de Breyne, Sylvain; Horvat, Branka; Grigorov, Boyan; Gerlier, Denis

2016-08-01

Nonsegmented negative-stranded RNA viruses, or members of the order Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have coevolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes the L protein through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-dimethylaminoethylamino-17-demethoxygeldanamycin as specific inhibitors of cellular heat shock protein 90 (HSP90), we found that efficient chaperoning of L by HSP90 requires P in the measles, Nipah, and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L, as evidenced by a luciferase reporter insert fused within measles virus L. HSP90 acts as a true chaperon; its activity is transient and dispensable for the activity of measles and Nipah virus polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order. Viruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus, successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, while P protein binds to the N terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded, and functional L proteins. Once formed, the mature P+L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved in several members of the Mononegavirales order, including the Nipah and vesicular stomatitis viruses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The level of heat shock protein 90 in pig Longissimus dorsi muscle and its relationship with meat pH and quality.

PubMed

Zhang, Muhan; Wang, Daoying; Geng, Zhiming; Bian, Huan; Liu, Fang; Zhu, Yongzhi; Xu, Weimin

2014-12-15

The 90 kDa heat shock protein (HSP90) is a molecular chaperone that participates in various cellular processes, the role and significance of HSP90 in postmortem muscle though remains unclear. In the present study, pig Longissimus dorsi muscles, categorized into three pH groups, were tested for HSP90 levels and meat quality parameters (i.e. water holding capacity, colour, tenderness and lipid oxidation). The muscles with a high initial pH (pHi) group (pH>6.4) possessing the greatest water holding capacity and lightness, contained the highest HSP90 level, followed by intermediate (6.0-6.4) and low pHi groups (pH<6.0). Statistical analysis indicated HSP90 level was significantly and negatively correlated with cooking loss, drip loss, and lightness (r=-0.797, -0.785, -0.604, respectively, P<0.01). The results suggest that HSP90 may play a crucial role in water retention of meat and may be involved in postmortem meat quality development. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris.

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 methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). 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. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

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 methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). 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. Ours results show that at 3% methanol −30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol −10 °C and 1% and at methanol −10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris. PMID:25242930

In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90.

PubMed

Iki, Taichiro; Yoshikawa, Manabu; Nishikiori, Masaki; Jaudal, Mauren C; Matsumoto-Yokoyama, Eiko; Mitsuhara, Ichiro; Meshi, Tetsuo; Ishikawa, Masayuki

2010-07-30

RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed. Copyright 2010 Elsevier Inc. All rights reserved.

Image-guided photo-therapeutic nanoporphyrin synergized HSP90 inhibitor in patient-derived xenograft bladder cancer model.

PubMed

Long, Qilai; Lin, Tzu-Yin; Huang, Yee; Li, Xiaocen; Ma, Ai-Hong; Zhang, Hongyong; Carney, Randy; Airhart, Susan; Lam, Kit S; deVere White, Ralph W; Pan, Chong-Xian; Li, Yuanpei

2018-04-01

Photodynamic therapy is a promising and effective non-invasive therapeutic approach for the treatment of bladder cancers. Therapies targeting HSP90 have the advantage of tumor cell selectivity and have shown great preclinical efficacy. In this study, we evaluated a novel multifunctional nanoporphyrin platform loaded with an HSP90 inhibitor 17AAG (NP-AAG) for use as a multi-modality therapy against bladder cancer. NP-AAG was efficiently accumulated and retained at bladder cancer patient-derived xenograft (PDX) over 7 days. PDX tumors could be synergistically eradicated with a single intravenous injection of NP-AAG followed by multiple light treatments within 7 days. NP-AAG mediated treatment could not only specifically deliver 17AAG and produce heat and reactive oxygen species, but also more effectively inhibit essential bladder cancer essential signaling molecules like Akt, Src, and Erk, as well as HIF-1α induced by photo-therapy. This multifunctional nanoplatform has high clinical relevance and could dramatically improve management for bladder cancers with minimal toxicity. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

[The dynamic mitochondria-nuclear redistribution of FKBP51 during the process of adipocyte differentiation is regulated by PKA].

PubMed

Toneatto, Judith; Charó, Nancy L; Susperreguy, Sebastián; Piwien-Pilipuk, Graciela

2013-01-01

Glucocorticoids play an important role in adipogenesis via the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90-Hsp70 and a high molecular weight immunophilin FKBP51 or FKBP52. We have found that FKBP51 level of expression progressively increases, FKBP52 decreases, whereas Hsp90, Hsp70, and p23 remain unchanged when 3T3-L1 preadipocytes differentiate. Interestingly, FKBP51 translocates from mitochondria to the nucleus at the onset of adipogenesis. FKBP51 transiently concentrates in the nuclear lamina, at a time that this nuclear compartment undergoes its reorganization. FKBP51 nuclear localization is transient, after 48 h it cycles back to mitochondria. We found that the dynamic FKBP51 mitochondrial-nuclear shuttling is regulated by glucocorticoids and mainly on cAMP-PKA signaling since PKA inhibition by myristoilated-PKI, abrogated FKBP51 nuclear translocation induced by 3-isobutyl-1-methylxanthine (IBMX). It has been reported that PKA interacts with GR in a ligand dependent manner potentiating its transcriptional capacity. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced nuclear translocation of FKBP51, therefore PKA may exert a dual role in the control of GR. In summary, the presence of FKBP51 in the nucleus may be critical for GR transcriptional control, and possibly for the control of other transcription factors that are not members of the nuclear receptor family but are regulated by PKA signaling pathway, when transcription has to be strictly controlled to succeed in the acquisition of the adipocyte phenotype.

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. Copyright 2010 Elsevier Inc. All rights reserved.

The Roles of Heat Shock Proteins 70 and 90 in Exopalaemon carinicauda After WSSV and Vibrio anguillarum Challenges

NASA Astrophysics Data System (ADS)

Li, Jitao; Li, Jian; Duan, Yafei; Chen, Ping; Liu, Ping

2018-04-01

Heat shock proteins (HSPs), such as HSP70 and HSP90, are a suite of highly conserved proteins produced in all cellular organisms when they are exposed to stresses. In aquatic animals, they have been proved to play important roles in response to environmental pollutants and particularly in the non-specific immune responses to pathogen infections. In the present study, the expression profiles of HSP70 and HSP90 genes in hemocytes and hepatopancreas from the ridgetail white prawn Exopalaemon carinicauda infected with WSSV and Vibrio anguillarum were detected using reverse transcription polymerase chain reaction (RT-PCR). After WSSV challenge, the expression level of HSP 70 gene transcripts in the hemocytes and hepatopancreas increased to peak level at 6 h and 48 h, respectively. HSP90 gene transcripts in hemocytes and hepatopancreas were up-regulated significantly at 12 h and 6 h, respectively. During V. anguillarum challenge, the mRNA content of HSP70 gene in hemocytes and hepatopancreas increased significantly at 12 h and 6 h post-infection, respectively. The expression level of HSP90 gene both in hemocytes and hepatopancreas were up-regulated in the first 3 h. The expression patterns of HSP70 and HSP90 genes in hemocytes and hepatopancreas showed temporal and spatial differences after challenged with WSSV and V. anguillarum. The results suggested that HSPs might be involved in immune responses to pathogens challenge in E. carinicauda.

The roles of heat shock proteins 70 and 90 in Exopalaemon carinicauda after WSSV and Vibrio anguillarum challenges

NASA Astrophysics Data System (ADS)

Li, Jitao; Li, Jian; Duan, Yafei; Chen, Ping; Liu, Ping

2017-12-01

Heat shock proteins (HSPs), such as HSP70 and HSP90, are a suite of highly conserved proteins produced in all cellular organisms when they are exposed to stresses. In aquatic animals, they have been proved to play important roles in response to environmental pollutants and particularly in the non-specific immune responses to pathogen infections. In the present study, the expression profiles of HSP70 and HSP90 genes in hemocytes and hepatopancreas from the ridgetail white prawn Exopalaemon carinicauda infected with WSSV and Vibrio anguillarum were detected using reverse transcription polymerase chain reaction (RT-PCR). After WSSV challenge, the expression level of HSP 70 gene transcripts in the hemocytes and hepatopancreas increased to peak level at 6 h and 48 h, respectively. HSP90 gene transcripts in hemocytes and hepatopancreas were up-regulated significantly at 12 h and 6 h, respectively. During V. anguillarum challenge, the mRNA content of HSP70 gene in hemocytes and hepatopancreas increased significantly at 12 h and 6 h post-infection, respectively. The expression level of HSP90 gene both in hemocytes and hepatopancreas were up-regulated in the first 3 h. The expression patterns of HSP70 and HSP90 genes in hemocytes and hepatopancreas showed temporal and spatial differences after challenged with WSSV and V. anguillarum. The results suggested that HSPs might be involved in immune responses to pathogens challenge in E. carinicauda.

Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension.

PubMed

Afolayan, Adeleye J; Eis, Annie; Alexander, Maxwell; Michalkiewicz, Teresa; Teng, Ru-Jeng; Lakshminrusimha, Satyan; Konduri, Girija G

2016-01-01

Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen. Copyright © 2016 the American Physiological Society.

Glutamine reduces myocardial cell apoptosis in a rat model of sepsis by promoting expression of heat shock protein 90.

PubMed

Li, Wanxia; Tao, Shaoyu; Wu, Qinghua; Wu, Tao; Tao, Ran; Fan, Jun

2017-12-01

Myocardial cell injury and cardiac myocyte apoptosis are associated with sepsis. Glutamine (Gln) has been reported to repair myocardial cell injury. The aim of this study was to explore the role of Gln on cardiac myocytes in a cecal ligation and puncture (CLP) model of sepsis in Wistar rats. Following induction of sepsis in a CLP rat model, viral encoding heat shock protein 90 (Hsp90) gene and Hsp90dsDNA were designed to express and knockdown Hsp90, respectively. Rat cardiac tissues were examined histologically, and apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein, Hsp90, p53 upregulated modulator of apoptosis, and p53 was measured by western blotting and real-time polymerase chain reaction. Caspase-3, caspase-8, and caspase-9 were detected by enzyme-linked immunosorbent assay. Rat cardiac myocyte damage induced by CLP was reduced by Gln treatment and Hsp90 overexpression, and these changes were reversed by Hsp90 knockdown. Bcl-2 expression, Bcl-2-associated X protein, p53, p53 upregulated modulator of apoptosis, caspase-8, caspase-9, and caspase-3 activities were significantly upregulated in the CLP model, which were reduced by Gln treatment and Hsp90 overexpression. Gln reduced apoptosis of cardiac myocytes in a rat model of sepsis, by promoting Hsp90 expression. Further studies are needed to determine the possible therapeutic action of Gln in sepsis in human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

HSP90 and pCREB alterations are linked to mancozeb-dependent behavioral and neurodegenerative effects in a marine teleost

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zizza, Merylin

The pesticide mancozeb (mz) is recognized as a potent inducer of oxidative stress due to its ability to catalyze the production of reactive oxygen species plus inhibiting mitochondrial respiration thus becoming an environmental risk for neurodegenerative diseases. Despite numerous toxicological studies on mz have been directed to mammals, attention on marine fish is still lacking. Thus, it was our intention to evaluate neurobehavioral activities of ornate wrasses (Thalassoma pavo) exposed to 0.2 mg/l of mz after a preliminary screening test (0.07–0.3 mg/l). Treated fish exhibited an evident (p < 0.001) latency to reach T-maze arms (> 1000%) while exploratory attitudesmore » (total arm entries) diminished (− 50%; p < 0.05) versus controls during spontaneous exploration tests. Moreover, they showed evident enhancements (+ 111%) of immobility in the cylinder test. Contextually, strong (− 88%; p < 0.01) reductions of permanence in light zone of the Light/Dark apparatus along with diminished crossings (− 65%) were also detected. Conversely, wrasses displayed evident enhancements (160%) of risk assessment consisting of fast entries in the dark side of this apparatus. From a molecular point of view, a notable activation (p < 0.005) of the brain transcription factor pCREB occurred during mz-exposure. Similarly, in situ hybridization supplied increased HSP90 mRNAs in most brain areas such as the lateral part of the dorsal telencephalon (Dl; + 68%) and valvula of the cerebellum (VCe; + 35%) that also revealed evident argyrophilic signals. Overall, these first indications suggest a possible protective role of the early biomarkers pCREB and HSP90 against fish toxicity. - Highlights: • Fish exposed to mancozeb exhibited an evident latency to reach T-maze arms. • Mancozeb caused immobility and reduction of explorative attitudes. • Fish exposed to mancozeb showed anxiogenic performances in the Light/Dark apparatus. • The brain of fish exposed to mancozeb supplied pCREB plus HSP90 mRNA up-regulations. • Some brain areas of fish exposed to mancozeb revealed an evident neurodegeneration.« less

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.

Hsp90 Orchestrates Transcriptional Regulation by Hsf1 and Cell Wall Remodelling by MAPK Signalling during Thermal Adaptation in a Pathogenic Yeast

PubMed Central

Leach, Michelle D.; Budge, Susan; Walker, Louise; Munro, Carol; Cowen, Leah E.; Brown, Alistair J. P.

