Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks. PMID:24922508

Computational modeling of allosteric regulation in the hsp90 chaperones: a statistical ensemble analysis of protein structure networks and allosteric communications.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-06-01

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks.

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.

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

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

PubMed

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

2016-08-01

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

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

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

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

PubMed

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

2014-02-01

The term molecular chaperone was first used to describe the ability of nucleoplasmin to prevent the aggregation of histones with DNA during the assembly of nucleosomes. Subsequently, the name was extended to proteins that mediate the post-translational assembly of oligomeric complexes protecting them from denaturation and/or aggregation. Hsp90 is a 90-kDa molecular chaperone that represents the major soluble protein of the cell. In contrast to most conventional chaperones, Hsp90 functions as a refined sensor of protein function and its principal role in the cell is to facilitate biological activity to properly folded client proteins that already have a preserved tertiary structure. Consequently, Hsp90 is related to basic cell functions such as cytoplasmic transport of soluble proteins, translocation of client proteins to organelles, and regulation of the biological activity of key signaling factors such as protein kinases, ubiquitin ligases, steroid receptors, cell cycle regulators, and transcription factors. A growing amount of evidence links the protective action of this molecular chaperone to mechanisms related to posttranslational modifications of soluble nuclear factors as well as histones. In this article, we discuss some aspects of the regulatory action of Hsp90 on transcriptional regulation and how this effect could have impacted genetic assimilation mechanism in some organisms. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

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

Li, Jingzhi; Qian, Xinguo; Hu, Junbin

2010-11-03

The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away,more » and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.« less

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

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

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

PubMed Central

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

2015-01-01

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

2.4 Å resolution crystal structure of human TRAP1 NM , the Hsp90 paralog in the mitochondrial matrix

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

Sung, Nuri; Lee, Jungsoon; Kim, Ji-Hyun

2016-07-13

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

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

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.

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.

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

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

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.

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

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.

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.

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.

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

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.

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.

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

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

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

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.

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.

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.

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

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

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

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

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.

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.

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

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

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.

Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

PubMed Central

Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K.; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P.; Abell, Benjamin M.; Nabok, Alexei

2011-01-01

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. PMID:21767504

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

PubMed Central

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

2012-01-01

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

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

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

Study of receptor-chaperone interactions using the optical technique of spectroscopic ellipsometry.

PubMed

Kriechbaumer, Verena; Tsargorodskaya, Anna; Mustafa, Mohd K; Vinogradova, Tatiana; Lacey, Joanne; Smith, David P; Abell, Benjamin M; Nabok, Alexei

2011-07-20

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation.

PubMed

Geller, Ron; Pechmann, Sebastian; Acevedo, Ashley; Andino, Raul; Frydman, Judith

2018-05-03

Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

The FKBP51 Glucocorticoid Receptor Co-Chaperone: Regulation, Function, and Implications in Health and Disease.

PubMed

Fries, Gabriel R; Gassen, Nils C; Rein, Theo

2017-12-05

Among the chaperones and co-chaperones regulating the glucocorticoid receptor (GR), FK506 binding protein (FKBP) 51 is the most intensely investigated across different disciplines. This review provides an update on the role of the different co-chaperones of Hsp70 and Hsp90 in the regulation of GR function. The development leading to the focus on FKBP51 is outlined. Further, a survey of the vast literature on the mechanism and function of FKBP51 is provided. This includes its structure and biochemical function, its regulation on different levels-transcription, post-transcription, and post-translation-and its function in signaling pathways. The evidence portraying FKBP51 as a scaffolding protein organizing protein complexes rather than a chaperone contributing to the folding of individual proteins is collated. Finally, FKBP51's involvement in physiology and disease is outlined, and the promising efforts in developing drugs targeting FKBP51 are discussed.

Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90

PubMed Central

Zara, Vincenzo; Ferramosca, Alessandra; Robitaille-Foucher, Philippe; Palmieri, Ferdinando; Young, Jason C.

2016-01-01

Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment. PMID:19143589

Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90.

PubMed

Zara, Vincenzo; Ferramosca, Alessandra; Robitaille-Foucher, Philippe; Palmieri, Ferdinando; Young, Jason C

2009-04-15

Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment.

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.

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

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.

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

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.

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.

Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18

PubMed Central

Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Ray, Sougata Sinha; Biswas, Ashis

2015-01-01

Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31–43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25–43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min-1. Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18. PMID:26098662

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 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.

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.

Apg-2 has a chaperone-like activity similar to Hsp110 and is overexpressed in hepatocellular carcinomas.

PubMed

Gotoh, Kazuhisa; Nonoguchi, Kohsuke; Higashitsuji, Hiroaki; Kaneko, Yoshiyuki; Sakurai, Toshiharu; Sumitomo, Yasuhiko; Itoh, Katsuhiko; Subjeck, John R; Fujita, Jun

2004-02-27

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. We constructed subtracted cDNA libraries enriched with genes overexpressed in HCCs. Among the 17 genes identified were molecular chaperones, Hsp110, Hsp90B, and Hsp70-1. Expression of the Hsp110 family members was further analyzed, and increased transcript levels of Hsp110 and Apg-2, but not Apg-1, were found in 12 and 14, respectively, of 18 HCCs. Immunohistochemical analysis demonstrated the overexpression of the proteins in tumor cells. Apg-2 had chaperone ability similar to Hsp110 in a thermal denaturation assay using luciferase, and showed anti-apoptotic activity. These results suggest that the Hsp110 family members play important roles in hepatocarcinogenesis through their chaperoning activities.

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

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.

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.

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.

Hsp90 is an essential regulator of EphA2 receptor stability and signaling: Implications for cancer cell migration and metastasis

PubMed Central

Annamalai, Balasubramaniam; Liu, Xueguang; Gopal, Udhayakumar; Isaacs, Jennifer

2011-01-01

A subset of Eph receptors and their corresponding ligands are commonly expressed in tumor cells, where they mediate biological processes such as cell migration and adhesion, while their expression in endothelial cells promotes angiogenesis. In particular, the tumor-specific upregulation of EphA2 confers properties of increased cellular motility, invasiveness, tumor angiogenesis, and tumor progression, and its overexpression correlates with poor prognosis in several cancer types. The cellular chaperone Hsp90 also plays a significant role in regulating cell migration and angiogenesis, although the full repertoire of motility driving proteins dependent upon Hsp90 function remain poorly defined. We explored the hypothesis that Hsp90 may regulate the activity of EphA2 and examined the potential relationship between EphA2 receptor signaling and chaperone function. We demonstrate that geldanamycin (GA), an Hsp90 antagonist, dramatically destabilizes newly synthesized EphA2 protein and diminishes receptor levels in a proteasome-dependent pathway. In addition, GA treatment impairs EphA2 signaling, as evidenced by a decrease in ligand-dependent receptor phosphorylation and subsequent cell rounding. Therefore, Hsp90 exerts a dual role in regulating the stability of nascent EphA2 protein, and maintaining the signaling capacity of the mature receptor. Our findings also suggest that the GA-dependent mitigation of EphA2 signaling in receptor-overexpressing cancer cells may be sufficient to recapitulate the anti-motility effects of this drug. Finally, the identification of a pharmacologic approach to suppress EphA2 expression and signaling highlights the attractive possibility that Hsp90 inhibitors may have clinical utility in antagonizing EphA2-dependent tumorigenic progression. PMID:19567782

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.

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

Regulation of AR Degradation and Function by Ubiquitylation

DTIC Science & Technology

2015-10-01

ubiquitylation and degradation remain to be established. Newly synthesized AR associates with an HSP90 chaperone complex, and an HSP90 associated E3 ubiquitin ...clearly additional cytoplasmic and/or nuclear ubiquitin ligases that regulate the normal turnover and degradation of the liganded AR. Indeed, multiple... ubiquitin ligases have been reported to interact with AR and regulate its transcriptional activities and/or degradation. Moreover, previous studies

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

Heat shock protein 83 plays pleiotropic roles in embryogenesis, longevity, and fecundity of the pea aphid Acyrthosiphon pisum.

PubMed

Will, Torsten; Schmidtberg, Henrike; Skaljac, Marisa; Vilcinskas, Andreas

2017-01-01

Heat shock protein 83 (HSP83) is homologous to the chaperone HSP90. It has pleiotropic functions in Drosophila melanogaster, including the control of longevity and fecundity, and facilitates morphological evolution by buffering cryptic deleterious mutations in wild populations. In the pea aphid Acyrthosiphon pisum, HSP83 expression is moderately induced by bacterial infection but upregulated more strongly in response to heat stress and fungal infection. Stress-inducible heat shock proteins are of considerable evolutionary and ecological importance because they are known to buffer environmental variation and to influence fitness under non-optimal conditions. To investigate the functions of HSP83 in viviparous aphids, we used RNA interference to attenuate its expression and studied the impact on complex parameters. The RNA interference (RNAi)-mediated depletion of HSP83 expression in A. pisum reduced both longevity and fecundity, suggesting this chaperone has an evolutionarily conserved function in insects. Surprisingly, HSP83 depletion reduced the number of viviparous offspring while simultaneously increasing the number of premature nymphs developing in the ovaries, suggesting an unexpected role in aphid embryogenesis and eclosion. The present study indicates that reduced HSP83 expression in A. pisum reveals both functional similarities and differences compared with its reported roles in holometabolous insects. Its impact on aphid lifespan, fecundity, and embryogenesis suggests a function that determines their fitness. This could be achieved by targeting different client proteins, recruiting distinct co-chaperones or transposon activation.