2012-01-01

Thermal adaptation is essential in all organisms. In yeasts, the heat shock response is commanded by the heat shock transcription factor Hsf1. Here we have integrated unbiased genetic screens with directed molecular dissection to demonstrate that multiple signalling cascades contribute to thermal adaptation in the pathogenic yeast Candida albicans. We show that the molecular chaperone heat shock protein 90 (Hsp90) interacts with and down-regulates Hsf1 thereby modulating short term thermal adaptation. In the longer term, thermal adaptation depends on key MAP kinase signalling pathways that are associated with cell wall remodelling: the Hog1, Mkc1 and Cek1 pathways. We demonstrate that these pathways are differentially activated and display cross talk during heat shock. As a result ambient temperature significantly affects the resistance of C. albicans cells to cell wall stresses (Calcofluor White and Congo Red), but not osmotic stress (NaCl). We also show that the inactivation of MAP kinase signalling disrupts this cross talk between thermal and cell wall adaptation. Critically, Hsp90 coordinates this cross talk. Genetic and pharmacological inhibition of Hsp90 disrupts the Hsf1-Hsp90 regulatory circuit thereby disturbing HSP gene regulation and reducing the resistance of C. albicans to proteotoxic stresses. Hsp90 depletion also affects cell wall biogenesis by impairing the activation of its client proteins Mkc1 and Hog1, as well as Cek1, which we implicate as a new Hsp90 client in this study. Therefore Hsp90 modulates the short term Hsf1-mediated activation of the classic heat shock response, coordinating this response with long term thermal adaptation via Mkc1- Hog1- and Cek1-mediated cell wall remodelling. PMID:23300438

The human escort protein Hep binds to the ATPase domain of mitochondrial hsp70 and regulates ATP hydrolysis.

PubMed

Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J

2008-09-19

Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.

The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis*

PubMed Central

Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J.

2008-01-01

Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8 ± 0.2 × 10-4 s-1) at 25 °C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain. PMID:18632665

Molecular chaperone complexes with antagonizing activities regulate stability and activity of the tumor suppressor LKB1.

PubMed

Gaude, H; Aznar, N; Delay, A; Bres, A; Buchet-Poyau, K; Caillat, C; Vigouroux, A; Rogon, C; Woods, A; Vanacker, J-M; Höhfeld, J; Perret, C; Meyer, P; Billaud, M; Forcet, C

2012-03-22

LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90-Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1-STRAD and LKB1-Hsp90-Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1-Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90-Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90-Cdc37 and Hsp/Hsc70-CHIP, finely control the cellular level of LKB1 protein.

Cloning of heat shock protein genes (hsp70, hsc70 and hsp90) and their expression in response to larval diapause and thermal stress in the wheat blossom midge, Sitodiplosis mosellana.

PubMed

Cheng, Weining; Li, Dan; Wang, Yue; Liu, Yang; Zhu-Salzman, Keyan

2016-12-01

Sitodiplosis mosellana Géhin, one of the most important pests of wheat, undergoes obligatory diapause as a larva to survive unfavorable temperature extremes during hot summers and cold winters. To explore the potential roles of heat shock proteins (hsp) in this process, we cloned full-length cDNAs of hsp70, hsc70 and hsp90 from S. mosellana larvae, and examined their expression in response to diapause and short-term temperature stresses. Three hsps included all signature sequences of corresponding protein family and EEVD motifs. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known classification of insects. Expression of hsp70 and hsp90 were highly induced by diapause, particularly pronounced during summer and winter. Interestingly, hsp70 was more strongly expressed in summer than in winter whereas hsp90 displayed the opposite pattern. Abundance of hsc70 mRNA was comparable prior to and during diapauses and was highly up-regulated when insects began to enter the stage of post-diapause quiescence. Heat-stressed over-summering larvae (⩾30°C) or cold-stressed over-wintering larvae (⩽0°C) could further elevate expression of these three genes, but temperature extremes i.e. as high as 45°C or as low as -15°C failed to trigger such expression patterns. Notably, hsp70 was most sensitive to heat stress and hsp90 was most sensitive to cold stress. These results suggested that hsp70 and hsp90 play key roles in diapause maintenance and thermal stress; the former may be more prominent contributor to heat tolerance and the latter for cold tolerance. In contrast, hsc70 most likely is involved in developmental transition from diapause to post-diapause quiescence, and thus may serve as a molecular marker to predict diapause termination. Copyright © 2016 Elsevier Ltd. All rights reserved.

Large scale free energy calculations for blind predictions of protein-ligand binding: the D3R Grand Challenge 2015.

PubMed

Deng, Nanjie; Flynn, William F; Xia, Junchao; Vijayan, R S K; Zhang, Baofeng; He, Peng; Mentes, Ahmet; Gallicchio, Emilio; Levy, Ronald M

2016-09-01

We describe binding free energy calculations in the D3R Grand Challenge 2015 for blind prediction of the binding affinities of 180 ligands to Hsp90. The present D3R challenge was built around experimental datasets involving Heat shock protein (Hsp) 90, an ATP-dependent molecular chaperone which is an important anticancer drug target. The Hsp90 ATP binding site is known to be a challenging target for accurate calculations of ligand binding affinities because of the ligand-dependent conformational changes in the binding site, the presence of ordered waters and the broad chemical diversity of ligands that can bind at this site. Our primary focus here is to distinguish binders from nonbinders. Large scale absolute binding free energy calculations that cover over 3000 protein-ligand complexes were performed using the BEDAM method starting from docked structures generated by Glide docking. Although the ligand dataset in this study resembles an intermediate to late stage lead optimization project while the BEDAM method is mainly developed for early stage virtual screening of hit molecules, the BEDAM binding free energy scoring has resulted in a moderate enrichment of ligand screening against this challenging drug target. Results show that, using a statistical mechanics based free energy method like BEDAM starting from docked poses offers better enrichment than classical docking scoring functions and rescoring methods like Prime MM-GBSA for the Hsp90 data set in this blind challenge. Importantly, among the three methods tested here, only the mean value of the BEDAM binding free energy scores is able to separate the large group of binders from the small group of nonbinders with a gap of 2.4 kcal/mol. None of the three methods that we have tested provided accurate ranking of the affinities of the 147 active compounds. We discuss the possible sources of errors in the binding free energy calculations. The study suggests that BEDAM can be used strategically to discriminate binders from nonbinders in virtual screening and to more accurately predict the ligand binding modes prior to the more computationally expensive FEP calculations of binding affinity.

Large scale free energy calculations for blind predictions of protein-ligand binding: the D3R Grand Challenge 2015

NASA Astrophysics Data System (ADS)

Deng, Nanjie; Flynn, William F.; Xia, Junchao; Vijayan, R. S. K.; Zhang, Baofeng; He, Peng; Mentes, Ahmet; Gallicchio, Emilio; Levy, Ronald M.

2016-09-01

We describe binding free energy calculations in the D3R Grand Challenge 2015 for blind prediction of the binding affinities of 180 ligands to Hsp90. The present D3R challenge was built around experimental datasets involving Heat shock protein (Hsp) 90, an ATP-dependent molecular chaperone which is an important anticancer drug target. The Hsp90 ATP binding site is known to be a challenging target for accurate calculations of ligand binding affinities because of the ligand-dependent conformational changes in the binding site, the presence of ordered waters and the broad chemical diversity of ligands that can bind at this site. Our primary focus here is to distinguish binders from nonbinders. Large scale absolute binding free energy calculations that cover over 3000 protein-ligand complexes were performed using the BEDAM method starting from docked structures generated by Glide docking. Although the ligand dataset in this study resembles an intermediate to late stage lead optimization project while the BEDAM method is mainly developed for early stage virtual screening of hit molecules, the BEDAM binding free energy scoring has resulted in a moderate enrichment of ligand screening against this challenging drug target. Results show that, using a statistical mechanics based free energy method like BEDAM starting from docked poses offers better enrichment than classical docking scoring functions and rescoring methods like Prime MM-GBSA for the Hsp90 data set in this blind challenge. Importantly, among the three methods tested here, only the mean value of the BEDAM binding free energy scores is able to separate the large group of binders from the small group of nonbinders with a gap of 2.4 kcal/mol. None of the three methods that we have tested provided accurate ranking of the affinities of the 147 active compounds. We discuss the possible sources of errors in the binding free energy calculations. The study suggests that BEDAM can be used strategically to discriminate binders from nonbinders in virtual screening and to more accurately predict the ligand binding modes prior to the more computationally expensive FEP calculations of binding affinity.

Dexamethasone treatment decreases replication of viral hemorrhagic septicemia virus in Epithelioma papulosum cyprini cells.

PubMed

Kim, Min Sun; Lee, Su Jin; Choi, Seung Hyuk; Kang, Yue Jai; Kim, Ki Hong

2017-05-01

The expression of Mx1 in EPC cells after treatment with poly(I:C) or infection with viral hemorrhagic septicemia virus (VHSV) was significantly suppressed by treatment with dexamethasone. However, the titer of VHSV did not increase but instead decreased after dexamethasone treatment. This suggests that dexamethasone not only downregulates type I IFN but also affects certain factors that are necessary for VHSV replication. An important effect of HSP90 on replication of RNA viruses and downregulation of HSP90 by glucocorticoids have been reported. In this study, dexamethasone downregulated HSP90α expression in EPC cells that were stimulated with poly(I:C) or infected with VHSV. Furthermore, cells treated with an HSP90 inhibitor, geldanamycin, showed significantly decreased titers of VHSV, suggesting that HSP90 may be an important host component involved in VHSV replication, and HSP90 inhibition might be one of the causes for the observed reduction in viral titer caused by dexamethasone treatment.

Variation of heat shock protein gene expression in the brain of cold-induced pulmonary hypertensive chickens.

PubMed

Hassanpour, H; Khosravi Alekoohi, Z; Madreseh, S; Bahadoran, S; Nasiri, L

2016-10-01

Quantitative real-time PCR was carried out to evaluate gene expression of heat shock proteins (HSP) (HSP27, HSP56, HSP60, HSP70, HSP90 and ubiquitin) in the brain (hindbrain, midbrain, forebrain) of chickens with cold-induced pulmonary hypertension. The ratio of the right ventricle to the total ventricle (index of pulmonary hypertension in chickens) was increased in the cold-induced pulmonary hypertensive chickens at 42 d of age compared with control. The HSP genes were expressed in the three parts of the brain in the two experimental groups. In the hindbrain of cold-induced pulmonary hypertensive chickens, the relative gene expression of HSP27, HSP60, HSP70 and HSP90 was decreased while gene expression of HSP56 and ubiquitin was increased compared with controls. In the midbrain of cold induced-pulmonary hypertensive chickens, the expression of HSP56, HSP60, HSP70 and ubiquitin genes was increased compared with controls while HSP27 and HSP90 were decreased. In the forebrain of cold induced-pulmonary hypertensive chickens, the expression of HSP56, HSP60, HSP70 and ubiquitin genes was increased while the expression of the HSP27 gene was decreased compared with controls. It is concluded that overexpression of HSPs in the forebrain and midbrain probably delays the pathological process of cold stress whereas diminished expression of HSP genes in the hindbrain may affect the normal function of brain centres in this area to exacerbate pulmonary hypertension.

Heat shock protein 90 inhibitors in the treatment of cancer: current status and future directions.

PubMed

Jhaveri, Komal; Ochiana, Stefan O; Dunphy, Mark Ps; Gerecitano, John F; Corben, Adriana D; Peter, Radu I; Janjigian, Yelena Y; Gomes-DaGama, Erica M; Koren, John; Modi, Shanu; Chiosis, Gabriela

2014-05-01

Heat shock protein 90 (HSP90) serves as a critical facilitator for oncogene addiction. There has been augmenting enthusiasm in pursuing HSP90 as an anticancer strategy. In fact, since the initial serendipitous discovery that geldanamycin (GM) inhibits HSP90, the field has rapidly moved from proof-of-concept clinical studies with GM derivatives to novel second-generation inhibitors. The authors highlight the current status of the second-generation HSP90 inhibitors in clinical development. Herein, the authors note the lessons learned from the completed clinical trials of first- and second-generation inhibitors and describe various assays attempting to serve for a more rational implementation of these agents to cancer treatment. Finally, the authors discuss the future perspectives for this promising class of agents. The knowledge gained thus far provides perhaps only a glimpse at the potential of HSP90 for which there is still much work to be done. Lessons from the clinical trials suggest that HSP90 therapy would advance at a faster pace if patient selection and tumor pharmacokinetics of these drugs were better understood and applied to their clinical development. It is also evident that combining HSP90 inhibitors with other potent anticancer therapies holds great promise not only due to synergistic antitumor activity but also due to the potential of prolonging or preventing the development of drug resistance.

Nitric oxide and heat shock protein 90 co-regulate temperature-induced bleaching in the soft coral Eunicea fusca

NASA Astrophysics Data System (ADS)

Ross, Cliff

2014-06-01

Coral bleaching represents a complex physiological process that is affected not only by environmental conditions but by the dynamic internal cellular biology of symbiotic dinoflagellates ( Symbiodinium spp.) and their cnidarian hosts. Recently, nitric oxide (NO) has emerged as a key molecule involved with the expulsion of Symbiodinium from host cnidarian cells. However, the site of production remains under debate, and the corresponding signaling pathways within and between host and endosymbiont remain elusive. In this study, using freshly isolated Symbiodinium from the soft coral Eunicea fusca, I demonstrate that thermally induced stress causes an upregulation in Symbiodinium heat shock protein 90 (Hsp90). In turn, Hsp90 shows a concomitant ability to enhance the activity of a constitutively expressed isoform of NO synthase. The resulting production of NO constitutes a signaling molecule capable of inducing Symbiodinium expulsion. Using nitric oxide synthase (NOS) and Hsp90 polyclonal antibodies, thermal stress-induced Hsp90 was shown to co-immunoprecipitate with a constitutive isoform of NOS. The specific blocking of Hsp90 activity, with the Hsp90 inhibitor geldanamycin, was capable of inhibiting NO production implicating the involvement of a coordinated regulatory system. These results have strong evolutionary implications for Hsp90-NOS chaperone complexes among biological kingdoms and provide evidence for a new functional role in symbiotic associations.

Effect of tributyltin chloride (TBT-Cl) exposure on expression of HSP90β1 in the river pufferfish (Takifugu obscurus): Evidences for its immunologic function involving in exploring process.

PubMed

Dong-Po, Xu; Di-An, Fang; Chang-Sheng, Zhao; Shu-Lun, Jiang; Hao-Yuan, Hu

2018-08-05

HSP90β1 (known as glyco-protein 96, GP96) is a vital endoplasmic reticulum (ER) depended chaperonin among the HSPs (heat shock proteins) family. Furthermore, it always processes and presents antigen of the tumor and keeps balance for the intracellular environment. In the present study, we explored the effect of tributyltin chloride (TBT-Cl) exposure on HSP90β1 expression in river pufferfish, Takifugu obscurus. The full length of To-HSP90β1 was gained with 2775 bp in length, with an ORF (open reading frame) encoding an 803 aa polypeptide. A phylogenetic tree was constructed and showed the close relationship to other fish species. The HSP90β1 mRNA transcript was expressed in all tissues investigated with higher level in the gill and liver. After the acute and chronic exposure of TBT-Cl, the To-HSP90β1 mRNA transcript significantly was up-regulated in gills. Moreover, the histology study indicated the different injury degree of TBT-Cl in liver and gill. Immunohistochemistry (IHC) staining results implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-HSP90β1 to TBT-Cl exposure. All the results indicated that HSP90β1 may be involved in the resistance to the invasion of TBT-Cl for keeping autoimmune homeostasis. Copyright © 2018. Published by Elsevier B.V.

Synthesis and evaluation of coumermycin A1 analogues that inhibit the Hsp90 protein folding machinery.

PubMed

Burlison, Joseph A; Blagg, Brian S J

2006-10-12

[structure: see text] The coumarin antibiotics are not only potent inhibitors of DNA gyrase but also represent the most effective C-terminal inhibitors of 90 kDa heat shock proteins (Hsp90) reported thus far. In contrast to the N-terminal ATP-binding site, little is known about the Hsp90 C-terminus. In addition, very limited structure-activity relationships exist between this class of natural products and Hsp90. In this letter, the syntheses of dimeric coumarin analogues are presented along with their inhibitory values in breast cancer cell lines.

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

PubMed Central

Conconi, M; Petropoulos, I; Emod, I; Turlin, E; Biville, F; Friguet, B

1998-01-01

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP. PMID:9657982

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

PubMed

Conconi, M; Petropoulos, I; Emod, I; Turlin, E; Biville, F; Friguet, B

1998-07-15

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.

Heat shock proteins stimulate APOBEC-3-mediated cytidine deamination in the hepatitis B virus.

PubMed

Chen, Zhigang; Eggerman, Thomas L; Bocharov, Alexander V; Baranova, Irina N; Vishnyakova, Tatyana G; Kurlander, Roger; Patterson, Amy P

2017-08-11

Apolipoprotein B mRNA-editing enzyme catalytic subunit 3 (APOBEC-3) enzymes are cytidine deaminases that are broadly and constitutively expressed. They are often up-regulated during carcinogenesis and candidate genes for causing the major single-base substitution in cancer-associated DNA mutations. Moreover, APOBEC-3s are involved in host innate immunity against many viruses. However, how APOBEC-3 mutational activity is regulated in normal and pathological conditions remains largely unknown. Heat shock protein levels are often elevated in both carcinogenesis and viral infection and are associated with DNA mutations. Here, using mutational analyses of hepatitis B virus (HBV), we found that Hsp90 stimulates deamination activity of APOBEC-3G (A3G), A3B, and A3C during co-expression in human liver HepG2 cells. Hsp90 directly stimulated A3G deamination activity when the purified proteins were used in in vitro reactions. Hsp40, -60, and -70 also had variable stimulatory effects in the cellular assay, but not in vitro Sequencing analyses further demonstrated that Hsp90 increased both A3G cytosine mutation efficiency on HBV DNA and total HBV mutation frequency. In addition, Hsp90 shifted A3G's cytosine region selection in HBV DNA and increased A3G's 5' nucleoside preference for deoxycytidine (5'-CC). Furthermore, the Hsp90 inhibitor 17- N -allylamino-17-demethoxygeldanamycin dose dependently inhibited A3G and A3B mutational activity on HBV viral DNA. Hsp90 knockdown by siRNA or by Hsp90 active-site mutation also decreased A3G activity. These results indicate that heat shock proteins, in particular Hsp90, stimulate APOBEC-3-mediated DNA deamination activity, suggesting a potential physiological role in carcinogenesis and viral innate immunity.

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

PubMed

Young, Christopher N J; 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 Dmd(mdx) 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 Ca(2+) 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.

Genetic and Genomic Architecture of the Evolution of Resistance to Antifungal Drug Combinations

PubMed Central

Hill, Jessica A.; Ammar, Ron; Torti, Dax; Nislow, Corey; Cowen, Leah E.

2013-01-01

The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast Saccharomyces cerevisiae and the leading human fungal pathogen Candida albicans with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, PDR1, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, MOT3, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in LCB1, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the C. albicans lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance. PMID:23593013

Improved pose and affinity predictions using different protocols tailored on the basis of data availability

NASA Astrophysics Data System (ADS)

Prathipati, Philip; Nagao, Chioko; Ahmad, Shandar; Mizuguchi, Kenji

2016-09-01

The D3R 2015 grand drug design challenge provided a set of blinded challenges for evaluating the applicability of our protocols for pose and affinity prediction. In the present study, we report the application of two different strategies for the two D3R protein targets HSP90 and MAP4K4. HSP90 is a well-studied target system with numerous co-crystal structures and SAR data. Furthermore the D3R HSP90 test compounds showed high structural similarity to existing HSP90 inhibitors in BindingDB. Thus, we adopted an integrated docking and scoring approach involving a combination of both pharmacophoric and heavy atom similarity alignments, local minimization and quantitative structure activity relationships modeling, resulting in the reasonable prediction of pose [with the root mean square deviation (RMSD) values of 1.75 Å for mean pose 1, 1.417 Å for the mean best pose and 1.85 Å for the mean all poses] and affinity (ROC AUC = 0.702 at 7.5 pIC50 cut-off and R = 0.45 for 180 compounds). The second protein, MAP4K4, represents a novel system with limited SAR and co-crystal structure data and little structural similarity of the D3R MAP4K4 test compounds to known MAP4K4 ligands. For this system, we implemented an exhaustive pose and affinity prediction protocol involving docking and scoring using the PLANTS software which considers side chain flexibility together with protein-ligand fingerprints analysis assisting in pose prioritization. This protocol through fares poorly in pose prediction (with the RMSD values of 4.346 Å for mean pose 1, 4.69 Å for mean best pose and 4.75 Å for mean all poses) and produced reasonable affinity prediction (AUC = 0.728 at 7.5 pIC50 cut-off and R = 0.67 for 18 compounds, ranked 1st among 80 submissions).

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

Combined inhibition of heat shock proteins 90 and 70 leads to simultaneous degradation of the oncogenic signaling proteins involved in muscle invasive bladder cancer

PubMed Central

Cavanaugh, Alice; Juengst, Brendon; Sheridan, Kathleen; Danella, John F.; Williams, Heinric

2015-01-01

Heat shock protein 90 (HSP90) plays a critical role in the survival of cancer cells including muscle invasive bladder cancer (MIBC). The addiction of tumor cells to HSP90 has promoted the development of numerous HSP90 inhibitors and their use in clinical trials. This study evaluated the role of inhibiting HSP90 using STA9090 (STA) alone or in combination with the HSP70 inhibitor VER155008 (VER) in several human MIBC cell lines. While both STA and VER inhibited MIBC cell growth and migration and promoted apoptosis, combination therapy was more effective. Therefore, the signaling pathways involved in MIBC were systematically interrogated following STA and/or VER treatments. STA and not VER reduced the expression of proteins in the p53/Rb, PI3K and SWI/SWF pathways. Interestingly, STA was not as effective as VER or combination therapy in degrading proteins involved in the histone modification pathway such as KDM6A (demethylase) and EP300 (acetyltransferase) as predicted by The Cancer Genome Atlas (TCGA) data. This data suggests that dual HSP90 and HSP70 inhibition can simultaneously disrupt the key signaling pathways in MIBC. PMID:26556859

Cloning of the heat shock protein 90 and 70 genes from the beet armyworm, Spodoptera exigua, and expression characteristics in relation to thermal stress and development

USDA-ARS?s Scientific Manuscript database

Two full-length complementary DNAs (cDNAs) of heat shock protein (HSP) genes (Se-hsp90 and Se-hsp70) were cloned from the beet armyworm, Spodoptera exigua, and their expression was investigated in relation to cold shock, heat shock, and development. The open reading frames of Se-hsp90 and Sehsp70 ar...

Epigenetic effects of inhibition of heat shock protein 90 (HSP90) in human pancreatic and colon cancer.

PubMed

Nagaraju, Ganji Purnachandra; Wu, Christina; Merchant, Neha; Chen, Zhengjia; Lesinski, Gregory B; El-Rayes, Bassel F

2017-08-28

Silencing of tumor suppressor and DNA repair genes through methylation plays a role in cancer development, growth and response to therapy in colorectal and pancreatic cancers. Heat shock protein 90 (HSP90) regulates transcription of DNA methyltransferase enzymes (DNMT). In addition, DNMTs are client proteins of HSP90. The aim of this study is to evaluate the effects of HSP90 inhibition on DNA methylation in colorectal and pancreatic cancer cell lines. Our data shows that inhibition of HSP90 using ganetespib resulted in downregulation of mRNA and protein expression of DNMT1, DNMT3A, and DNMT3B in HT-29 and MIA PaCa-2 cell lines. This in turn was associated with a drop in the fraction of methylated cytosine residues and re-expression of silenced genes including MLH-1, P16 and SPARC. These effects were validated in HT-29 tumors implanted subcutaneously in mice following in vivo administration of ganetespib. This work demonstrates the effectiveness of ganetespib, an HSP90 inhibitor in modulating DNA methylation through downregulation of DNMT expression. Copyright © 2017 Elsevier B.V. All rights reserved.

Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

PubMed Central

Finka, Andrija; Mattoo, Rayees U. H.

2010-01-01

Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0216-8) contains supplementary material, which is available to authorized users. PMID:20694844

Identification, Characterization and Expression Profiling of Stress-Related Genes in Easter Lily (Lilium formolongi)

PubMed Central

Howlader, Jewel; Park, Jong-In; Robin, Arif Hasan Khan; Sumi, Kanij Rukshana; Nou, Ill-Sup

2017-01-01

Biotic and abiotic stresses are the major causes of crop loss in lily worldwide. In this study, we retrieved 12 defense-related expressed sequence tags (ESTs) from the NCBI database and cloned, characterized, and established seven of these genes as stress-induced genes in Lilium formolongi. Using rapid amplification of cDNA ends PCR (RACE-PCR), we successfully cloned seven full-length mRNA sequences from L. formolongi line Sinnapal lily. Based on the presence of highly conserved characteristic domains and phylogenetic analysis using reference protein sequences, we provided new nomenclature for the seven nucleotide and protein sequences and submitted them to GenBank. The real-time quantitative PCR (qPCR) relative expression analysis of these seven genes, including LfHsp70-1, LfHsp70-2, LfHsp70-3, LfHsp90, LfUb, LfCyt-b5, and LfRab, demonstrated that they were differentially expressed in all organs examined, possibly indicating functional redundancy. We also investigated the qPCR relative expression levels under two biotic and four abiotic stress conditions. All seven genes were induced by Botrytis cinerea treatment, and all genes except LfHsp70-3 and LfHsp90 were induced by Botrytis elliptica treatment; these genes might be associated with disease tolerance mechanisms in L. formolongi. In addition, LfHsp70-1, LfHsp70-2, LfHsp70-3, LfHsp90, LfUb, and LfCyt-b5 were induced by heat treatment, LfHsp70-1, LfHsp70-2, LfHsp70-3, LfHsp90, and LfCyt-b5 were induced by cold treatment, and LfHsp70-1, LfHsp70-2, LfHsp70-3, LfHsp90, LfCy-b5, and LfRab were induced by drought and salt stress, indicating their likely association with tolerance to these stress conditions. The stress-induced candidate genes identified in this study provide a basis for further functional analysis and the development of stress-resistant L. formolongi cultivars.

PET imaging of Hsp90 expression in pancreatic cancer using a new 64Cu-labeled dimeric Sansalvamide A decapeptide.

PubMed

Wang, Xiaohui; Zhang, Jun; Wu, Hubing; Li, Yumin; Conti, Peter S; Chen, Kai

2018-04-24

Heat shock protein 90 (Hsp90) plays a vital role in the progress of malignant disease and elevated Hsp90 expression has been reported in pancreatic cancer. In this study, we radiolabeled a dimeric Sansalvamide A derivative (Di-San A1) with 64 Cu, and evaluated the feasibility of using 64 Cu-Di-San A1 for PET imaging of Hsp90 expression in a mouse model of pancreatic cancer. A macrocyclic chelator NOTA (1,4,7-triazacyclononane-1,4,7-trisacetic acid) was conjugated to Di-San A1. 64 Cu-Di-San A1 was successfully prepared in a radiochemical yield > 97% with a radiochemical purity > 98%. 64 Cu-Di-San A1 is stable in PBS and mouse serum with > 92% of parent probe intact after 4 h incubation. The cell binding and uptake revealed that 64 Cu-Di-San A1 binds to Hsp90-positive PL45 pancreatic cancer cells, and the binding can be effectively blocked by an Hsp90 inhibitor (17AAG). For microPET study, 64 Cu-Di-San A1 shows good in vivo performance in terms of tumor uptake in nude mice bearing PL45 tumors. The Hsp90-specific tumor activity accumulation of 64 Cu-Di-San A1 was further demonstrated by significant reduction of PL45 tumor uptake with a pre-injected blocking dose of 17AAG. The ex vivo PET imaging and biodistribution results were consistent with the quantitative analysis of PET imaging, demonstrating good tumor-to-muscle ratio (5.35 ± 0.46) of 64 Cu-Di-San A1 at 4 h post-injection in PL45 tumor mouse xenografts. 64 Cu-Di-San A1 allows PET imaging of Hsp90 expression in PL45 tumors, which may provide a non-invasive method to quantitatively characterize Hsp90 expression in pancreatic cancer.