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.

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

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.

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.

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.

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.

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.

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.

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.

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

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.

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

Spotlight on the microbes that produce heat shock protein 90-targeting antibiotics.

PubMed

Piper, Peter W; Millson, Stefan H

2012-12-12

Heat shock protein 90 (Hsp90) is a promising cancer drug target as a molecular chaperone critical for stabilization and activation of several of the oncoproteins that drive cancer progression. Its actions depend upon its essential ATPase, an activity fortuitously inhibited with a very high degree of selectivity by natural antibiotics: notably the actinomycete-derived benzoquinone ansamycins (e.g. geldanamycin) and certain fungal-derived resorcyclic acid lactones (e.g. radicicol). The molecular interactions made by these antibiotics when bound within the ADP/ATP-binding site of Hsp90 have served as templates for the development of several synthetic Hsp90 inhibitor drugs. Much attention now focuses on the clinical trials of these drugs. However, because microbes have evolved antibiotics to target Hsp90, it is probable that they often exploit Hsp90 inhibition when interacting with each other and with plants. Fungi known to produce Hsp90 inhibitors include mycoparasitic, as well as plant-pathogenic, endophytic and mycorrhizal species. The Hsp90 chaperone may, therefore, be a prominent target in establishing a number of mycoparasitic (interfungal), fungal pathogen-plant and symbiotic fungus-plant relationships. Furthermore the Hsp90 family proteins of the microbes that produce Hsp90 inhibitor antibiotics are able to reveal how drug resistance can arise by amino acid changes in the highly conserved ADP/ATP-binding site of Hsp90.

Prion-specific Hsp40 function: The role of the auxilin homolog Swa2

PubMed Central

Oliver, Emily E.; Troisi, Elizabeth M.; Hines, Justin K.

2017-01-01

ABSTRACT Yeast prions are protein-based genetic elements that propagate through cell populations via cytosolic transfer from mother to daughter cell. Molecular chaperone proteins including Hsp70, the Hsp40/J-protein Sis1, and Hsp104 are required for continued prion propagation, however the specific requirements of chaperone proteins differ for various prions. We recently reported that Swa2, the yeast homolog of the mammalian protein auxilin, is specifically required for the propagation of the prion [URE3].1 [URE3] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain of Swa2, but does not require Swa2 clathrin binding. We concluded that the TPR domain determines the specificity of the genetic interaction between Swa2 and [URE3], and that this domain likely interacts with one or more proteins with a C-terminal EEVD motif. Here we extend that analysis to incorporate additional data that supports this hypothesis. We also present new data eliminating Hsp104 as the relevant Swa2 binding partner and discuss our findings in the context of other recent work involving Hsp90. Based on these findings, we propose a new model for Swa2's involvement in [URE3] propagation in which Swa2 and Hsp90 mediate the formation of a multi-protein complex that increases the number of sites available for Hsp104 disaggregation. PMID:28574745

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

A S52P mutation in the 'α-crystallin domain' of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function.

PubMed

Nandi, Sandip K; Rehna, Elengikal A A; Panda, Alok K; Shiburaj, Sugathan; Dharmalingam, Kuppamuthu; Biswas, Ashis

2013-12-01

Mycobacterium leprae HSP18 is a small heat shock protein (sHSP). It is a major immunodominant antigen of M. leprae pathogen. Previously, we have reported the existence of two M. leprae HSP18 variants in various leprotic patients. One of the variants has serine at position 52, whereas the other one has proline at the same position. We have also reported that HSP18 having proline at position 52 (HSP18P(52)) is a nonameric protein and exhibits chaperone function. However, the structural and functional characterization of wild-type HSP18 having serine at position 52 (HSP18S(52)) is yet to be explored. Furthermore, the implications of the S52P mutation on the structure and chaperone function of HSP18 are not well understood. Therefore, we cloned and purified these two HSP18 variants. We found that HSP18S(52) is also a molecular chaperone and an oligomeric protein. Intrinsic tryptophan fluorescence and far-UV CD measurements revealed that the S52P mutation altered the tertiary and secondary structure of HSP18. This point mutation also reduced the oligomeric assembly and decreased the surface hydrophobicity of HSP18, as revealed by HPLC and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding studies, respectively. Mutant protein was less stable against thermal and chemical denaturation and was more susceptible towards tryptic cleavage than wild-type HSP18. HSP18P(52) had lower chaperone function and was less effective in protecting thermal killing of Escherichia coli than HSP18S(52). Taken together, our data suggest that serine 52 is important for the larger oligomerization and chaperone function of HSP18. Because both variants differ in stability and function, they may have different roles in the survival of M. leprae in infected hosts. © 2013 FEBS.

Effect of Hypergravity on the Level of Heat Shock Proteins 70 and 90 in Pea Seedlings

NASA Astrophysics Data System (ADS)

Kozeko, Liudmyla; Kordyum, Elizabeth

2009-01-01

Exposure to hypergravity induces significant changes in gene expression of plants which are indicative of stress conditions. A substantial part of the general stress response is up-regulation of heat shock proteins (Hsp) which function as molecular chaperones. The objective of this research was to test the possible changes in the Hsp70 and Hsp90 level in response to short-term hypergravity exposure. In this study 5-day-old etiolated pea seedlings were exposed to centrifuge-induced hypergravity (3-14 g) for 15 min and 1 h and a part of the seedlings was sampled at 1.5 and 24 h after the exposures. Western blot analysis showed time-dependent changes in Hsp70 and Hsp90 levels: an increase under hypergravity and a tendency towards recovery of the normal content during re-adaptation. The quantity and time of their expression was correlated with the g-force level. These data suggest that short-term hypergravity acts as a stress which could increase the risk of protein denaturation and aggregation. Molecular chaperons induced during the stress may have an essential role in counteracting this risk.

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.

Multi-chaperone function modulation and association with cytoskeletal proteins are key features of the function of AIP in the pituitary gland

PubMed Central

Hernández-Ramírez, Laura C.; Morgan, Rhodri M.L.; Barry, Sayka; D’Acquisto, Fulvio; Prodromou, Chrisostomos; Korbonits, Márta

2018-01-01

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-specific function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant findings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was confirmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/stability of multiple chaperone client proteins could be perturbed by a deficient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identified. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our findings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP. PMID:29507682

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

Chaperone activity of human small heat shock protein-GST fusion proteins.

PubMed

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

Interaction of ATP with a small heat shock protein from Mycobacterium leprae: effect on its structure and function.

PubMed

Nandi, Sandip Kumar; Chakraborty, Ayon; Panda, Alok Kumar; Ray, Sougata Sinha; Kar, Rajiv Kumar; Bhunia, Anirban; Biswas, Ashis

2015-03-01

Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of "HSP18-ATP" interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.

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

PubMed

Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

2016-05-01

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

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.

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

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.

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

PubMed Central

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

2012-01-01

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

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

PubMed

Finka, Andrija; Goloubinoff, Pierre

2013-09-01

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10% of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

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.

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

ATP-dependent molecular chaperones in plastids--More complex than expected.

PubMed

Trösch, Raphael; Mühlhaus, Timo; Schroda, Michael; Willmund, Felix

2015-09-01

Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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.

Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae.

PubMed

Valaviciute, Monika; Norkiene, Milda; Goda, Karolis; Slibinskas, Rimantas; Gedvilaite, Alma

2016-07-01

A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis.

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.

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

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.

Interaction of ATP with a Small Heat Shock Protein from Mycobacterium leprae: Effect on Its Structure and Function

PubMed Central

Nandi, Sandip Kumar; Chakraborty, Ayon; Panda, Alok Kumar; Sinha Ray, Sougata; Kar, Rajiv Kumar; Bhunia, Anirban; Biswas, Ashis

2015-01-01

Adenosine-5’-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of “HSP18-ATP” interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts. PMID:25811190

From hatching to dispatching: the multiple cellular roles of the Hsp70 molecular chaperone machinery.

PubMed

Meimaridou, Eirini; Gooljar, Sakina B; Chapple, J Paul

2009-01-01

Molecular chaperones are best recognized for their roles in de novo protein folding and the cellular response to stress. However, many molecular chaperones, and in particular the Hsp70 chaperone machinery, have multiple diverse cellular functions. At the molecular level, chaperones are mediators of protein conformational change. To facilitate conformational change of client/substrate proteins, in manifold contexts, chaperone power must be closely regulated and harnessed to specific cellular locales--this is controlled by cochaperones. This review considers specialized functions of the Hsp70 chaperone machinery mediated by its cochaperones. We focus on vesicular trafficking, protein degradation and a potential role in G protein-coupled receptor processing.

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

Comparison of the carboxy-terminal DP-repeat region in the co-chaperones Hop and Hip

PubMed Central

Nelson, Gregory M.; Huffman, Holly; Smith, David F.