Identification of the plant compound geraniin as a novel Hsp90 inhibitor.

PubMed

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.

Could the FDA-approved anti-HIV PR inhibitors be promising anticancer agents? An answer from enhanced docking approach and molecular dynamics analyses.

PubMed

Arodola, Olayide A; Soliman, Mahmoud E S

2015-01-01

Based on experimental data, the anticancer activity of nelfinavir (NFV), a US Food and Drug Administration (FDA)-approved HIV-1 protease inhibitor (PI), was reported. Nevertheless, the mechanism of action of NFV is yet to be verified. It was hypothesized that the anticancer activity of NFV is due to its inhibitory effect on heat shock protein 90 (Hsp90), a promising target for anticancer therapy. Such findings prompted us to investigate the potential anticancer activity of all other FDA-approved HIV-1 PIs against human Hsp90. To accomplish this, "loop docking" - an enhanced in-house developed molecular docking approach - followed by molecular dynamic simulations and postdynamic analyses were performed to elaborate on the binding mechanism and relative binding affinities of nine FDA-approved HIV-1 PIs against human Hsp90. Due to the lack of the X-ray crystal structure of human Hsp90, homology modeling was performed to create its 3D structure for subsequent simulations. Results showed that NFV has better binding affinity (ΔG =-9.2 kcal/mol) when compared with other PIs: this is in a reasonable accordance with the experimental data (IC50 3.1 μM). Indinavir, saquinavir, and ritonavir have close binding affinity to NFV (ΔG =-9.0, -8.6, and -8.5 kcal/mol, respectively). Per-residue interaction energy decomposition analysis showed that hydrophobic interaction (most importantly with Val534 and Met602) played the most predominant role in drug binding. To further validate the docking outcome, 5 ns molecular dynamic simulations were performed in order to assess the stability of the docked complexes. To our knowledge, this is the first account of detailed computational investigations aimed to investigate the potential anticancer activity and the binding mechanism of the FDA-approved HIV PIs binding to human Hsp90. Information gained from this study should also provide a route map toward the design, optimization, and further experimental investigation of potential derivatives of PIs to treat HER2+ breast cancer.

PRDM14 directly interacts with heat shock proteins HSP90α and glucose-regulated protein 78.

PubMed

Moriya, Chiharu; Taniguchi, Hiroaki; Nagatoishi, Satoru; Igarashi, Hisayoshi; Tsumoto, Kouhei; Imai, Kohzoh

2018-02-01

PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem-like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple-negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2). We obtained 13 candidates that were pulled down with PRDM14 in TNBC HCC1937 cells and identified them by mass spectrometry. Two candidates-glucose-regulated protein 78 (GRP78) and heat shock protein 90-α (HSP90α)-were confirmed in immunoprecipitation assay in two TNBC cell lines (HCC1937 and MDA-MB231). Surface plasmon resonance analysis using GST-PRDM14 showed that these two proteins directly interacted with PRDM14 and that the interactions required the C-terminal region of PRDM14, which includes zinc finger motifs. We also confirmed the interactions in living cells by NanoLuc luciferase-based bioluminescence resonance energy transfer (NanoBRET) assay. Moreover, HSP90 inhibitors (17DMAG and HSP990) significantly decreased breast cancer stem-like CD24 -  CD44 + and side population (SP) cells in HCC1937 cells, but not in PRDM14 knockdown HCC1937 cells. The combination of the GRP78 inhibitor HA15 and PRDM14 knockdown significantly decreased cell proliferation and SP cell number in HCC1937 cells. These results suggest that HSP90α and GRP78 interact with PRDM14 and participate in cancer regulation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

An In Vivo Photo-Cross-Linking Approach Reveals a Homodimerization Domain of Aha1 in S. cerevisiae

PubMed Central

Berg, Michael; Michalowski, Annette; Palzer, Silke; Rupp, Steffen; Sohn, Kai

2014-01-01

Protein-protein interactions play an essential role in almost any biological processes. Therefore, there is a particular need for methods which describe the interactions of a defined target protein in its physiological context. Here we report a method to photo-cross-link interacting proteins in S. cerevisiae by using the non-canonical amino acid p-azido-L-phenylalanine (pAzpa). Based on the expanded genetic code the photoreactive non-canonical amino acid pAzpa was site-specifically incorporated at eight positions into a domain of Aha1 that was previously described to bind Hsp90 in vitro to function as a cochaperone of Hsp90 and activates its ATPase activity. In vivo photo-cross-linking to the cognate binding partner of Aha1 was carried out by irradiation of mutant strains with UV light (365 nm) to induce covalent intermolecular bonds. Surprisingly, an interaction between Aha1 and Hsp90 was not detected, although, we could confirm binding of suppressed pAzpa containing Aha1 to Hsp90 by native co-immunoprecipitation. However, a homodimer consisting of two covalently crosslinked Aha1 monomers was identified by mass spectrometry. This homodimer could also be confirmed using p-benzoyl-L-phenylalanine, another photoreactive non-canonical amino acid. Crosslinking was highly specific as it was dependent on irradiation using UV light, the exact position of the non-canonical amino acid in the protein sequence as well as on the addition of the non-canonical amino acid to the growth medium. Therefore it seems possible that an interaction of Aha1 with Hsp90 takes place at different positions than previously described in vitro highlighting the importance of in vivo techniques to study protein-protein interactions. Accordingly, the expanded genetic code can easily be applied to other S. cerevisiae proteins to study their interaction under physiological relevant conditions in vivo. PMID:24614167

Identification of an HSP90 modulated multi-step process for ERBB2 degradation in breast cancer cells

PubMed Central

Birocchi, Filippo; Gagliani, Maria Cristina; Tacchetti, Carlo; Cortese, Katia

2016-01-01

The receptor tyrosine kinase ERBB2 interacts with HSP90 and is overexpressed in aggressive breast cancers. Therapeutic HSP90 inhibitors, i.e. Geldanamycin (GA), target ERBB2 to degradation. We have previously shown that HSP90 is responsible for the missorting of recycling ERBB2 to degradation compartments. In this study, we used biochemical, immunofluorescence and electron microscopy techniques to demonstrate that in SKBR3 human breast cancer cells, GA strongly induces polyubiquitination and internalization of the full-length p185-ERBB2, and promotes its cleavage, with the formation of a p116-ERBB2 form in EEA1-positive endosomes (EE). p116-ERBB2 corresponds to a non-ubiquitinated, signaling-impaired, membrane-bound fragment, which is readily sorted to lysosomes and degraded. To define the sequence of events leading to p116-ERBB2 degradation, we first blocked the EE maturation/trafficking to late endosomes/lysosomes with wortmannin, and found an increase in GA-dependent formation of p116-ERBB2; we then inhibited the proteasome activity with MG-132 or lactacystin, and observed an efficient block of p185-ERBB2 cleavage, and its accumulation in EE, suggesting that p185-ERBB2 polyubiquitination is necessary for proteasome-dependent p116-ERBB2 generation occurring in EE. As polyubiquitination has also been implicated in autophagy-mediated degradation of ERBB2 under different experimental conditions, we exploited this possibility and demonstrate that GA strongly inhibits early autophagy, and reduces the levels of the autophagy markers atg5-12 and LC3-II, irrespective of GA-induced ERBB2 polyubiquitination, ruling out a GA-dependent autophagic degradation of ERBB2. In conclusion, we propose that HSP90 inhibition fosters ERBB2 polyubiquitination and proteasome-dependent generation of a non-ubiquitinated and inactive p116-ERBB2 form in EE, which is trafficked from altered EE to lysosomes. PMID:27863425

Dual inhibition of chaperoning process by taxifolin: molecular dynamics simulation study.

PubMed

Verma, Sharad; Singh, Amit; Mishra, Abha

2012-07-01

Hsp90 (heat shock protein 90), a molecular chaperone, stabilizes more than 200 mutated and over expressed oncogenic proteins in cancer development. Cdc37 (cell division cycle protein 37), a co-chaperone of Hsp90, has been found to facilitate the maturation of protein kinases by acting as an adaptor and load these kinases onto the Hsp90 complex. Taxifolin (a natural phytochemical) was found to bind at ATP-binding site of Hsp90 and stabilized the inactive "open" or "lid-up" conformation as evidenced by molecular dynamic simulation. Furthermore, taxifolin was found to bind to interface of Hsp90 and Cdc37 complex and disrupt the interaction of residues of both proteins which were essential for the formation of active super-chaperone complex. Thus, taxifolin was found to act as an inhibitor of chaperoning process and may play a potential role in the cancer chemotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

PubMed Central

Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

2017-01-01

Heat shock protein 90 (Hsp90) contains amino (N)–terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states. PMID:28036294

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET

PubMed Central

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-01-01

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions. PMID:23624933

The conformational dynamics of the mitochondrial Hsp70 chaperone.

PubMed

Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana

2010-04-09

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments. 2010 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark

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)more » 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.« less

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

PubMed

Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

2015-12-29

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

PubMed Central

Ding, Yan; Liu, Zixing; Desai, Shruti; Zhao, Yuhua; Liu, Hao; Pannell, Lewis K; Yi, Hong; Wright, Elizabeth R; Owen, Laurie B; Dean-Colomb, Windy; Fodstad, Oystein; Lu, Jianrong; LeDoux, Susan P; Wilson, Glenn L; Tan, Ming

2012-01-01

It is well known that ErbB2, a receptor tyrosine kinase, localizes on the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through the association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to ErbB2 targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. PMID:23232401

Comparative analysis of the interaction of HSPs in dendritic cells, macrophages, RGM-1 cells infected by Helicobacter pylori

PubMed Central

Yao, Yongliang; Wu, Jianhong; Gu, Tao; Cheng, Yang; Li, Guangxin

2016-01-01

Helicobacter pylori may cause chronic gastritis, even gastric cancer, however, antigen-presenting cells (APCs) are most important immune cells involved in the induction and expression of the underlying inflammatory responses to resist H. pylori. To study the interaction of HSPs in dendritic cells (DCs), macrophages and RGM-1 cells infected with H. pylori, HSP-27, HSP-60, HSP-70 and HSP-90 proteins were analyzed in the mucosa tissue or serum of gastritis patients caused by H. pylori, and in cell supernatant of DCs, macrophages, RGM-1 infected by H. pylori, or in above host cells. We found that HSP-27, HSP-60, HSP-70 and HSP-90 decreased in gastric epithelial cells, but increased significantly in DCs, macrophages. Meanwhile, inflammation associated proteins iNOS-2 and COX-2 were participated in the expression of HSPs in the process of host cells defensing against H. pylori infection. These findings contribute to understand the functions of HSP-27, HSP-60, HSP-70 and HSP-90 in H. pylori infection APCs and gastric epithelial cells indicating that HSPs would be diagnostic markers for H. pylori infection. PMID:27830002

Microarray-based screening of heat shock protein inhibitors.

PubMed

Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

2014-06-20

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

Heat shock proteins 70 and 90 from Clonorchis sinensis induce Th1 response and stimulate antibody production.

PubMed

Chung, Eun Joo; Jeong, Young-Il; Lee, Myoung-Ro; Kim, Yu Jung; Lee, Sang-Eun; Cho, Shin-Hyeong; Lee, Won-Ja; Park, Mi-Yeoun; Ju, Jung-Won

2017-03-01

Heat shock proteins (HSPs) are found in all prokaryotes and most compartments of eukaryotic cells. Members of the HSP family mediate immune responses to tissue damage or cellular stress. However, little is known about the immune response induced by the oriental liver fluke, Clonorchis sinensis, even though this organism is carcinogenic to humans. We address this issue in the present study in mouse bone marrow dendritic cells (mBMDCs), using recombinant HSP70 and 90 from C. sinensis (rCsHSP70 and rCsHSP90). rCsHSP70 and rCsHSP90 were produced in an E. coli system. Purified recombinant proteins were treated in BMDCs isolated from C57BL/6 mice. T cells were isolated from Balb/c mice and co-cultured with activated mBMDCs. Expression of surface molecules was measured by flow cytometry and cytokine secretion was quantified using ELISA. C57BL/6 mice were divided into four groups, including peptide alone, peptide/Freund's adjuvant, peptide/CsHSP70, peptide/CsHSP90, and were immunized intraperitoneally three times. Two weeks after final immunization, antibodies against peptide were measured using ELISA. Both proteins induced a dose-dependent upregulation in major histocompatibility complex and co-stimulatory molecule expression and increased secretion of pro-inflammatory cytokines including interleukin (IL)-1β, -6, and -12p70 and tumor necrosis factor-α in mBMDCs. Furthermore, when allogenic T cells were incubated with mBMDCs activated by rCsHSP70 and rCsHSP90, the helper T cell (Th)1 cytokine interferon-γ was up-regulated whereas the level of the Th2 cytokine IL-4 was unchanged. These results indicate that rCsHSPs predominantly induce a Th1 response. Over and above these results, we also demonstrated that the production of peptide-specific antibodies can be activated after immunization via in vitro peptide binding with rCsHSP70 or rCsHSP90. This study showed for the first time that the HSP or HSP/peptide complexes of C. sinensis could be considered as a more effective vaccine against C. sinensis infection as results of the activator of host immune response as well as the adjuvant for antigenic peptide conjugate to induce peptide-specific antibody response in mice.

A Bipartite Interaction between Hsp70 and CHIP Regulates Ubiquitination of Chaperoned Client Proteins

DOE PAGES

Zhang, Huaqun; Amick, Joseph; Chakravarti, Ritu; ...

2015-02-12

The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. Here, we demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required formore » proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.« less

Arabidopsis HSP90 protein modulates RPP4-mediated temperature-dependent cell death and defense responses.