2003-01-01

Functional steroid receptor complexes are assembled and maintained by an ordered pathway of interactions involving multiple components of the cellular chaperone machinery. Two of these components, Hop and Hip, serve as co-chaperones to the major heat shock proteins (Hsps), Hsp70 and Hsp90, and participate in intermediate stages of receptor assembly. In an effort to better understand the functions of Hop and Hip in the assembly process, we focused on a region of similarity located near the C-terminus of each co-chaperone. Contained within this region is a repeated sequence motif we have termed the DP repeat. Earlier mutagenesis studies implicated the DP repeat of either Hop or Hip in Hsp70 binding and in normal assembly of the co-chaperones with progesterone receptor (PR) complexes. We report here that the DP repeat lies within a protease-resistant domain that extends to or is near the C-terminus of both co-chaperones. Point mutations in the DP repeats render the C-terminal regions hypersensitive to proteolysis. In addition, a Hop DP mutant displays altered proteolytic digestion patterns, which suggest that the DP-repeat region influences the folding of other Hop domains. Although the respective DP regions of Hop and Hip share sequence and structural similarities, they are not functionally interchangeable. Moreover, a double-point mutation within the second DP-repeat unit of Hop that converts this to the sequence found in Hip disrupts Hop function; however, the corresponding mutation in Hip does not alter its function. We conclude that the DP repeats are important structural elements within a C-terminal domain, which is important for Hop and Hip function. PMID:14627198

Comparison of the carboxy-terminal DP-repeat region in the co-chaperones Hop and Hip.

PubMed

Nelson, Gregory M; Huffman, Holly; Smith, David F

2003-01-01

Functional steroid receptor complexes are assembled and maintained by an ordered pathway of interactions involving multiple components of the cellular chaperone machinery. Two of these components, Hop and Hip, serve as co-chaperones to the major heat shock proteins (Hsps), Hsp70 and Hsp90, and participate in intermediate stages of receptor assembly. In an effort to better understand the functions of Hop and Hip in the assembly process, we focused on a region of similarity located near the C-terminus of each co-chaperone. Contained within this region is a repeated sequence motif we have termed the DP repeat. Earlier mutagenesis studies implicated the DP repeat of either Hop or Hip in Hsp70 binding and in normal assembly of the co-chaperones with progesterone receptor (PR) complexes. We report here that the DP repeat lies within a protease-resistant domain that extends to or is near the C-terminus of both co-chaperones. Point mutations in the DP repeats render the C-terminal regions hypersensitive to proteolysis. In addition, a Hop DP mutant displays altered proteolytic digestion patterns, which suggest that the DP-repeat region influences the folding of other Hop domains. Although the respective DP regions of Hop and Hip share sequence and structural similarities, they are not functionally interchangeable. Moreover, a double-point mutation within the second DP-repeat unit of Hop that converts this to the sequence found in Hip disrupts Hop function; however, the corresponding mutation in Hip does not alter its function. We conclude that the DP repeats are important structural elements within a C-terminal domain, which is important for Hop and Hip function.

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.

Structural characterization of tetranortriterpenes from Pseudrocedrela kotschyi and Trichilia emetica and study of their activity towards the chaperone Hsp90.

PubMed

Piaz, Fabrizio Dal; Malafronte, Nicola; Romano, Adriana; Gallotta, Dario; Belisario, Maria Antonietta; Bifulco, Giuseppe; Gualtieri, Maria Josefine; Sanogo, Rokia; Tommasi, Nunziatina De; Pisano, Claudio

2012-03-01

Investigation of roots extracts Pseudrocedrela kotschyi and Trichilia emetica led to identification of 5 limonoid derivatives, Kotschyins D-H, and 11 known compounds. Their structures were elucidated by extensive 1D and 2D NMR experiments in conjunction with mass spectrometry. A surface plasmon resonance (SPR) approach was adopted to screen their Hsp90 binding capability and kotschyin D showed a significant affinity for the chaperone. Therefore, the characterization of the biological activity of kotschyin D by means of a panel of chemical and biological approaches, including limited proteolysis, molecular docking and biochemical and cellular assays, was performed. Our result indicated this compound as a type of client selective Hsp90 inhibitor, directly binding to the middle domain of the protein and possibly preventing its interaction with the activator of Hsp90 ATPase 1 (Aha1). Copyright © 2011 Elsevier Ltd. All rights reserved.

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2015-01-01

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones.

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

PubMed Central

Stetz, Gabrielle; Verkhivker, Gennady M.

2015-01-01

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones. PMID:26619280

Chaperones and protein folding in the archaea.

PubMed

Large, Andrew T; Goldberg, Martin D; Lund, Peter A

2009-02-01

A survey of archaeal genomes for the presence of homologues of bacterial and eukaryotic chaperones reveals several interesting features. All archaea contain chaperonins, also known as Hsp60s (where Hsp is heat-shock protein). These are more similar to the type II chaperonins found in the eukaryotic cytosol than to the type I chaperonins found in bacteria, mitochondria and chloroplasts, although some archaea also contain type I chaperonin homologues, presumably acquired by horizontal gene transfer. Most archaea contain several genes for these proteins. Our studies on the type II chaperonins of the genetically tractable archaeon Haloferax volcanii have shown that only one of the three genes has to be present for the organisms to grow, but that there is some evidence for functional specialization between the different chaperonin proteins. All archaea also possess genes for prefoldin proteins and for small heat-shock proteins, but they generally lack genes for Hsp90 and Hsp100 homologues. Genes for Hsp70 (DnaK) and Hsp40 (DnaJ) homologues are only found in a subset of archaea. Thus chaperone-assisted protein folding in archaea is likely to display some unique features when compared with that in eukaryotes and bacteria, and there may be important differences in the process between euryarchaea and crenarchaea.

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.

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

What we know about ST13, a co-factor of heat shock protein, or a tumor suppressor?*

PubMed Central

Shi, Zheng-zheng; Zhang, Jia-wei; Zheng, Shu

2007-01-01

This article is to summarize the molecular and functional analysis of the gene “suppression of tumorigenicity 13” (ST13). ST13 is in fact the gene encoding Hsp70 interacting protein (Hip), a co-factor (co-chaperone) of the 70-kDa heat shock proteins (Hsc/Hsp70). By collaborating with other positive co-factors such as Hsp40 and the Hsp70-Hsp90 organizing protein (Hop), or competing with negative co-factors such as Bcl2-associated athanogen 1 (Bag1), Hip may facilitate the chaperone function of Hsc/Hsp70 in protein folding and repair, and in controlling the activity of regulatory proteins such as steroid receptors and regulators of proliferation or apoptosis. Although the nomenclature of ST13 implies a role in the suppression of tumorigenicity (ST), to date available experimental data are not sufficient to support its role in cancer development, except for the possible down-regulation of ST13 in gastric and colorectal cancers. Further investigation of this gene at the physiological level would benefit our understanding of diseases such as endocrinological disorders, cancer, and neurodegeneration commonly associated with protein misfolding. PMID:17323428

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.

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.

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

Fe-S Cluster Hsp70 Chaperones: The ATPase Cycle and Protein Interactions.

PubMed

Dutkiewicz, Rafal; Nowak, Malgorzata; Craig, Elizabeth A; Marszalek, Jaroslaw

2017-01-01

Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of cellular structures, and disassembly of protein complexes. The biogenesis of iron-sulfur (Fe-S) clusters is one of the critical cellular processes that require Hsp70/J-protein action. Fe-S clusters are ubiquitous cofactors critical for activity of proteins performing diverse functions in, for example, metabolism, RNA/DNA transactions, and environmental sensing. This biogenesis process can be divided into two sequential steps: first, the assembly of an Fe-S cluster on a conserved scaffold protein, and second, the transfer of the cluster from the scaffold to a recipient protein. The second step involves Hsp70/J-protein chaperones. Via binding to the scaffold, chaperones enable cluster transfer to recipient proteins. In eukaryotic cells mitochondria have a key role in Fe-S cluster biogenesis. In this review, we focus on methods that enabled us to dissect protein interactions critical for the function of Hsp70/J-protein chaperones in the mitochondrial process of Fe-S cluster biogenesis in the yeast Saccharomyces cerevisiae. © 2017 Elsevier Inc. All rights reserved.

Cullin 5: A Destabilizing Force for Some Oncogenes | Center for Cancer Research

Cancer.gov

Cancer can result when cellular processes such as proliferation and cell death go haywire. Among the many mechanisms in place to regulate these critical processes are molecular chaperones, which help proteins attain their proper functional shape and also regulate protein degradation through the cell’s recycling program, called the ubiquitin/proteasome system. One molecular chaperone, heat shock protein 90 (Hsp90), is of particular interest to cancer researchers because many of its target proteins—sometimes called client proteins—have been implicated in the maintenance and progression of a number of cancers.

BAG2 Interferes with CHIP-Mediated Ubiquitination of HSP72.

PubMed

Schönbühler, Bianca; Schmitt, Verena; Huesmann, Heike; Kern, Andreas; Gamerdinger, Martin; Behl, Christian

2016-12-30

The maintenance of cellular proteostasis is dependent on molecular chaperones and protein degradation pathways. Chaperones facilitate protein folding, maturation, and degradation, and the particular fate of a misfolded protein is determined by the interaction of chaperones with co-chaperones. The co-factor CHIP (C-terminus of HSP70-inteacting protein, STUB1) ubiquitinates chaperone substrates and directs proteins to the cellular degradation systems. The activity of CHIP is regulated by two co-chaperones, BAG2 and HSPBP1, which are potent inhibitors of the E3 ubiquitin ligase activity. Here, we examined the functional correlation of HSP72, CHIP, and BAG2, employing human primary fibroblasts. We showed that HSP72 is a substrate of CHIP and that BAG2 efficiently prevented the ubiquitination of HSP72 in young cells as well as aged cells. Aging is associated with a decline in proteostasis and we observed increased protein levels of CHIP as well as BAG2 in senescent cells. Interestingly, the ubiquitination of HSP72 was strongly reduced during aging, which revealed that BAG2 functionally counteracted the increased levels of CHIP. Interestingly, HSPBP1 protein levels were down-regulated during aging. The data presented here demonstrates that the co-chaperone BAG2 influences HSP72 protein levels and is an important modulator of the ubiquitination activity of CHIP in young as well as aged cells.

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

The small-molecule kinase inhibitor D11 counteracts 17-AAG-mediated up-regulation of HSP70 in brain cancer cells

PubMed Central

Schaefer, Susanne; Svenstrup, Tina H.

2017-01-01

Many types of cancer express high levels of heat shock proteins (HSPs) that are molecular chaperones regulating protein folding and stability ensuring protection of cells from potentially lethal stress. HSPs in cancer cells promote survival, growth and spreading even in situations of growth factors deprivation by associating with oncogenic proteins responsible for cell transformation. Hence, it is not surprising that the identification of potent inhibitors of HSPs, notably HSP90, has been the primary research focus, in recent years. Exposure of cancer cells to HSP90 inhibitors, including 17-AAG, has been shown to cause resistance to chemotherapeutic treatment mostly attributable to induction of the heat shock response and increased cellular levels of pro-survival chaperones. In this study, we show that treatment of glioblastoma cells with 17-AAG leads to HSP90 inhibition indicated by loss of stability of the EGFR client protein, and significant increase in HSP70 expression. Conversely, co-treatment with the small-molecule kinase inhibitor D11 leads to suppression of the heat shock response and inhibition of HSF1 transcriptional activity. Beside HSP70, Western blot and differential mRNA expression analysis reveal that combination treatment causes strong down-regulation of the small chaperone protein HSP27. Finally, we demonstrate that incubation of cells with both agents leads to enhanced cytotoxicity and significantly high levels of LC3-II suggesting autophagy induction. Taken together, results reported here support the notion that including D11 in future treatment regimens based on HSP90 inhibition can potentially overcome acquired resistance induced by the heat shock response in brain cancer cells. PMID:28542269

The Hsp90 mosaic: a picture emerges

PubMed Central

Mayer, Matthias P; Prodromou, Chrisostomos; Frydman, Judith

2012-01-01

Hsp90s, molecular chaperones critically involved in many essential cellular processes, were the focus of a recent international conference held in Seeon, Germany. The scope of the conference ranged from structural and mechanistic insights all the way to medical applications. PMID:19125165

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

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family.

PubMed

Shaner, Lance; Trott, Amy; Goeckeler, Jennifer L; Brodsky, Jeffrey L; Morano, Kevin A

2004-05-21

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

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

Natural Iminosugar (+)-Lentiginosine Inhibits ATPase and Chaperone Activity of Hsp90

PubMed Central

Dal Piaz, Fabrizio; Vassallo, Antonio; Chini, Maria Giovanna; Cordero, Franca M.; Cardona, Francesca; Pisano, Claudio; Bifulco, Giuseppe; De Tommasi, Nunziatina; Brandi, Alberto

2012-01-01

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. The relevance of Hsp90 as a therapeutic target for numerous diseases states has prompted the identification and optimization of novel Hsp90 inhibitors as an emerging therapeutic strategy. We performed a screening aimed to identify novel Hsp90 inhibitors among several natural compounds and we focused on the iminosugar (+)-lentiginosine, a natural amyloglucosidases inhibitor, for its peculiar bioactivity profile. Characterization of Hsp90 inhibition was performed using a panel of chemical and biological approaches, including limited proteolysis, biochemical and cellular assays. Our result suggested that the middle domain of Hsp90, as opposed to its ATP-binding pocket, is a promising binding site for new classes of Hsp90 inhibitors with multi-target anti-cancer potential. PMID:22916240

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

Hsp100/ClpB Chaperone Function and Mechanism

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

Vierling, Elizabeth

2015-01-27

The supported research investigated the mechanism of action of a unique class of molecular chaperones in higher plants, the Hsp100/ClpB proteins, with the ultimate goal of defining how these chaperones influence plant growth, development, stress tolerance and productivity. Molecular chaperones are essential effectors of cellular “protein quality control”, which comprises processes that ensure the proper folding, localization, activation and turnover of proteins. Hsp100/ClpB proteins are required for temperature acclimation in plants, optimal seed yield, and proper chloroplast development. The model plant Arabidopsis thaliana and genetic and molecular approaches were used to investigate two of the three members of the Hsp100/ClpBmore » proteins in plants, cytosolic AtHsp101 and chloroplast-localized AtClpB-p. Investigating the chaperone activity of the Hsp100/ClpB proteins addresses DOE goals in that this activity impacts how “plants generate and assemble components” as well as “allowing for their self repair”. Additionally, Hsp100/ClpB protein function in plants is directly required for optimal “utilization of biological energy” and is involved in “mechanisms that control the architecture of energy transduction systems”.« less

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

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

PubMed

Nakamoto, Hitoshi; Fujita, Kensaku; Ohtaki, Aguru; Watanabe, Satoru; Narumi, Shoichi; Maruyama, Takahiro; Suenaga, Emi; Misono, Tomoko S; Kumar, Penmetcha K R; Goloubinoff, Pierre; Yoshikawa, Hirofumi

2014-02-28

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

PubMed Central

Riggs, Daniel L.; Roberts, Patricia J.; Chirillo, Samantha C.; Cheung-Flynn, Joyce; Prapapanich, Viravan; Ratajczak, Thomas; Gaber, Richard; Picard, Didier; Smith, David F.

2003-01-01

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52. PMID:12606580

Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70.

PubMed

Colvin, Teresa A; Gabai, Vladimir L; Sherman, Michael Y

2014-01-01

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.

Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

PubMed

Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M

2017-07-01

The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

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.

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

Prospects of engineering thermotolerance in crops through modulation of heat stress transcription factor and heat shock protein networks.

PubMed

Fragkostefanakis, Sotirios; Röth, Sascha; Schleiff, Enrico; Scharf, Klaus-Dieter

2015-09-01

Cell survival under high temperature conditions involves the activation of heat stress response (HSR), which in principle is highly conserved among different organisms, but shows remarkable complexity and unique features in plant systems. The transcriptional reprogramming at higher temperatures is controlled by the activity of the heat stress transcription factors (Hsfs). Hsfs allow the transcriptional activation of HSR genes, among which heat shock proteins (Hsps) are best characterized. Hsps belong to multigene families encoding for molecular chaperones involved in various processes including maintenance of protein homeostasis as a requisite for optimal development and survival under stress conditions. Hsfs form complex networks to activate downstream responses, but are concomitantly subjected to cell-type-dependent feedback regulation through factor-specific physical and functional interactions with chaperones belonging to Hsp90, Hsp70 and small Hsp families. There is increasing evidence that the originally assumed specialized function of Hsf/chaperone networks in the HSR turns out to be a complex central stress response system that is involved in the regulation of a broad variety of other stress responses and may also have substantial impact on various developmental processes. Understanding in detail the function of such regulatory networks is prerequisite for sustained improvement of thermotolerance in important agricultural crops. © 2014 John Wiley & Sons Ltd.

Hsp70 Protein Complexes as Drug Targets

PubMed Central

Assimon, Victoria A.; Gillies, Anne T.; Rauch, Jennifer N.; Gestwicki, Jason E.

2013-01-01

Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70’s interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, such as pro-folding, pro-degradation and pro-trafficking. Thus, a promising strategy may be to block protein-protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to those goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology. PMID:22920901

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.

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.

The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90.

PubMed

Wang, J; Guo, W; Long, C; Zhou, H; Wang, H; Sun, X

2016-03-01

Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction.

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

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

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

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

Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity

PubMed Central

Sastry, M S R; Zhou, Weibin; Baneyx, François

2009-01-01

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging. PMID:19517531

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.

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

BAG3 Is a Modular, Scaffolding Protein that physically Links Heat Shock Protein 70 (Hsp70) to the Small Heat Shock Proteins.