PubMed

Bao, Fei; Huang, Xiaozhen; Zhu, Chipan; Zhang, Xiaoyan; Li, Xin; Yang, Shuhua

2014-06-01

Plant defense responses are regulated by temperature. In Arabidopsis, the chilling-sensitive mutant chs2-1 (rpp4-1d) contains a gain-of-function mutation in the TIR-NB-LRR (Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat) gene, RPP4 (RECOGNITION OF PERONOSPORA PARASITICA 4), which leads to constitutive activation of the defense response at low temperatures. Here, we identified and characterized two suppressors of rpp4-1d from a genetic screen, hsp90.2 and hsp90.3, which carry point mutations in the cytosolic heat shock proteins HSP90.2 and HSP90.3, respectively. The hsp90 mutants suppressed the chilling sensitivity of rpp4-1d, including seedling lethality, activation of the defense responses and cell death under chilling stress. The hsp90 mutants exhibited compromised RPM1 (RESISTANCE TO PSEUDOMONAS MACULICOLA 1)-, RPS4 (RESISTANCE TO P. SYRINGAE 4)- and RPP4-mediated pathogen resistance. The wild-type RPP4 and the mutated form rpp4 could interact with HSP90 to form a protein complex. Furthermore, RPP4 and rpp4 proteins accumulated in the cytoplasm and nucleus at normal temperatures, whereas the nuclear accumulation of the mutated rpp4 was decreased at low temperatures. Genetic analysis of the intragenic suppressors of rpp4-1d revealed the important functions of the NB-ARC and LRR domains of RPP4 in temperature-dependent defense signaling. In addition, the rpp4-1d-induced chilling sensitivity was largely independent of the WRKY70 or MOS (modifier of snc1) genes. [Correction added after online publication 11 March 2013: the expansions of TIR-NB-LRR and RPS4 were amended] This study reveals that Arabidopsis HSP90 regulates RPP4-mediated temperature-dependent cell death and defense responses. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

HSP90 Inhibition Suppresses Lipopolysaccharide-Induced Lung Inflammation In Vivo

PubMed Central

Lilja, Andrew; Weeden, Clare E.; McArthur, Kate; Nguyen, Thao; Donald, Alastair; Wong, Zi Xin; Dousha, Lovisa; Bozinovski, Steve; Vlahos, Ross; Burns, Christopher J.; Asselin-Labat, Marie-Liesse; Anderson, Gary P.

2015-01-01

Inflammation is an important component of cancer diathesis and treatment-refractory inflammation is a feature of many chronic degenerative lung diseases. HSP90 is a 90kDa protein which functions as an ATP-dependent molecular chaperone that regulates the signalling conformation and expression of multiple protein client proteins especially oncogenic mediators. HSP90 inhibitors are in clinical development as cancer therapies but the myeleosuppressive and neutropenic effect of first generation geldanamycin-class inhibitors has confounded studies on the effects on HSP90 inhibitors on inflammation. To address this we assessed the ability of Ganetespib, a non-geldanamycin HSP90 blocker, to suppress lipopolysaccharide (LPS)-induced cellular infiltrates, proteases and inflammatory mediator and transcriptional profiles. Ganetespib (10–100mg/kg, i.v.) did not directly cause myelosuppression, as assessed by video micrography and basal blood cell count, but it strongly and dose-dependently suppressed LPS-induced neutrophil mobilization into blood and neutrophil- and mononuclear cell-rich steroid-refractory lung inflammation. Ganetespib also suppressed B cell and NK cell accumulation, inflammatory cytokine and chemokine induction and MMP9 levels. These data identify non-myelosuppresssive HSP90 inhibitors as potential therapies for inflammatory diseases refractory to conventional therapy, in particular those of the lung. PMID:25615645

Chronology of UPR activation in skeletal muscle adaptations to chronic contractile activity

PubMed Central

Memme, Jonathan M.; Oliveira, Ashley N.

2016-01-01

The mitochondrial and endoplasmic reticulum unfolded protein responses (UPRmt and UPRER) are important for cellular homeostasis during stimulus-induced increases in protein synthesis. Exercise triggers the synthesis of mitochondrial proteins, regulated in part by peroxisome proliferator activator receptor-γ coactivator 1α (PGC-1α). To investigate the role of the UPR in exercise-induced adaptations, we subjected rats to 3 h of chronic contractile activity (CCA) for 1, 2, 3, 5, or 7 days followed by 3 h of recovery. Mitochondrial biogenesis signaling, through PGC-1α mRNA, increased 14-fold after 1 day of CCA. This resulted in 10–32% increases in cytochrome c oxidase activity, indicative of mitochondrial content, between days 3 and 7, as well as increases in the autophagic degradation of p62 and microtubule-associated proteins 1A/1B light chain 3A (LC3)-II protein. Before these adaptations, the UPRER transcripts activating transcription factor-4, spliced X-box-binding protein 1, and binding immunoglobulin protein were elevated (1.3- to 3.8-fold) at days 1–3, while CCAAT/enhancer-binding protein homologous protein (CHOP) and chaperones binding immunoglobulin protein and heat shock protein (HSP) 70 were elevated at mRNA and protein levels (1.5- to 3.9-fold) at days 1–7 of CCA. The mitochondrial chaperones 10-kDa chaperonin, HSP60, and 75-kDa mitochondrial HSP, the protease ATP-dependent Clp protease proteolytic subunit, and the regulatory protein sirtuin-3 of the UPRmt were concurrently induced 10–80% between days 1 and 7. To test the role of the UPR in CCA-induced remodeling, we treated animals with the endoplasmic reticulum stress suppressor tauroursodeoxycholic acid and subjected them to 2 or 7 days of CCA. Tauroursodeoxycholic acid attenuated CHOP and HSP70 protein induction; however, this failed to impact mitochondrial remodeling. Our data indicate that signaling to the UPR is rapidly activated following acute contractile activity, that this is attenuated with repeated bouts, and that the UPR is involved in chronic adaptations to CCA; however, this appears to be independent of CHOP signaling. PMID:27122157

Sulforaphane attenuates EGFR signaling in NSCLC cells.

PubMed

Chen, Chi-Yuan; Yu, Zhu-Yun; Chuang, Yen-Shu; Huang, Rui-Mei; Wang, Tzu-Chien V

2015-06-03

EGFR, a receptor tyrosine kinase (RTK), is frequently overexpressed and mutated in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) have been widely used in the treatment of many cancers, including NSCLC. However, intrinsic and acquired resistance to TKI remains a common obstacle. One strategy that may help overcome EGFR-TKI resistance is to target EGFR for degradation. As EGFR is a client protein of heat-shock protein 90 (HSP90) and sulforaphane is known to functionally regulate HSP90, we hypothesized that sulforaphane could attenuate EGFR-related signaling and potentially be used to treat NSCLC. Our study revealed that sulforaphane displayed antitumor activity against NSCLC cells both in vitro and in vivo. The sensitivity of NSCLC cells to sulforaphane appeared to positively correlate with the inhibition of EGFR-related signaling, which was attributed to the increased proteasomal degradation of EGFR. Combined treatment of NSCLC cells with sulforaphane plus another HSP90 inhibitor (17-AAG) enhanced the inhibition of EGFR-related signaling both in vitro and in vivo. We have shown that sulforaphane is a novel inhibitory modulator of EGFR expression and is effective in inhibiting the tumor growth of EGFR-TKI-resistant NSCLC cells. Our findings suggest that sulforaphane should be further explored for its potential clinical applications against NSCLC.

Effects of Thermal Stress on the mRNA Expression of SOD, HSP90, and HSP70 in the Spotted Sea Bass ( Lateolabrax maculatus)

NASA Astrophysics Data System (ADS)

Shin, Moon-Kyeong; Park, Ho-Ra; Yeo, Won-Jun; Han, Kyung-Nam

2018-03-01

The aim of this study was to elucidate the molecular mechanisms underlying the thermal stress response in the spotted sea bass ( Lateolabrax maculatus). Spotted sea basses were exposed to 4 different water temperatures (20, 22, 24, and 28°C) in increasing increments of 2°C/h from 18°C (control) for different time periods (0, 6, 12, 24, 48, 72, and 96 h). Subsequently, 3 tissues (liver, muscle, and gill) were isolated, and the levels of SOD, HSP90, and HSP70 mRNA were assessed. SOD mRNA expression was maintained at baseline levels of control fish at all water temperatures in the liver, while muscle and gill tissue showed an increase followed by a decrease over each certain time with higher water temperature. HSP90 mRNA expression increased in the liver at ≤ 24°C over time, but maintained baseline expression at 28°C. In muscle, HSP90 mRNA expression gradually increased at all water temperatures, but increased and then decreased at ≥ 24°C in gill tissue. HSP70 mRNA expression exhibited an increase and then a decrease in liver tissue at 28°C, but mainly showed similar expression patterns to HSP90 in all tissues. These results suggest the activity of a defense mechanism using SOD, HSP90, and HSP70 in the spotted sea bass upon rapid increases in water temperature, where the expression of these genes indicated differences between tissues in the extent of the defense mechanisms. Also, these results indicate that high water temperature and long-term thermal stress exposure can inhibit physiological defense mechanisms.

Chemical tools to investigate mechanisms associated with HSP90 and HSP70 in disease

PubMed Central

Shrestha, Liza; Patel, Hardik J.; Chiosis, Gabriela

2016-01-01

The chaperome is a large and diverse protein machinery composed of chaperone proteins and a variety of helpers, such as the co-chaperones, folding enzymes and scaffolding and adapter proteins. Heat shock protein 90s and 70s (HSP90s and HSP70s), the most abundant chaperome members in human cells, are also the most complex. As we have learned to appreciate, their functions are context dependent and manifested through a variety of conformations that each recruit a subset of co-chaperone, scaffolding and folding proteins and which are further diversified by the post-translational modifications each carry, making their study through classic genetic and biochemical techniques quite a challenge. Chemical biology tools and techniques have been developed over the years to help decipher the complexities of the HSPs and this review will provide an overview of such efforts with focus on HSP90 and HSP70. PMID:26933742

Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Suyeun; Jang, Yeogil; Paik, Donggi

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan.more » - Highlights: • Ubiquitous Nmdmc overexpression enhanced lifespan and stress tolerance. • Nmdmc overexpression in the fat body extended longevity. • Fat body-specific Nmdmc overexpression increased oxidative stress resistance. • Nmdmc overexpression decreased Hsp22 transcript levels and ROS. • Nmdmc overexpression increased mitochondrial DNA copy number.« less

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.

Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90.

PubMed

Burlison, Joseph A; Neckers, Len; Smith, Andrew B; Maxwell, Anthony; Blagg, Brian S J

2006-12-06

Novobiocin is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. Recent studies have shown that novobiocin binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. In an effort to develop more efficacious inhibitors of the C-terminal binding site, a library of novobiocin analogues was prepared and initial structure-activity relationships revealed. These data suggested that the 4-hydroxy moiety of the coumarin ring and the 3'-carbamate of the noviose appendage were detrimental to Hsp90 inhibitory activity. In an effort to confirm these findings, 4-deshydroxy novobiocin (DHN1) and 3'-descarbamoyl-4-deshydroxynovobiocin (DHN2) were prepared and evaluated against Hsp90. Both compounds were significantly more potent than the natural product, and DHN2 proved to be more active than DHN1. In an effort to determine whether these moieties are important for DNA gyrase inhibition, these compounds were tested for their ability to inhibit DNA gyrase and found to exhibit significant reduction in gyrase activity. Thus, we have established the first set of compounds that clearly differentiate between the C-terminus of Hsp90 and DNA gyrase, converted a well-established gyrase inhibitor into a selective Hsp90 inhibitor, and confirmed essential structure-activity relationships for the coumermycin family of antibiotics.

Role for Heat Shock Protein 90α in the Proliferation and Migration of HaCaT Cells and in the Deep Second-Degree Burn Wound Healing in Mice

PubMed Central

Li, Na; Li, Xiaoqiang; Han, Fei; Su, Linlin; Hu, Dahai

2014-01-01

Inflammation, proliferation, and tissue remodeling are essential steps for wound healing. The hypoxic wound microenvironment promotes cell migration through a hypoxia—heat shock protein 90 alpha (Hsp90α)—low density lipoprotein receptor-related protein-1 (LRP-1) autocrine loop. To elucidate the role of this autocrine loop on burn wound healing, we investigated the expression profile of Hsp90α at the edge of burn wounds and found a transient increase in both mRNA and protein levels. Experiments performed with a human keratinocyte cell line—HaCaT also confirmed above results. 17-dimethylaminoethylamino-17demethoxygeldanamycin hydrochloride (17-DMAG), an Hsp90α inhibitor, was used to further evaluate the function of Hsp90α in wound healing. Consistently, topical application of Hsp90α in the early stage of deep second-degree burn wounds led to reduced inflammation and increased tissue granulation, with a concomitant reduction in the size of the wound at each time point tested (p<0.05). Consequently, epidermal cells at the wound margin progressed more rapidly causing an expedited healing process. In conclusion, these results provided a rationale for the therapeutic effect of Hsp90α on the burn wound management. PMID:25111496

Interspecific- and acclimation-induced variation in levels of heat-shock proteins 70 (hsp70) and 90 (hsp90) and heat-shock transcription factor-1 (HSF1) in congeneric marine snails (genus Tegula): implications for regulation of hsp gene expression.