PubMed

Rauch, Jennifer N; Tse, Eric; Freilich, Rebecca; Mok, Sue-Ann; Makley, Leah N; Southworth, Daniel R; Gestwicki, Jason E

2017-01-06

Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that are important for binding and stabilizing unfolded proteins. In this task, the sHsps have been proposed to coordinate with ATP-dependent chaperones, including heat shock protein 70 (Hsp70). However, it is not yet clear how these two important components of the chaperone network are linked. We report that the Hsp70 co-chaperone, BAG3, is a modular, scaffolding factor to bring together sHsps and Hsp70s. Using domain deletions and point mutations, we found that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), αB-crystallin (HspB5), Hsp22 (HspB8), and Hsp20 (HspB6). BAG3 does not appear to be a passive scaffolding factor; rather, its binding promoted de-oligomerization of Hsp27, likely by competing for the self-interactions that normally stabilize large oligomers. BAG3 bound to Hsp70 at the same time as Hsp22, Hsp27, or αB-crystallin, suggesting that it might physically bring the chaperone families together into a complex. Indeed, addition of BAG3 coordinated the ability of Hsp22 and Hsp70 to refold denatured luciferase in vitro. Together, these results suggest that BAG3 physically and functionally links Hsp70 and sHsps. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of changes in gene regulatory patterns, elicited by perturbations of the Hsp90 molecular chaperone complex, by visualizing multiple experiments with an animation

PubMed Central

2011-01-01

Background To make sense out of gene expression profiles, such analyses must be pushed beyond the mere listing of affected genes. For example, if a group of genes persistently display similar changes in expression levels under particular experimental conditions, and the proteins encoded by these genes interact and function in the same cellular compartments, this could be taken as very strong indicators for co-regulated protein complexes. One of the key requirements is having appropriate tools to detect such regulatory patterns. Results We have analyzed the global adaptations in gene expression patterns in the budding yeast when the Hsp90 molecular chaperone complex is perturbed either pharmacologically or genetically. We integrated these results with publicly accessible expression, protein-protein interaction and intracellular localization data. But most importantly, all experimental conditions were simultaneously and dynamically visualized with an animation. This critically facilitated the detection of patterns of gene expression changes that suggested underlying regulatory networks that a standard analysis by pairwise comparison and clustering could not have revealed. Conclusions The results of the animation-assisted detection of changes in gene regulatory patterns make predictions about the potential roles of Hsp90 and its co-chaperone p23 in regulating whole sets of genes. The simultaneous dynamic visualization of microarray experiments, represented in networks built by integrating one's own experimental with publicly accessible data, represents a powerful discovery tool that allows the generation of new interpretations and hypotheses. PMID:21672238

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

The enhancement of antiproliferative and proapoptotic activity of HDAC inhibitors by curcumin is mediated by Hsp90 inhibition.

PubMed

Giommarelli, Chiara; Zuco, Valentina; Favini, Enrica; Pisano, Claudio; Dal Piaz, Fabrizio; De Tommasi, Nunziatina; Zunino, Franco

2010-03-01

Curcumin, a natural polyphenol, has been described to exhibit effects on signaling pathways, leading to induction of apoptosis. In this study, we observed that curcumin inhibited Hsp90 activity causing depletion of client proteins implicated in survival pathways. Based on this observation, this study was designed to investigate the cellular effects of curcumin combination with the pan-HDAC inhibitors, vorinostat and panobinostat, which induce hyperacetylation of Hsp90, resulting in inhibition of its chaperone function. The results showed that, at subtoxic concentrations, curcumin markedly sensitized tumor cells to vorinostat- and panobinostat-induced growth inhibition and apoptosis. The sensitization was associated with persistent depletion of Hsp90 client proteins (EGFR, Raf-1, Akt, and survivin). In conclusion, our findings document a novel mechanism of action of curcumin and support the therapeutic potential of curcumin/HDAC inhibitors combination, because the synergistic interaction was observed at pharmacologically achievable concentrations, which were ineffective when each drug was used alone.

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

PubMed

Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A

2017-10-01

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

Alternative modes of client binding enable functional plasticity of Hsp70

NASA Astrophysics Data System (ADS)

Mashaghi, Alireza; Bezrukavnikov, Sergey; Minde, David P.; Wentink, Anne S.; Kityk, Roman; Zachmann-Brand, Beate; Mayer, Matthias P.; Kramer, Günter; Bukau, Bernd; Tans, Sander J.

2016-11-01

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

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.

The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors

PubMed Central

Demand, Jens; Lüders, Jens; Höhfeld, Jörg

1998-01-01

The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70’s cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell. PMID:9528774

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.

The HSP70 family and cancer

PubMed Central

Murphy, Maureen E.

2013-01-01

The HSP70 family of heat shock proteins consists of molecular chaperones of approximately 70kDa in size that serve critical roles in protein homeostasis. These adenosine triphosphatases unfold misfolded or denatured proteins and can keep these proteins in an unfolded, folding-competent state. They also protect nascently translating proteins, promote the cellular or organellar transport of proteins, reduce proteotoxic protein aggregates and serve general housekeeping roles in maintaining protein homeostasis. The HSP70 family is the most conserved in evolution, and all eukaryotes contain multiple members. Some members of this family serve specific organellar- or tissue-specific functions; however, in many cases, these members can function redundantly. Overall, the HSP70 family of proteins can be thought of as a potent buffering system for cellular stress, either from extrinsic (physiological, viral and environmental) or intrinsic (replicative or oncogenic) stimuli. As such, this family serves a critical survival function in the cell. Not surprisingly, cancer cells rely heavily on this buffering system for survival. The overwhelming majority of human tumors overexpress HSP70 family members, and expression of these proteins is typically a marker for poor prognosis. With the proof of principle that inhibitors of the HSP90 chaperone have emerged as important anticancer agents, intense focus has now been placed on the potential for HSP70 inhibitors to assume a role as a significant chemotherapeutic avenue. In this review, the history, regulation, mechanism of action and role in cancer of the HSP70 family are reviewed. Additionally, the promise of pharmacologically targeting this protein for cancer therapy is addressed. PMID:23563090

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.

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.

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

Hsp90 prevents phenotypic variation by suppressing the mutagenic activity of transposons.

PubMed

Specchia, Valeria; Piacentini, Lucia; Tritto, Patrizia; Fanti, Laura; D'Alessandro, Rosalba; Palumbo, Gioacchino; Pimpinelli, Sergio; Bozzetti, Maria P

2010-02-04

The canalization concept describes the resistance of a developmental process to phenotypic variation, regardless of genetic and environmental perturbations, owing to the existence of buffering mechanisms. Severe perturbations, which overcome such buffering mechanisms, produce altered phenotypes that can be heritable and can themselves be canalized by a genetic assimilation process. An important implication of this concept is that the buffering mechanism could be genetically controlled. Recent studies on Hsp90, a protein involved in several cellular processes and development pathways, indicate that it is a possible molecular mechanism for canalization and genetic assimilation. In both flies and plants, mutations in the Hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden genetic variability. Thus, Hsp90 chaperone machinery may be an evolutionarily conserved buffering mechanism of phenotypic variance, which provides the genetic material for natural selection. Here we offer an additional, perhaps alternative, explanation for proposals of a concrete mechanism underlying canalization. We show that, in Drosophila, functional alterations of Hsp90 affect the Piwi-interacting RNA (piRNA; a class of germ-line-specific small RNAs) silencing mechanism leading to transposon activation and the induction of morphological mutants. This indicates that Hsp90 mutations can generate new variation by transposon-mediated 'canonical' mutagenesis.

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.

Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity

PubMed Central

Lelj-Garolla, Barbara; Mauk, A Grant

2012-01-01

The small heat shock protein 27 (Hsp27 or HSPB1) is an oligomeric molecular chaperone in vitro that is associated with several neuromuscular, neurological, and neoplastic diseases. Although aspects of Hsp27 biology are increasingly well known, understanding of the structural basis for these involvements or of the functional properties of the protein remains limited. As all 11 human small heat shock proteins (sHsps) possess an α-crystallin domain, their varied functional and physiological characteristics must arise from contributions of their nonconserved sequences. To evaluate the role of two such sequences in Hsp27, we have studied three Hsp27 truncation variants to assess the functional contributions of the nonconserved N- and C-terminal sequences. The N-terminal variants Δ1–14 and Δ1–24 exhibit little chaperone activity, somewhat slower but temperature-dependent subunit exchange kinetics, and temperature-independent self-association with formation of smaller oligomers than wild-type Hsp27. The C-terminal truncation variants exhibit chaperone activity at 40 °C but none at 20 °C, limited subunit exchange, and temperature-independent self-association with an oligomer distribution at 40 °C that is very similar to that of wild-type Hsp27. We conclude that more of the N-terminal sequence than simply the WPDF domain is essential in the formation of larger, native-like oligomers after binding of substrate and/or in binding of Hsp27 to unfolding peptides. On the other hand, the intrinsically flexible C-terminal region drives subunit exchange and thermally-induced unfolding, both of which are essential to chaperone activity at low temperature and are linked to the temperature dependence of Hsp27 self-association. PMID:22057845

Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

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

Lee, Jungsoon; Sung, Nuri; Mercado, Jonathan M.

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conficting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of anmore » N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We fnd that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specifc substrates, such as yeast prions, which likely depends on a direct N domain interaction.« less

Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

DOE PAGES

Lee, Jungsoon; Sung, Nuri; Mercado, Jonathan M.; ...