PubMed

Tomanek, Lars; Somero, George N

2002-03-01

In our previous studies of heat-shock protein (hsp) expression in congeneric marine gastropods of the genus Tegula, we observed interspecific and acclimation-induced variation in the temperatures at which heat-shock gene expression is induced (T(on)). To investigate the factors responsible for these inter- and intraspecific differences in T(on), we tested the predictions of the 'cellular thermometer' model for the transcriptional regulation of hsp expression. According to this model, hsps not active in chaperoning unfolded proteins bind to a transcription factor, heat-shock factor-1 (HSF1), thereby reducing the levels of free HSF1 that are available to bind to the heat-shock element, a regulatory element upstream of hsp genes. Under stress, hsps bind to denatured proteins, releasing HSF1, which can now activate hsp gene transcription. Thus, elevated levels of heat-shock proteins of the 40, 70 and 90 kDa families (hsp 40, hsp70 and hsp90, respectively) would be predicted to elevate T(on). Conversely, elevated levels of HSF1 would be predicted to decrease T(on). Following laboratory acclimation to 13, 18 and 23 degrees C, we used solid-phase immunochemistry (western analysis) to quantify endogenous levels of two hsp70 isoforms (hsp74 and hsp72), hsp90 and HSF1 in the low- to mid-intertidal species Tegula funebralis and in two subtidal to low-intertidal congeners, T. brunnea and T. montereyi. We found higher endogenous levels of hsp72 (a strongly heat-induced isoform) at 13 and 18 degrees C in T. funebralis in comparison with T. brunnea and T. montereyi. However, T. funebralis also had higher levels of HSF1 than its congeners. The higher levels of HSF1 in T. funebralis cannot, within the framework of the cellular thermometer model, account for the higher T(on) observed for this species, although they may explain why T. funebralis is able to induce the heat-shock response more rapidly than T. brunnea. However, the cellular thermometer model does appear to explain the cause of the increases in T(on) that occurred during warm acclimation of the two subtidal species, in which warm acclimation was accompanied by increased levels of hsp72, hsp74 and hsp90, whereas levels of HSF1 remained stable. T. funebralis, which experiences greater heat stress than its subtidal congeners, consistently had higher ratios of hsp72 to hsp74 than its congeners, although the sum of levels of the two isoforms was similar for all three species except at the highest acclimation temperature (23 degrees C). The ratio of hsp72 to hsp74 may provide a more accurate estimate of environmental heat stress than the total concentrations of both hsp70 isoforms.

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

TaHsfA6f is a transcriptional activator that regulates a suite of heat stress protection genes in wheat (Triticum aestivum L.) including previously unknown Hsf targets

PubMed Central

Xue, Gang-Ping; Drenth, Janneke; McIntyre, C. Lynne

2015-01-01

Heat stress is a significant environmental factor adversely affecting crop yield. Crop adaptation to high-temperature environments requires transcriptional reprogramming of a suite of genes involved in heat stress protection. This study investigated the role of TaHsfA6f, a member of the A6 subclass of heat shock transcription factors, in the regulation of heat stress protection genes in Triticum aestivum (bread wheat), a poorly understood phenomenon in this crop species. Expression analysis showed that TaHsfA6f was expressed constitutively in green organs but was markedly up-regulated during heat stress. Overexpression of TaHsfA6f in transgenic wheat using a drought-inducible promoter resulted in up-regulation of heat shock proteins (HSPs) and a number of other heat stress protection genes that included some previously unknown Hsf target genes such as Golgi anti-apoptotic protein (GAAP) and the large isoform of Rubisco activase. Transgenic wheat plants overexpressing TaHsfA6f showed improved thermotolerance. Transactivation assays showed that TaHsfA6f activated the expression of reporter genes driven by the promoters of several HSP genes (TaHSP16.8, TaHSP17, TaHSP17.3, and TaHSP90.1-A1) as well as TaGAAP and TaRof1 (a co-chaperone) under non-stress conditions. DNA binding analysis revealed the presence of high-affinity TaHsfA6f-binding heat shock element-like motifs in the promoters of these six genes. Promoter truncation and mutagenesis analyses identified TaHsfA6f-binding elements that were responsible for transactivation of TaHSP90.1-A1 and TaGAAP by TaHsfA6f. These data suggest that TaHsfA6f is a transcriptional activator that directly regulates TaHSP, TaGAAP, and TaRof1 genes in wheat and its gene regulatory network has a positive impact on thermotolerance. PMID:25428996

TaHsfA6f is a transcriptional activator that regulates a suite of heat stress protection genes in wheat (Triticum aestivum L.) including previously unknown Hsf targets.

PubMed

Xue, Gang-Ping; Drenth, Janneke; McIntyre, C Lynne

2015-02-01

Heat stress is a significant environmental factor adversely affecting crop yield. Crop adaptation to high-temperature environments requires transcriptional reprogramming of a suite of genes involved in heat stress protection. This study investigated the role of TaHsfA6f, a member of the A6 subclass of heat shock transcription factors, in the regulation of heat stress protection genes in Triticum aestivum (bread wheat), a poorly understood phenomenon in this crop species. Expression analysis showed that TaHsfA6f was expressed constitutively in green organs but was markedly up-regulated during heat stress. Overexpression of TaHsfA6f in transgenic wheat using a drought-inducible promoter resulted in up-regulation of heat shock proteins (HSPs) and a number of other heat stress protection genes that included some previously unknown Hsf target genes such as Golgi anti-apoptotic protein (GAAP) and the large isoform of Rubisco activase. Transgenic wheat plants overexpressing TaHsfA6f showed improved thermotolerance. Transactivation assays showed that TaHsfA6f activated the expression of reporter genes driven by the promoters of several HSP genes (TaHSP16.8, TaHSP17, TaHSP17.3, and TaHSP90.1-A1) as well as TaGAAP and TaRof1 (a co-chaperone) under non-stress conditions. DNA binding analysis revealed the presence of high-affinity TaHsfA6f-binding heat shock element-like motifs in the promoters of these six genes. Promoter truncation and mutagenesis analyses identified TaHsfA6f-binding elements that were responsible for transactivation of TaHSP90.1-A1 and TaGAAP by TaHsfA6f. These data suggest that TaHsfA6f is a transcriptional activator that directly regulates TaHSP, TaGAAP, and TaRof1 genes in wheat and its gene regulatory network has a positive impact on thermotolerance. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

A non-toxic Hsp90 inhibitor protects neurons from Abeta-induced toxicity.

PubMed

Ansar, Sabah; Burlison, Joseph A; Hadden, M Kyle; Yu, Xiao Ming; Desino, Kelly E; Bean, Jennifer; Neckers, Len; Audus, Ken L; Michaelis, Mary L; Blagg, Brian S J

2007-04-01

The molecular chaperones have been implicated in numerous neurodegenerative disorders in which the defining pathology is misfolded proteins and the accumulation of protein aggregates. In Alzheimer's disease, hyperphosphorylation of tau protein results in its dissociation from microtubules and the formation of pathogenic aggregates. An inverse relationship was demonstrated between Hsp90/Hsp70 levels and aggregated tau, suggesting that Hsp90 inhibitors that upregulate these chaperones could provide neuroprotection. We recently identified a small molecule novobiocin analogue, A4 that induces Hsp90 overexpression at low nanomolar concentrations and sought to test its neuroprotective properties. A4 protected neurons against Abeta-induced toxicity at low nanomolar concentrations that paralleled its ability to upregulate Hsp70 expression. A4 exhibited no cytotoxicity in neuronal cells at the highest concentration tested, 10 microM, thus providing a large therapeutic window for neuroprotection. In addition, A4 was transported across BMECs in vitro, suggesting the compound may permeate the blood-brain barrier in vivo. Taken together, these data establish A4, a C-terminal inhibitor of Hsp90, as a potent lead for the development of a novel class of compounds to treat Alzheimer's disease.

Spontaneous assembly of HSP90 inhibitors at water/octanol interface: A molecular dynamics simulation study

NASA Astrophysics Data System (ADS)

Zolghadr, Amin Reza; Boroomand, Samaneh

2017-02-01

Drug absorption at an acceptable dose depends on the pair of solubility and permeability. There are many potent therapeutics that are not active in vivo, presumably due to the lack of capability to cross the cell membrane. Molecular dynamics simulation of radicicol, diol-radicicol, cyclopropane-radicicol and 17-DMAG were performed at water/octanol interface to suggest interfacial activity as a physico-chemical characteristic of these heat shock protein 90 (HSP90) inhibitors. We have observed that orally active HSP90 inhibitors form aggregates at the water/octanol and DPPC-lipid/water interfaces by starting from an initial configuration with HSP90 inhibitors embedded in the water matrix.

Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

PubMed Central

Kodys, Karen; Bala, Shashi; Szabo, Gyongyi

2014-01-01

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies. PMID:25275643

The Forkhead Transcription Factor Hcm1 Promotes Mitochondrial Biogenesis and Stress Resistance in Yeast*

PubMed Central

Rodriguez-Colman, Maria José; Reverter-Branchat, Gemma; Sorolla, M. Alba; Tamarit, Jordi; Ros, Joaquim; Cabiscol, Elisa

2010-01-01

In Saccharomyces cerevisiae, the forkhead transcription factor Hcm1 is involved in chromosome segregation, spindle pole dynamics, and budding. We found that Hcm1 interacts with the histone deacetylase Sir2 and shifts from cytoplasm to the nucleus in the G1/S phase or in response to oxidative stress stimuli. The nuclear localization of Hcm1 depends on the activity of Sir2 as revealed by activators and inhibitors of the sirtuins and the Δsir2 mutant. Hcm1-overexpressing cells display more mitochondria that can be attributed to increased amounts of Abf2, a protein involved in mitochondrial biogenesis. These cells also show higher rates of oxygen consumption and improved resistance to oxidative stress that would be explained by increased catalase and Sod2 activities and molecular chaperones such as Hsp26, Hsp30, and members of Hsp70 family. Microarray analyses also reveal increased expression of genes involved in mitochondrial energy pathways and those allowing the transition from the exponential to the stationary phase. Taken together, these results describe a new and relevant role of Hcm1 for mitochondrial functions, suggesting that this transcription factor would participate in the adaptation of cells from fermentative to respiratory metabolism. PMID:20847055

The association of SNPs in Hsp90β gene 5' flanking region with thermo tolerance traits and tissue mRNA expression in two chicken breeds.

PubMed

Chen, Zhuo-Yu; Gan, Jian-Kang; Xiao, Xiong; Jiang, Li-Yan; Zhang, Xi-Quan; Luo, Qing-Bin

2013-09-01

Thermo stress induces heat shock proteins (HSPs) expression and HSP90 family is one of them that has been reported to involve in cellular protection against heat stress. But whether there is any association of genetic variation in the Hsp90β gene in chicken with thermo tolerance is still unknown. Direct sequencing was used to detect possible SNPs in Hsp90β gene 5' flanking region in 3 chicken breeds (n = 663). Six mutations, among which 2 SNPs were chosen and genotypes were analyzed with PCR-RFLP method, were found in Hsp90β gene in these 3 chicken breeds. Association analysis indicated that SNP of C.-141G>A in the 5' flanking region of the Hsp90β gene in chicken had some effect on thermo tolerance traits, which may be a potential molecular marker of thermo tolerance, and the genotype GG was the thermo tolerance genotype. Hsp90β gene mRNA expression in different tissues detected by quantitative real-time PCR assay were demonstrated to be tissue dependent, implying that different tissues have distinct sensibilities to thermo stress. Besides, it was shown time specific and varieties differences. The expression of Hsp90β mRNA in Lingshan chickens in some tissues including heart, liver, brain and spleen were significantly higher or lower than that of White Recessive Rock (WRR). In this study, we presume that these mutations could be used in marker assisted selection for anti-heat stress chickens in our breeding program, and WRR were vulnerable to tropical thermo stress whereas Lingshan chickens were well adapted.

Key role of heat shock protein 90 in leptin‐induced STAT3 activation and feeding regulation

PubMed Central

Kohda, Toshiko; Matsuzaki, Syu; Ishiguchi, Mizuho; Kuwamura, Ayaka; Akita, Tomoyuki; Tanaka, Junko

2016-01-01

Abstract Background and Purpose Leptin, an important regulator of the energy balance, acts on the brain to inhibit feeding. However, the mechanisms involved in leptin signalling have not yet been fully elucidated. Heat shock protein 90 (HSP90) is a molecular chaperone that is involved in regulating cellular homeostasis. In the present study, we investigated the possible involvement of HSP90 in leptin signal transduction. Experimental Approach HEK293 and SH‐SY5Y cell lines stably transfected with the Ob‐Rb leptin receptor (HEK293 Ob‐Rb, SH‐SY5Y Ob‐Rb) were used in the present study. Phosphorylation of JAK2 and STAT3 was analysed by western blotting. An HSP90 inhibitor was administered i.c.v. into rats and their food intake was analysed. Key Results The knock‐down of HSP90 in the HEK293 Ob‐Rb cell line attenuated leptin‐induced JAK2 and STAT3 signalling. Moreover, leptin‐induced JAK2/STAT3 phosphorylation was markedly attenuated by the HSP90 inhibitors geldanamycin, radicicol and novobiocin. However, these effects were not mediated through previously known factors, which are known to be involved in the development of leptin resistance, such as suppressor of cytokine signalling 3 or endoplasmic reticulum stress. The infusion of an HSP90 inhibitor into the CNS blunted the anorexigenic actions of leptin in rats (male Wister rat). Conclusions and Implications HSP90 may be a novel factor involved in leptin‐mediated signalling that is linked to anorexia. PMID:27205876

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

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.

Cardiac-specific knockout of ETA receptor mitigates low ambient temperature-induced cardiac hypertrophy and contractile dysfunction

PubMed Central

Zhang, Yingmei; Li, Linlin; Hua, Yinan; Nunn, Jennifer M.; Dong, Feng; Yanagisawa, Masashi; Ren, Jun

2012-01-01

Cold exposure is associated with oxidative stress and cardiac dysfunction. The endothelin (ET) system, which plays a key role in myocardial homeostasis, may participate in cold exposure-induced cardiovascular dysfunction. This study was designed to examine the role of ET-1 in cold stress-induced cardiac geometric and contractile responses. Wild-type (WT) and ETA receptor knockout (ETAKO) mice were assigned to normal or cold exposure (4°C) environment for 2 and 5 weeks prior to evaluation of cardiac geometry, contractile, and intracellular Ca2+ properties. Levels of the temperature sensor transient receptor potential vanilloid (TRPV1), mitochondrial proteins for biogenesis and oxidative phosphorylation, including UCP2, HSP90, and PGC1α were evaluated. Cold stress triggered cardiac hypertrophy, depressed myocardial contractile capacity, including fractional shortening, peak shortening, and maximal velocity of shortening/relengthening, reduced intracellular Ca2+ release, prolonged intracellular Ca2+ decay and relengthening duration, generation of ROS and superoxide, as well as apoptosis, the effects of which were blunted by ETAKO. Western blotting revealed downregulated TRPV1 and PGC1α as well as upregulated UCP2 and activation of GSK3β, GATA4, and CREB in cold-stressed WT mouse hearts, which were obliterated by ETAKO. Levels of HSP90, an essential regulator for thermotolerance, were unchanged. The TRPV1 agonist SA13353 attenuated whereas TRPV1 antagonist capsazepine mimicked cold stress- or ET-1-induced cardiac anomalies. The GSK3β inhibitor SB216763 ablated cold stress-induced cardiac contractile (but not remodeling) changes and ET-1-induced TRPV1 downregulation. These data suggest that ETAKO protects against cold exposure-induced cardiac remodeling and dysfunction mediated through TRPV1 and mitochondrial function. PMID:22442497

Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride.