2017-09-11

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conficting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of anmore » N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We fnd that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specifc substrates, such as yeast prions, which likely depends on a direct N domain interaction.« less

Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis

PubMed Central

Yoshida, Soichiro; Tsutsumi, Shinji; Muhlebach, Guillaume; Sourbier, Carole; Lee, Min-Jung; Lee, Sunmin; Vartholomaiou, Evangelia; Tatokoro, Manabu; Beebe, Kristin; Miyajima, Naoto; Mohney, Robert P.; Chen, Yang; Hasumi, Hisashi; Xu, Wanping; Fukushima, Hiroshi; Nakamura, Ken; Koga, Fumitaka; Kihara, Kazunori; Trepel, Jane; Picard, Didier; Neckers, Leonard

2013-01-01

TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor. PMID:23564345

Structure and function of Hip, an attenuator of the Hsp70 chaperone cycle.

PubMed

Li, Zhuo; Hartl, F Ulrich; Bracher, Andreas

2013-08-01

The Hsp70-interacting protein, Hip, cooperates with the chaperone Hsp70 in protein folding and prevention of aggregation. Hsp70 interacts with non-native protein substrates in an ATP-dependent reaction cycle regulated by J-domain proteins and nucleotide exchange factors (NEFs). Hip is thought to delay substrate release by slowing ADP dissociation from Hsp70. Here we present crystal structures of the dimerization domain and the tetratricopeptide repeat (TPR) domain of rat Hip. As shown in a cocrystal structure, the TPR core of Hip interacts with the Hsp70 ATPase domain through an extensive interface, to form a bracket that locks ADP in the binding cleft. Hip and NEF binding to Hsp70 are mutually exclusive, and thus Hip attenuates active cycling of Hsp70-substrate complexes. This mechanism explains how Hip enhances aggregation prevention by Hsp70 and facilitates transfer of specific proteins to downstream chaperones or the proteasome.

Heat shock protein antagonists in early stage clinical trials for NSCLC.

PubMed

Hendriks, Lizza E L; Dingemans, Anne-Marie C

2017-05-01

Cancer cells have a higher need of chaperones than normal cells to prevent the toxic effects of intracellular protein misfolding and aggregation. Heat shock proteins (Hsps) belong to these chaperones; they are classified into families according to molecular size. Hsps are upregulated in many cancers and inhibition can inhibit tumor growth by destabilizing proteins necessary for tumor survival. In non-small cell lung cancer (NSCLC), there are three different Hsp antagonist classes that are in (early) clinical trials: Hsp90, Hsp70 and Hsp27 inhibitors. Areas covered: The rationale to use Hsp inhibitors in NSCLC will be summarized and phase I-III trials will be reviewed. Expert opinion: Several Hsp90 inhibitors have been tested in phase I-III trials, until now none was positive in unselected NSCLC; therefore development of AUY922, ganetespib and retaspimycin was halted. Results seem more promising in molecularly selected patients, especially in ALK-rearranged NSCLC. Hsp27 is overexpressed in squamous NSCLC and is a mechanism of chemotherapy resistance. The Hsp27 inhibitor apatorsen is now tested in squamous NSCLC. No phase II/III data are known for Hsp70 inhibitors. Combination of Hsp inhibitors with heat shock transcription factor 1 inhibitors or focal adhesion kinase inhibitors might be of interest for future trials.

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

Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study

PubMed Central

Gupta, Saurabh; Rao, Atmakuri Ramakrishna; Varadwaj, Pritish Kumar; De, Sachinandan; Mohapatra, Trilochan

2015-01-01

Heat shock protein 70 (HSP70) is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70) has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state) and with HSP70 protein of E. coli 70kDa DnaK (close state) and relaxed them for 100 nanoseconds (ns) using all-atom Molecular Dynamics (MD) Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD) in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA) and Minimum Distance Matrix (MDM). The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD) and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel. PMID:26313938

Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study.

PubMed

Gupta, Saurabh; Rao, Atmakuri Ramakrishna; Varadwaj, Pritish Kumar; De, Sachinandan; Mohapatra, Trilochan

2015-01-01

Heat shock protein 70 (HSP70) is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70) has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state) and with HSP70 protein of E. coli 70kDa DnaK (close state) and relaxed them for 100 nanoseconds (ns) using all-atom Molecular Dynamics (MD) Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD) in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA) and Minimum Distance Matrix (MDM). The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD) and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel.

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 Allosteric Control Mechanisms in Heat Shock Protein 70 (Hsp70).

PubMed

Li, Xiaokai; Shao, Hao; Taylor, Isabelle R; Gestwicki, Jason E

2016-01-01

Heat shock protein 70 (Hsp70) is a molecular chaperone that plays critical roles in protein homeostasis. Hsp70's chaperone activity is coordinated by intra-molecular interactions between its two domains, as well as inter-molecular interactions between Hsp70 and its co-chaperones. Each of these contacts represents a potential opportunity for the development of chemical inhibitors. To illustrate this concept, we review three classes of recently identified molecules that bind distinct pockets on Hsp70. Although all three compounds share the ability to interrupt core biochemical functions of Hsp70, they stabilize different conformers. Accordingly, each compound appears to interrupt a specific subset of inter- and intra-molecular interactions. Thus, an accurate definition of an Hsp70 inhibitor may require a particularly detailed understanding of the molecule's binding site and its effects on protein-protein interactions.

Inhibitors of the HSP90 molecular chaperone: attacking the master regulator in cancer.

PubMed

McDonald, Edward; Workman, Paul; Jones, Keith

2006-01-01

The heat shock protein 90 (HSP90) chaperones represent some 1-2% of all cellular protein and are key players in protein quality control in cells. They are over expressed in many human cancers and the fact that many oncogenic proteins are clients has prompted much recent research on HSP90 inhibitors as new cancer therapeutics. A brief introduction is followed by a detailed review of the various classes of inhibitors, both natural product-based and synthetic, that have emerged over the last decade. The natural products geldanamycin, radicicol and novobiocin have provided the start points for new drugs in this area and their medicinal chemistry is reviewed, including the exciting recent results emerging from clinical trials using geldanamycin analogues. The detailed understanding of the binding mode of these compounds to HSP90 has been significantly enhanced by X-ray crystallography of HSP90 constructs co-crystallised with various ligands. Efforts to replace the natural product inhibitors with more drug-like synthetic compounds have mushroomed over the last 4 years. The purines and the 3,4-diarylpyrazoles have proven to be the most successful and their medicinal chemistry is reviewed with particular emphasis on structure-based design. Protein/ligand co-crystal structures have shown that conserved water molecules in the active site are a vital part of the hydrogen-bonding network established on binding both natural product and synthetic inhibitors. Medicinal chemists have used this information to develop high affinity lead compounds. Recent research provides the platform for exciting developments in the area of HSP90 inhibition over the next few years.

Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore.

PubMed

Galigniana, Mario D; Echeverría, Pablo C; Erlejman, Alejandra G; Piwien-Pilipuk, Graciela

2010-01-01

In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR) -domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor "transformation") is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore.

Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope1[OPEN

PubMed Central

Tanabe, Noriaki; Clarke, Adrian K.

2016-01-01

The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone’s interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import. PMID:26586836

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

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

RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex.

PubMed

Martino, Fabrizio; Pal, Mohinder; Muñoz-Hernández, Hugo; Rodríguez, Carlos F; Núñez-Ramírez, Rafael; Gil-Carton, David; Degliesposti, Gianluca; Skehel, J Mark; Roe, S Mark; Prodromou, Chrisostomos; Pearl, Laurence H; Llorca, Oscar

2018-04-16

The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1-RUVBL2-RPAP3-PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins.

Pyrimidinone-Peptoid Hybrid Molecules with Distinct Effects on Molecular Chaperone Function and Cell Proliferation

PubMed Central

Wright, Christine M.; Chovatiya, Raj J.; Jameson, Nora E.; Turner, David M.; Zhu, Guangyu; Werner, Stefan; Huryn, Donna M.; Pipas, James M.; Day, Billy W.; Wipf, Peter; Brodsky, Jeffrey L.

2008-01-01

The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak, but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70. PMID:18164205

Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

PubMed

Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan

2017-06-07

Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.

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

Targeting Allosteric Control Mechanisms in Heat Shock Protein 70 (Hsp70)

PubMed Central

Li, Xiaokai; Shao, Hao; Taylor, Isabelle R.; Gestwicki, Jason E.

2017-01-01

Heat shock protein 70 (Hsp70) is a molecular chaperone that plays critical roles in protein homeostasis. Hsp70’s chaperone activity is coordinated by intra-molecular interactions between its two domains, as well as inter-molecular interactions between Hsp70 and its co-chaperones. Each of these contacts represents a potential opportunity for the development of chemical inhibitors. To illustrate this concept, we review three classes of recently identified molecules that bind distinct pockets on Hsp70. Although all three compounds share the ability to interrupt core biochemical functions of Hsp70, they stabilize different conformers. Accordingly, each compound appears to interrupt a specific subset of inter- and intra-molecular interactions. Thus, an accurate definition of an Hsp70 inhibitor may require a particularly detailed understanding of the molecule’s binding site and its effects on protein-protein interactions. PMID:27072701

Cooperative Regulation of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by Core Components of the Molecular Chaperone Machinery*

PubMed Central

Narayan, Vikram; Eckert, Mirjam; Zylicz, Alicja; Zylicz, Maciej; Ball, Kathryn L.

2009-01-01

Our understanding of the post-translational processes involved in regulating the interferon regulatory factor-1 (IRF-1) tumor suppressor protein is limited. The introduction of mutations within the C-terminal Mf1 domain (amino acids 301–325) impacts on IRF-1-mediated gene repression and growth suppression as well as the rate of IRF-1 degradation. However, nothing is known about the proteins that interact with this region to modulate IRF-1 function. A biochemical screen for Mf1-interacting proteins has identified an LXXLL motif that is required for binding of Hsp70 family members and cooperation with Hsp90 to regulate IRF-1 turnover and activity. These conclusions are supported by the finding that Hsp90 inhibitors suppress IRF-1-dependent transcription shortly after treatment, although at later time points inhibition of Hsp90 leads to an Hsp70-dependent depletion of nuclear IRF-1. Conversely, the half-life of IRF-1 is increased by Hsp90 in an ATPase-dependent manner leading to the accumulation of nuclear but not cytoplasmic IRF-1. This study begins to elucidate the role of the Mf1 domain of IRF-1 in orchestrating the recruitment of regulatory factors that can impact on both its turnover and transcriptional activity. PMID:19502235

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

A PQM-1-Mediated Response Triggers Transcellular Chaperone Signaling and Regulates Organismal Proteostasis.

PubMed

O'Brien, Daniel; Jones, Laura M; Good, Sarah; Miles, Jo; Vijayabaskar, M S; Aston, Rebecca; Smith, Catrin E; Westhead, David R; van Oosten-Hawle, Patricija

2018-06-26

In metazoans, tissues experiencing proteotoxic stress induce "transcellular chaperone signaling" (TCS) that activates molecular chaperones, such as hsp-90, in distal tissues. How this form of inter-tissue communication is mediated to upregulate systemic chaperone expression and whether it can be utilized to protect against protein misfolding diseases remain open questions. Using C. elegans, we identified key components of a systemic stress signaling pathway that links the innate immune response with proteostasis maintenance. We show that mild perturbation of proteostasis in the neurons or the intestine activates TCS via the GATA zinc-finger transcription factor PQM-1. PQM-1 coordinates neuron-activated TCS via the innate immunity-associated transmembrane protein CLEC-41, whereas intestine-activated TCS depends on the aspartic protease ASP-12. Both TCS pathways can induce hsp-90 in muscle cells and facilitate amelioration of Aβ 3-42 -associated toxicity. This may have powerful implications for the treatment of diseases related to proteostasis dysfunction. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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

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.

Ensemble-based modeling and rigidity decomposition of allosteric interaction networks and communication pathways in cyclin-dependent kinases: Differentiating kinase clients of the Hsp90-Cdc37 chaperone

PubMed Central

Stetz, Gabrielle; Tse, Amanda

2017-01-01

The overarching goal of delineating molecular principles underlying differentiation of protein kinase clients and chaperone-based modulation of kinase activity is fundamental to understanding activity of many oncogenic kinases that require chaperoning of Hsp70 and Hsp90 systems to attain a functionally competent active form. Despite structural similarities and common activation mechanisms shared by cyclin-dependent kinase (CDK) proteins, members of this family can exhibit vastly different chaperone preferences. The molecular determinants underlying chaperone dependencies of protein kinases are not fully understood as structurally similar kinases may often elicit distinct regulatory responses to the chaperone. The regulatory divergences observed for members of CDK family are of particular interest as functional diversification among these kinases may be related to variations in chaperone dependencies and can be exploited in drug discovery of personalized therapeutic agents. In this work, we report the results of a computational investigation of several members of CDK family (CDK5, CDK6, CDK9) that represented a broad repertoire of chaperone dependencies—from nonclient CDK5, to weak client CDK6, and strong client CDK9. By using molecular simulations of multiple crystal structures we characterized conformational ensembles and collective dynamics of CDK proteins. We found that the elevated dynamics of CDK9 can trigger imbalances in cooperative collective motions and reduce stability of the active fold, thus creating a cascade of favorable conditions for chaperone intervention. The ensemble-based modeling of residue interaction networks and community analysis determined how differences in modularity of allosteric networks and topography of communication pathways can be linked with the client status of CDK proteins. This analysis unveiled depleted modularity of the allosteric network in CDK9 that alters distribution of communication pathways and leads to impaired signaling in the client kinase. According to our results, these network features may uniquely define chaperone dependencies of CDK clients. The perturbation response scanning and rigidity decomposition approaches identified regulatory hotspots that mediate differences in stability and cooperativity of allosteric interaction networks in the CDK structures. By combining these synergistic approaches, our study revealed dynamic and network signatures that can differentiate kinase clients and rationalize subtle divergences in the activation mechanisms of CDK family members. The therapeutic implications of these results are illustrated by identifying structural hotspots of pathogenic mutations that preferentially target regions of the increased flexibility to enable modulation of activation changes. Our study offers a network-based perspective on dynamic kinase mechanisms and drug design by unravelling relationships between protein kinase dynamics, allosteric communications and chaperone dependencies. PMID:29095844

Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90

PubMed Central

Kazlauskas, Egidijus; Petrikaitė, Vilma; Michailovienė, Vilma; Revuckienė, Jurgita; Matulienė, Jurgita; Grinius, Leonas; Matulis, Daumantas

2012-01-01

The design of specific inhibitors against the Hsp90 chaperone and other enzyme relies on the detailed and correct understanding of both the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. Here we present a detailed thermodynamic study of binding of aryl-dihydroxyphenyl-thiadiazole inhibitor series to recombinant human Hsp90 alpha isozyme. The inhibitors are highly potent, with the intrinsic Kd approximately equal to 1 nM as determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Dissection of protonation contributions yielded the intrinsic thermodynamic parameters of binding, such as enthalpy, entropy, Gibbs free energy, and the heat capacity. The differences in binding thermodynamic parameters between the series of inhibitors revealed contributions of the functional groups, thus providing insight into molecular reasons for improved or diminished binding efficiency. The inhibitor binding to Hsp90 alpha primarily depended on a large favorable enthalpic contribution combined with the smaller favorable entropic contribution, thus suggesting that their binding was both enthalpically and entropically optimized. The enthalpy-entropy compensation phenomenon was highly evident when comparing the inhibitor binding enthalpies and entropies. This study illustrates how detailed thermodynamic analysis helps to understand energetic reasons for the binding efficiency and develop more potent inhibitors that could be applied for therapeutic use as Hsp90 inhibitors. PMID:22655030

Antarctic krill 454 pyrosequencing reveals chaperone and stress transcriptome.

PubMed

Clark, Melody S; Thorne, Michael A S; Toullec, Jean-Yves; Meng, Yan; Guan, Le Luo; Peck, Lloyd S; Moore, Stephen

2011-01-06

The Antarctic krill Euphausia superba is a keystone species in the Antarctic food chain. Not only is it a significant grazer of phytoplankton, but it is also a major food item for charismatic megafauna such as whales and seals and an important Southern Ocean fisheries crop. Ecological data suggest that this species is being affected by climate change and this will have considerable consequences for the balance of the Southern Ocean ecosystem. Hence, understanding how this organism functions is a priority area and will provide fundamental data for life history studies, energy budget calculations and food web models. The assembly of the 454 transcriptome of E. superba resulted in 22,177 contigs with an average size of 492bp (ranging between 137 and 8515bp). In depth analysis of the data revealed an extensive catalogue of the cellular chaperone systems and the major antioxidant proteins. Full length sequences were characterised for the chaperones HSP70, HSP90 and the super-oxide dismutase antioxidants, with the discovery of potentially novel duplications of these genes. The sequence data contained 41,470 microsatellites and 17,776 Single Nucleotide Polymorphisms (SNPs/INDELS), providing a resource for population and also gene function studies. This paper details the first 454 generated data for a pelagic Antarctic species or any pelagic crustacean globally. The classical "stress proteins", such as HSP70, HSP90, ferritin and GST were all highly expressed. These genes were shown to be over expressed in the transcriptomes of Antarctic notothenioid fish and hypothesized as adaptations to living in the cold, with the associated problems of decreased protein folding efficiency and increased vulnerability to damage by reactive oxygen species. Hence, these data will provide a major resource for future physiological work on krill, but in particular a suite of "stress" genes for studies understanding marine ectotherms' capacities to cope with environmental change.

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.

In vitro reconstitution of chaperone-mediated human RISC assembly.

PubMed

Naruse, Ken; Matsuura-Suzuki, Eriko; Watanabe, Mariko; Iwasaki, Shintaro; Tomari, Yukihide

2018-01-01

To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of Drosophila melanogaster RISC was recently reconstituted by Ago2, the Dicer-2/R2D2 heterodimer, and five chaperone proteins, the absence of a reconstitution system for mammalian RISC assembly has posed analytical challenges. Here we describe reconstitution of human RISC assembly using Ago2 and five recombinant chaperone proteins: Hsp90β, Hsc70, Hop, Dnaja2, and p23. Our data show that ATP hydrolysis by both Hsp90β and Hsc70 is required for RISC assembly of small RNA duplexes but not for that of single-stranded RNAs. The reconstitution system lays the groundwork for further studies of small RNA-mediated gene silencing in mammals. © 2018 Naruse et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

GRP94/gp96 in Cancer: Biology, Structure, Immunology, and Drug Development.