PubMed

Panneerselvam, Lakshmikanthan; Raghunath, Azhwar; Perumal, Ekambaram

2017-09-01

Acute fluoride (F - ) toxicity is known to cause severe cardiac complications and leads to sudden heart failure. Previously, we reported that increased myocardial oxidative damage, apoptosis, altered cytoskeleton and AMPK signaling proteins associated with energy deprivation in acute F - induced cardiac dysfunction. The present study was aimed to decipher the status of myocardial heat shock proteins (Hsps-Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, Hsp90) and heat shock transcription factor 1 (Hsf1) in acute F - -intoxicated rats. In order to study the expression of myocardial Hsps, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F - for 24 h. The expression levels of myocardial Hsps were determined using RT-PCR, western blotting, and immunohistochemical studies. Acute F - -intoxicated rats showed elevated levels of both the transcripts and protein expression of Hsf1, Hsp27, Hsp32, Hsp60, and Hsp70 when compared to control. In addition, the expression levels of Hsp40 and Hsp90 were significantly declined in a dose-dependent fashion in F - -treated animals. Our result suggests that differential expression of Hsps in the rat myocardium could serve as a balance between pro-survival and death signal during acute F - -induced heart failure.

HSP90, HSPA8, HIF-1 alpha and HSP70-2 polymorphisms in breast cancer: a case-control study.

PubMed

Zagouri, Flora; Sergentanis, Theodoros N; Gazouli, Maria; Tsigginou, Alexandra; Dimitrakakis, Constantine; Papaspyrou, Irene; Eleutherakis-Papaiakovou, Evaggelos; Chrysikos, Dimosthenis; Theodoropoulos, George; Zografos, George C; Antsaklis, Aris; Dimopoulos, Athanassios-Meletios; Papadimitriou, Christos A

2012-12-01

This case control study aims to investigate the role of HSP90 Gln488His (C > G), HSP70-2 P1/P2, HIF-1 alpha C1772T and HSPA8 intronic 1541-1542delGT polymorphisms as potential risk factors and/or prognostic markers for breast cancer. 113 consecutive incident cases of histologically confirmed ductal breast cancer and 124 healthy cases were recruited. The above mentioned polymorphisms were genotyped; multivariate logistic regression was performed. HSP90 GG (His/His) genotype was associated with elevated breast cancer risk. Similarly, the allele dose-response model pointed to increase in breast cancer risk per G allele. HSP70-2 P1/P2, HSPA8 intronic 1541-1542delGT and HIF-1 alpha polymorphisms were not associated with breast cancer risk, as evidenced by the dose-response allele models. The positive association between HSP90 G allele and breast cancer risk seemed to pertain to both premenopausal and postmenopausal women. With respect to survival analysis, none of the aforementioned polymorphisms was associated with either disease-free survival or overall survival. HSP90α Gln488His polymorphism seems to be a risk factor for breast cancer. On the other hand, our study did not point to excess risk conferred by HSPA8 1541-1542delGT, Hsp70-2 P1/P2 and HIF-1α C1772T.

Purification and comparison of heat shock protein 90 (Hsp90) in Candida albicans isolates from Malaysian and Iranian patients and infected mice.

PubMed

Khalili, V; Shokri, H; Khosravi, A R; Akim, A; Amri Saroukolaei, S

2016-06-01

The purposes of this study were to purify and compare the concentration ratios of heat shock protein 90 (Hsp90) in clinical isolates of Candida albicans (C. albicans) obtained from Malaysian and Iranian patients and infected mice. Hsp90 was extracted using glass beads and ultracentrifugation from yeast cells and purified by ion exchange chromatography (DEAE-cellulose) and followed by affinity chromatography (hydroxyapatite). Purity of Hsp90 was controlled by SDS-PAGE and its identification was realized by immunoblotting test. The graphs of ion exchange and affinity chromatography showed one peak in all C. albicans isolates obtained from both Malaysian and Iranian samples, infected mice and under high-thermal (42°C) and low-thermal (25°C) shock. In immunoblotting, the location of Hsp90 fragments was obtained around 47, 75 and 82kDa. The least average concentration ratios of Hsp90 were 0.350 and 0.240mg/g for Malaysian and Iranian isolates at 25°C, respectively, while the highest average concentration ratios of Hsp90 were 3.05 and 2.600mg/g for Malaysian and Iranian isolates at 42°C, respectively. There were differences in the ratio amount of Hsp90 between Malaysian isolates (1.01±0.07mg/g) and mice kidneys (1.23±0.28mg/g) as well as between Iranian isolates (0.70±0.19mg/g) and mice kidneys (1.00±0.28mg/g) (P<0.05). The results showed differences in all situations tested including Iranian and Malaysian isolates, samples treated with temperatures (25°C or 42°C) and before and after infecting the mice (37°C), indicating higher virulent nature of this yeast species in high temperature in human and animal models. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Cardiac-Specific Knockout of ETA Receptor Mitigates Paraquat-Induced Cardiac Contractile Dysfunction.

PubMed

Wang, Jiaxing; Lu, Songhe; Zheng, Qijun; Hu, Nan; Yu, Wenjun; Li, Na; Liu, Min; Gao, Beilei; Zhang, Guoyong; Zhang, Yingmei; Wang, Haichang

2016-07-01

Paraquat (1,1'-dim ethyl-4-4'-bipyridinium dichloride), a highly toxic quaternary ammonium herbicide widely used in agriculture, exerts potent toxic prooxidant effects resulting in multi-organ failure including the lung and heart although the underlying mechanism remains elusive. Recent evidence suggests possible involvement of endothelin system in paraquat-induced acute lung injury. This study was designed to examine the role of endothelin receptor A (ETA) in paraquat-induced cardiac contractile and mitochondrial injury. Wild-type (WT) and cardiac-specific ETA receptor knockout mice were challenged to paraquat (45 mg/kg, i.p.) for 48 h prior to the assessment of echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, as well as apoptosis and mitochondrial damage. Levels of the mitochondrial proteins for biogenesis and oxidative phosphorylation including UCP2, HSP90 and PGC1α were evaluated. Our results revealed that paraquat elicited cardiac enlargement, mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic and end-diastolic diameters as well as reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, overt apoptosis and mitochondrial damage. ETA receptor knockout itself failed to affect myocardial function, apoptosis, mitochondrial integrity and mitochondrial protein expression. However, ETA receptor knockout ablated or significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) defect, apoptosis and mitochondrial damage. Taken together, these findings revealed that endothelin system in particular the ETA receptor may be involved in paraquat-induced toxic myocardial contractile anomalies possibly related to apoptosis and mitochondrial damage.

Protein, carbohydrate, lipid concentrations and HSP 70-HSP 90 (stress protein) expression over an annual cycle: useful tools to detect feeding constraints in a benthic suspension feeder

NASA Astrophysics Data System (ADS)

Rossi, Sergio; Snyder, Mark J.; Gili, Josep-Marìa

2006-03-01

In the present paper we suggest an effect of seasonal variations in food availability on two ecophysiological parameters in a warm temperate benthic suspension feeder: the tissue concentrations of proteins, carbohydrates and lipids on the one hand, and the expression of stress proteins (HSP 70 and 90, inducible and/or constitutive) on the other hand. The concentrations of biomacromolecules have already been used to describe bentho-pelagic and reproductive processes, but this is the first time that stress protein expression is suggested to be directly related with food constraints in marine organisms. Paramuricea clavata (Cnidaria: Gorgonacea) express HSP 70 and 90 (constitutive and/or inducible) throughout the seasonal cycle, and HSP 70 levels are twice as high as the levels of HSP 90. In summer and autumn, when seston availability to suspension feeders was low, P. clavata showed low levels of carbohydrates and lipids, but high levels of HSPs expression. The levels of HSP 70 and 90 expression fit with negative exponential functions of carbohydrate and lipid concentrations. We suggest a direct effect of food availability on the studied ecophysiological parameters while the effect of temperature may be rather indirect. HSP expression as well as the tissue concentrations of carbohydrate and lipids may be used as biomarkers of environmental changes and seston availability to benthic suspension feeders.

Adjuvant effect of polysaccharide from fruits of Physalis alkekengi L. in DNA vaccine against systemic candidiasis.

PubMed

Yang, Huimin; Han, Shuying; Zhao, Danyang; Wang, Guiyun

2014-08-30

Adjuvant effect mediated by polysaccharide (PPSB) isolated from the fruits of Physalis alkekengi L. in DNA vaccine was evaluated in mice. Recombinant plasmid containing epitope C (LKVIRK) from heat shock protein 90 (HSP90) of Candida albicans (C. albican) was used as DNA vaccine (pD-HSP90C). The results indicated that PPSB significantly enhanced specific antibody titers IgG, IgG1, IgG2b, and concentration of IL-2 and IL-4 in sera of mice immunized with pD-HSP90C (p<0.05). More importantly, it was found that the mice immunized with pD-HSP90C/PPSB not only had fewer CFU (colony forming unites) in the kidneys than mice immunized with pD-HSP90C, but also a statistically significant higher survival rate over PBS-injected group (p<0.05) when the immunized mice were challenged with living C. albican cells. However, no statistically significant difference in survival rate was observed between pD-HSP90C-immunized group and PBS-injected group. Therefore, PPSB can be considered as a promising adjuvant eliciting both Th1 and Th2 responses to enhance the efficacy of DNA vaccines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster.

PubMed

Landis, Gary; Shen, Jie; Tower, John

2012-11-01

Gene expression changes in response to aging, heat stress, hyperoxia, hydrogen peroxide, and ionizing radiation were compared using microarrays. A set of 18 genes were up-regulated across all conditions, indicating a general stress response shared with aging, including the heat shock protein (Hsp) genes Hsp70, Hsp83 and l(2)efl, the glutathione-S-transferase gene GstD2, and the mitochondrial unfolded protein response (mUPR) gene ref(2)P. Selected gene expression changes were confirmed using quantitative PCR, Northern analysis and GstD-GFP reporter constructs. Certain genes were altered in only a subset of the conditions, for example, up-regulation of numerous developmental pathway and signaling genes in response to hydrogen peroxide. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hyperoxia, hydrogen peroxide, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging.

Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster

PubMed Central

Landis, Gary; Shen, Jie; Tower, John

2012-01-01

Gene expression changes in response to aging, heat stress, hyperoxia, hydrogen peroxide, and ionizing radiation were compared using microarrays. A set of 18 genes were up-regulated across all conditions, indicating a general stress response shared with aging, including the heat shock protein (Hsp) genes Hsp70, Hsp83 and l(2)efl, the glutathione-S-transferase gene GstD2, and the mitochondrial unfolded protein response (mUPR) gene ref(2)P. Selected gene expression changes were confirmed using quantitative PCR, Northern analysis and GstD-GFP reporter constructs. Certain genes were altered in only a subset of the conditions, for example, up-regulation of numerous developmental pathway and signaling genes in response to hydrogen peroxide. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hyperoxia, hydrogen peroxide, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging. PMID:23211361

Essential oil from Cymbopogon flexuosus as the potential inhibitor for HSP90.

PubMed

Gaonkar, Roopa; Shiralgi, Yallappa; Lakkappa, Dhananjaya B; Hegde, Gurumurthy

2018-01-01

The essential oil of Cymbopogon flexuosus or lemongrass oil (LO) is reported to have antibacterial, antifungal and anticancerous effects. HSP90 is one of the major chaperones responsible for the proper folding of cancer proteins. In this paper we show that the essential oil of C. flexuosus significantly suppresses the HSP90 gene expression. The cytotoxicity of the compounds was tested by MTT assay and the gene expression studies were carried out using HEK-293 and MCF-7 cells. Also we tested the efficacy of the major component of this essential oil viz. citral and geraniol in inhibiting the HSP90 expression. The oil was found to be more cytotoxic to MCF-7 cells with different IC 50 values for the oil (69.33 μg/mL), citral (140.7 μg/mL) and geraniol (117 μg/mL). The fold change of expression was calculated by RT-qPCR using ΔΔCt (2 ^-ΔΔCt ) method and it was 0.1 and 0.03 in MCF-7 cells at 80 μg/mL and 160 μg/mL of LO. Western blot results showed suppression of HSP90 protein expression and HSP90 - ATPase activity inhibition was also observed using LO. This study shows the anticancer mechanism exhibited by the essential oil of C. flexuosus is by the inhibition of the important chaperone protein HSP90.

Loss of Smyhc1 or Hsp90α1 Function Results in Different Effects on Myofibril Organization in Skeletal Muscles of Zebrafish Embryos

PubMed Central

Codina, Marta; Li, Junling; Gutiérrez, Joaquim; Kao, Joseph P. Y.; Du, Shao Jun

2010-01-01

Background Myofibrillogenesis requires the correct folding and assembly of sarcomeric proteins into highly organized sarcomeres. Heat shock protein 90α1 (Hsp90α1) has been implicated as a myosin chaperone that plays a key role in myofibrillogenesis. Knockdown or mutation of hsp90α1 resulted in complete disorganization of thick and thin filaments and M- and Z-line structures. It is not clear whether the disorganization of these sarcomeric structures is due to a direct effect from loss of Hsp90α1 function or indirectly through the disorganization of myosin thick filaments. Methodology/Principal Findings In this study, we carried out a loss-of-function analysis of myosin thick filaments via gene-specific knockdown or using a myosin ATPase inhibitor BTS (N-benzyl-p-toluene sulphonamide) in zebrafish embryos. We demonstrated that knockdown of myosin heavy chain 1 (myhc1) resulted in sarcomeric defects in the thick and thin filaments and defective alignment of Z-lines. Similarly, treating zebrafish embryos with BTS disrupted thick and thin filament organization, with little effect on the M- and Z-lines. In contrast, loss of Hsp90α1 function completely disrupted all sarcomeric structures including both thick and thin filaments as well as the M- and Z-lines. Conclusion/Significance Together, these studies indicate that the hsp90α1 mutant phenotype is not simply due to disruption of myosin folding and assembly, suggesting that Hsp90α1 may play a role in the assembly and organization of other sarcomeric structures. PMID:20049323

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 Central

Gao, Linlin

2013-01-01

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. PMID:24109220

Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development

PubMed Central

Moon, Robert W.; Whalley, David; Bowyer, Paul W.; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K.; Howell, Steven A.; Grainger, Munira; Jones, Hayley M.; Ansell, Keith H.; Chapman, Timothy M.; Taylor, Debra L.; Osborne, Simon A.; Baker, David A.; Tatu, Utpal

2015-01-01

Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. PMID:26711771

Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development.

PubMed

Green, Judith L; Moon, Robert W; Whalley, David; Bowyer, Paul W; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K; Howell, Steven A; Grainger, Munira; Jones, Hayley M; Ansell, Keith H; Chapman, Timothy M; Taylor, Debra L; Osborne, Simon A; Baker, David A; Tatu, Utpal; Holder, Anthony A

2015-12-28

Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. Copyright © 2016 Green et al.

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion.

PubMed

Garg, Manoj; Kanojia, Deepika; Seth, Amlesh; Kumar, Rajive; Gupta, Anju; Surolia, Avadhesha; Suri, Anil

2010-01-01

Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma.

Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensable in vivo and conserved in evolution.

PubMed

Ciesielski, Szymon J; Schilke, Brenda A; Osipiuk, Jerzy; Bigelow, Lance; Mulligan, Rory; Majewska, Julia; Joachimiak, Andrzej; Marszalek, Jaroslaw; Craig, Elizabeth A; Dutkiewicz, Rafal

2012-03-16

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70's ATPase activity. However, in apparent contradiction, we previously reported that an 8-fold decrease in Jac1's affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here, we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, we determined that a total of eight surface-exposed residues play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype, and a reduction in the activity of Fe-S cluster-containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensable role in the essential process of mitochondrial Fe-S cluster biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensible in vivo and conserved in evolution

PubMed Central

Ciesielski, Szymon; Schilke, Brenda; Osipiuk, Jerzy; Bigelow, Lance; Mulligan, Rory; Majewska, Julia; Joachimiak, Andrzej; Marszalek, Jaroslaw; Craig, Elizabeth A.; Dutkiewicz, Rafal

2012-01-01

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70’s ATPase activity. However, in apparent contradiction, we previously reported that an 8 fold decrease in Jac1’s affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, a total of eight surface exposed residues were determined to play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype and a reduction in the activity of Fe-S cluster containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensible role in the essential process of mitochondrial Fe-S cluster biogenesis. PMID:22306468

Potential contributions of heat shock proteins to temperature-dependent sex determination in the American alligator.

PubMed

Kohno, S; Katsu, Y; Urushitani, H; Ohta, Y; Iguchi, T; Guillette, L J

2010-01-01

Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be 'reversed' by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90alpha, HSP90beta, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90 alpha (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed. (c) 2009 S. Karger AG, Basel.

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.

Drug-Induced HSP90 Inhibition Alleviates Pain in Monoarthritic Rats and Alters the Expression of New Putative Pain Players at the DRG.

PubMed

Nascimento, Diana Sofia Marques; Potes, Catarina Soares; Soares, Miguel Luz; Ferreira, António Carlos; Malcangio, Marzia; Castro-Lopes, José Manuel; Neto, Fani Lourença Moreira

2018-05-01

Purinergic receptors (P2XRs) have been widely associated with pain states mostly due to their involvement in neuron-glia communication. Interestingly, we have previously shown that satellite glial cells (SGC), surrounding dorsal root ganglia (DRG) neurons, become activated and proliferate during monoarthritis (MA) in the rat. Here, we demonstrate that P2X7R expression increases in ipsilateral DRG after 1 week of disease, while P2X3R immunoreactivity decreases. We have also reported a significant induction of the activating transcriptional factor 3 (ATF3) in MA. In this study, we show that ATF3 knocked down in DRG cell cultures does not affect the expression of P2X7R, P2X3R, or glial fibrillary acidic protein (GFAP). We suggest that P2X7R negatively regulates P2X3R, which, however, is unlikely mediated by ATF3. Interestingly, we found that ATF3 knockdown in vitro induced significant decreases in the heat shock protein 90 (HSP90) expression. Thus, we evaluated in vivo the involvement of HSP90 in MA and demonstrated that the HSP90 messenger RNA levels increase in ipsilateral DRG of inflamed animals. We also show that HSP90 is mostly found in a cleaved form in this condition. Moreover, administration of a HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), attenuated MA-induced mechanical allodynia in the first hours. The drug also reversed the HSP90 upregulation and cleavage. 17-DMAG seemed to attenuate glial activation and neuronal sensitization (as inferred by downregulation of GFAP and P2X3R in ipsilateral DRG) which might correlate with the observed pain alleviation. Our data indicate a role of HSP90 in MA pathophysiology, but further investigation is necessary to clarify the underlying mechanisms.

Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro

PubMed Central

Chen, Chuandao; Liu, Shangjie; Huang, Runqing; Zhong, Mingqi; Wei, Chiju; Zhang, Yueling

2016-01-01

Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway. PMID:27007573

Heat shock protein 90 (HSP90) is overexpressed in p16-negative oropharyngeal squamous cell carcinoma, and its inhibition in vitro potentiates the effects of chemoradiation.

PubMed

Patel, Kirtesh; Wen, Jing; Magliocca, Kelly; Muller, Susan; Liu, Yuan; Chen, Zhuo Georgia; Saba, Nabil; Diaz, Roberto

2014-11-01

Cisplatin and radiation therapy remain the current standard for treating locally advanced SCCHN. Novel treatment approaches are needed, especially in patients with human papilloma virus (HPV)-negative disease who have worse outcomes despite multimodality therapy. Using our institutional review board approved database, we obtained twenty oropharyngeal squamous cell carcinoma (SCC) tissue samples: ten p16 positive, ten p16-negative. Because p16 expression is strongly associated with HPV positivity in oropharyngeal SCC, p16 status was used as a marker of HPV. We subsequently analyzed, via immunohistochemistry, heat shock protein 90 (HSP90) protein levels. Using HPV-positive and HPV-negative SCC cell lines, we compared baseline HSP90 expression levels and the effect of the HSP90 inhibitor ganetespib on viability and apoptosis. Clonogenic survival of HPV-negative cells treated with ganetespib, radiation therapy, and/or cisplatin was then investigated. We characterize the effects of ganetespib on proteins that are thought to drive DNA damage resistance in HPV-negative cells. HSP90 expression was significantly higher in p16-negative compared with p16-positive samples (p = 0.016) and in HPV-negative cell lines compared with positive cells. Ganetespib increased cytotoxicity and induced apoptosis in HPV-negative more than positive cells. Adding ganetespib to cisplatin and/or radiation therapy in HPV-negative cells further decreased clonogenic survival. Finally, ganetespib downregulated expressions of EGFR, ERK, AKT, p53, and HIF-1α. Ganetespib inhibited HPV-negative SCCHN viability and potentiated cell kill when combined with cisplatin or radiation therapy in vitro. With HSP90 expression higher in HPV-negative cells and in p16-negative patients, further exploration of the clinical activity of HSP90 inhibitors in SCCHN is warranted.

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.

Hsp90 can Accommodate the Simultaneous Binding of the FKBP52 and HOP Proteins

PubMed Central

Hildenbrand, Zacariah L.; Molugu, Sudheer K.; Herrera, Nadia; Ramirez, Citlally; Xiao, Chuan; Bernal, Ricardo A.

2011-01-01

The regulation of steroidogenic hormone receptor-mediated activity plays an important role in the development of hormone-dependent cancers. For example, during prostate carcinogenesis, the regulatory function played by the androgen receptor is often converted from a growth suppressor to an oncogene thus promoting prostate cancer cell survival and eventual metastasis. Within the cytoplasm, steroid hormone receptor activity is regulated by the Hsp90 chaperone in conjunction with a series of co-chaperone proteins. Collectively, Hsp90 and its binding associates form a large heteromeric complex that scaffold the fully mature receptor for binding with the respective hormone. To date our understanding of the interactions between Hsp90 with the various TPR domain-containing co-chaperone proteins is limited due to a lack of available structural information. Here we present the stable formation of Hsp902-FKBP521- HOP2 and Hsp902-FKBP521-p232-HOP2 complexes as detected by immunoprecipitation, time course dynamic light scattering and electron microscopy. The simultaneous binding of FKBP52 and HOP to the Hsp90 dimer provide direct evidence of a novel chaperone sub-complex that likely plays a transient role in the regulation of the fully mature steroid hormone receptor. PMID:21378414

Chaperone expression profiles correlate with distinct physiological states of Plasmodium falciparum in malaria patients

PubMed Central

2010-01-01

Background Molecular chaperones have been shown to be important in the growth of the malaria parasite Plasmodium falciparum and inhibition of chaperone function by pharmacological agents has been shown to abrogate parasite growth. A recent study has demonstrated that clinical isolates of the parasite have distinct physiological states, one of which resembles environmental stress response showing up-regulation of specific molecular chaperones. Methods Chaperone networks operational in the distinct physiological clusters in clinical malaria parasites were constructed using cytoscape by utilizing their clinical expression profiles. Results Molecular chaperones show distinct profiles in the previously defined physiologically distinct states. Further, expression profiles of the chaperones from different cellular compartments correlate with specific patient clusters. While cluster 1 parasites, representing a starvation response, show up-regulation of organellar chaperones, cluster 2 parasites, which resemble active growth based on glycolysis, show up-regulation of cytoplasmic chaperones. Interestingly, cytoplasmic Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, cluster in the same group. Detailed analysis of chaperone expression in the patient cluster 2 reveals up-regulation of the entire Hsp90-dependent pro-survival circuitries. In addition, cluster 2 also shows up-regulation of Plasmodium export element (PEXEL)-containing Hsp40s thought to have regulatory and host remodeling roles in the infected erythrocyte. Conclusion In all, this study demonstrates an intimate involvement of parasite-encoded chaperones, PfHsp90 in particular, in defining pathogenesis of malaria. PMID:20719001

Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

PubMed Central

Hu, J; Seeger, C

1996-01-01

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8577714

1H, 15N and 13C resonance assignments for free and IEEVD peptide-bound forms of the tetratricopeptide repeat domain from the human E3 ubiquitin ligase CHIP.

PubMed

Zhang, Huaqun; McGlone, Cameron; Mannion, Matthew M; Page, Richard C

2017-04-01

The ubiquitin ligase CHIP catalyzes covalent attachment of ubiquitin to unfolded proteins chaperoned by the heat shock proteins Hsp70/Hsc70 and Hsp90. CHIP interacts with Hsp70/Hsc70 and Hsp90 by binding of a C-terminal IEEVD motif found in Hsp70/Hsc70 and Hsp90 to the tetratricopeptide repeat (TPR) domain of CHIP. Although recruitment of heat shock proteins to CHIP via interaction with the CHIP-TPR domain is well established, alterations in structure and dynamics of CHIP upon binding are not well understood. In particular, the absence of a structure for CHIP-TPR in the free form presents a significant limitation upon studies seeking to rationally design inhibitors that may disrupt interactions between CHIP and heat shock proteins. Here we report the 1 H, 13 C, and 15 N backbone and side chain chemical shift assignments for CHIP-TPR in the free form, and backbone chemical shift assignments for CHIP-TPR in the IEEVD-bound form. The NMR resonance assignments will enable further studies examining the roles of dynamics and structure in regulating interactions between CHIP and the heat shock proteins Hsp70/Hsc70 and Hsp90.

Snail phenotypic variation and stress proteins: do different heat response strategies contribute to Waddington's widget in field populations?

PubMed

Köhler, Heinz-R; Lazzara, Raimondo; Dittbrenner, Nils; Capowiez, Yvan; Mazzia, Christophe; Triebskorn, Rita

2009-03-15

On the basis of studies with laboratory strains of Drosophila and Arabidopsis, it has been hypothesized that potential buffers to the expression of phenotypic morphological variation, such as Hsp90 and possibly Hsp70, represent important components of Waddington's widget, which may confer capacitive evolution. As studies on field populations of living organisms to test this hypothesis are lacking, we tested whether a heat response strategy involving high stress protein levels is associated with low morphological variation and vice versa, using four natural populations of Mediterranean pulmonate snails. In response to 8 hr of elevated temperatures, a population of Xeropicta derbentina with uniform shell pigmentation pattern showed remarkably high Hsp70 but low Hsp90 levels. In contrast, a highly variable population of Cernuella virgata kept both Hsp90 and Hsp70 levels low when held at diverse though environmentally relevant temperatures. Two other populations (Theba pisana and another X. derbentina population) with intermediate variation in shell pigmentation pattern were also intermediate in inducing Hsp70, though Hsp90 was maintained at a low level. The observed correlation of stress protein levels and coloration pattern variation provide the first indirect evidence for an association of stress proteins with Waddington's widget under natural conditions.