PubMed

Wu, Bill X; Hong, Feng; Zhang, Yongliang; Ansa-Addo, Ephraim; Li, Zihai

2016-01-01

As an endoplasmic reticulum heat-shock protein 90 (HSP90) paralog, GRP94 (glucose-regulated protein 94)/gp96 (hereafter referred to as GRP94) has been shown to be an essential master chaperone for multiple receptors including Toll-like receptors, Wnt coreceptors, and integrins. Clinically, expression of GRP94 correlates with advanced stage and poor survival in a variety of cancers. Recent preclinical studies have also revealed that GRP94 expression is closely linked to cancer growth and metastasis in melanoma, ovarian cancer, multiple myeloma, lung cancer, and inflammation-associated colon cancer. Thus, GRP94 is an attractive therapeutic target in a number of malignancies. The chaperone function of GRP94 depends on its ATPase domain, which is structurally distinct from HSP90, allowing design of highly selective GRP94-targeted inhibitors. In this chapter, we discuss the biology and structure-function relationship of GRP94. We also summarize the immunological roles of GRP94 based on the studies documented over the last two decades, as these pertain to tumorigenesis and cancer progression. Finally, the structure-based rationale for the design of selective small-molecule inhibitors of GRP94 and their potential application in the treatment of cancer are highlighted. © 2016 Elsevier Inc. All rights reserved.

A subclass of plant heat shock cognate 70 chaperones carries a motif that facilitates trafficking through plasmodesmata

PubMed Central

Aoki, Koh; Kragler, Friedrich; Xoconostle-Cázares, Beatriz; Lucas, William J.

2002-01-01

Plasmodesmata establish a pathway for the trafficking of non-cell-autonomously acting proteins and ribonucleoprotein complexes. Plasmodesmal enriched cell fractions and the contents of enucleate sieve elements, in the form of phloem sap, were used to isolate and characterize heat shock cognate 70 (Hsc70) chaperones associated with this cell-to-cell transport pathway. Three Cucurbita maxima Hsc70 chaperones were cloned and functional and sequence analysis led to the identification of a previously uncharacterized subclass of non-cell-autonomous chaperones. The highly conserved nature of the heat shock protein 70 (Hsp70) family, in conjunction with mutant analysis, permitted the characterization of a motif that allows these Hsc70 chaperones to engage the plasmodesmal non-cell-autonomous translocation machinery. Proof of concept that this motif is necessary for Hsp70 gain-of-movement function was obtained through the engineering of a human Hsp70 that acquired the capacity to traffic through plasmodesmata. These results are discussed in terms of the roles likely played by this subclass of Hsc70 chaperones in the trafficking of non-cell-autonomous proteins. PMID:12456884

Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity.

PubMed

Jovcevski, Blagojce; Kelly, Megan A; Rote, Anthea P; Berg, Tracey; Gastall, Heidi Y; Benesch, Justin L P; Aquilina, J Andrew; Ecroyd, Heath

2015-02-19

Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process1[OPEN

PubMed Central

Huang, Po-Kai; Chan, Po-Ting; Chen, Lih-Jen

2016-01-01

Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preprotein-translocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope. PMID:26676256

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.

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.

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.

Co-chaperone Hsp70/Hsp90-organizing protein (Hop) is required for transposon silencing and Piwi-interacting RNA (piRNA) biogenesis.

PubMed

Karam, Joseph A; Parikh, Rasesh Y; Nayak, Dhananjaya; Rosenkranz, David; Gangaraju, Vamsi K

2017-04-14

Piwi-interacting RNAs (piRNAs) are 26-30-nucleotide germ line-specific small non-coding RNAs that have evolutionarily conserved function in mobile genetic element (transposons) silencing and maintenance of genome integrity. Drosophila Hsp70/90-organizing protein homolog (Hop), a co-chaperone, interacts with piRNA-binding protein Piwi and mediates silencing of phenotypic variations. However, it is not known whether Hop has a direct role in piRNA biogenesis and transposon silencing. Here, we show that knockdown of Hop in the germ line nurse cells (GLKD) of Drosophila ovaries leads to activation of transposons. Hop GLKD females can lay eggs at the same rate as wild-type counterparts, but the eggs do not hatch into larvae. Hop GLKD leads to the accumulation of γ-H2Av foci in the germ line, indicating increased DNA damage in the ovary. We also show that Hop GLKD-induced transposon up-regulation is due to inefficient piRNA biogenesis. Based on these results, we conclude that Hop is a critical component of the piRNA pathway and that it maintains genome integrity by silencing transposons. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Hsp40 function in yeast prion propagation: Amyloid diversity necessitates chaperone functional complexity.

PubMed

Sporn, Zachary A; Hines, Justin K

2015-01-01

Yeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as 'strains' in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called 'variants', dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI(+)] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1. Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.

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.

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.

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.

Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry

PubMed Central

Kriechbaumer, Verena; Nabok, Alexei; Mustafa, Mohd K.; Al-Ammar, Rukaiah; Tsargorodskaya, Anna; Smith, David P.; Abell, Ben M.

2012-01-01

Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins. PMID:22479632

A New Therapeutic Paradigm for Breast Cancer Exploiting Low Dose Estrogen-Induced Apoptosis

DTIC Science & Technology

2009-09-01

binds to the Hsp90, Hsp27 , and c-Fos promoters constitutively and modulates their expression. Cell Stress Chaperones, 12: 283-290...C. Yang, Radiological health risks for exploratory class missions in space. Acta Astronaut . 23, 227–231 (1991). 10. V. M. Petrov, Solar cosmic rays

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

PubMed Central

Quintá, HR; Galigniana, MD

2012-01-01

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

Redox-regulated chaperones.

PubMed

Kumsta, Caroline; Jakob, Ursula

2009-06-09

Redox regulation of stress proteins, such as molecular chaperones, guarantees an immediate response to oxidative stress conditions. This review focuses on the two major classes of redox-regulated chaperones, Hsp33 in bacteria and typical 2-Cys peroxiredoxins in eukaryotes. Both proteins employ redox-sensitive cysteines, whose oxidation status directly controls their affinity for unfolding proteins and therefore their chaperone function. We will first discuss Hsp33, whose oxidative stress-induced disulfide bond formation triggers the partial unfolding of the chaperone, which, in turn, leads to the exposure of a high-affinity binding site for unfolded proteins. This rapid mode of activation makes Hsp33 essential for protecting bacteria against severe oxidative stress conditions, such as hypochlorite (i.e., bleach) treatment, which leads to widespread protein unfolding and aggregation. We will compare Hsp33 to the highly abundant eukaryotic typical 2-Cys peroxiredoxin, whose oxidative stress-induced sulfinic acid formation turns the peroxidase into a molecular chaperone in vitro and presumably in vivo. These examples illustrate how proteins use reversible cysteine modifications to rapidly adjust to oxidative stress conditions and demonstrate that redox regulation plays a vital role in protecting organisms against reactive oxygen species-mediated cell death.

The Gln32Lys polymorphism in HSP22 of Zhikong scallop Chlamys farreri is associated with heat tolerance.

PubMed

Yang, Chuanyan; Zhang, Lei; Wang, Lingling; Zhang, Huan; Qiu, Limei; Siva, Vinu S; Song, Linsheng

2011-01-01

Heat shock protein 22 is a member of small heat shock proteins with molecular chaperone activity. Though their multiple functions have been well characterized, there is no report about the association between the polymorphisms of HSP22 and heat tolerance. Three single nucleotide polymorphisms were identified in HSP22 from scallop Chlamys farreri (CfHSP22), and the +94 C-A locus was found to be nonsynonymous. Three genotypes at locus +94, A/A, A/C and C/C, were revealed by using Bi-PASA PCR analysis, and their frequencies were 19.5%, 27.6% and 52.9% in the heat resistant stock, while 9.3%, 17.4% and 73.3% in the heat susceptible stock, respectively. The frequency differences of the three genotypes were significant (P<0.05) between the two stocks. After incubating at 30°C for 84 h, the cumulative mortality of scallops with +94 C/C genotype and +94 A/C genotypes was 95% and 90%, respectively, which was significantly higher (P<0.01) than that of scallops with +94 A/A genotype (70%). The molecular chaperone activity of two His-tagged fusion proteins, rCfHSP22Q with +94 C/C genotype and rCfHSP22K with +94 A/A genotype were analyzed by testing the ability of protecting citrate synthase (CS) against thermal inactivation in vitro. After incubated with rCfHSP22Q or rCfHSP22K at 38°C for 1 h, the activity of CS lost 50% and 45%, and then recovered to 89% and 95% of the original activity following 1 h restoration at 22°C, respectively, indicating that the mutation from Gln to Lys at this site might have an impact on molecular chaperone activities of CfHSP22. These results implied that the polymorphism at locus +94 of CfHSP22 was associated with heat tolerance of scallop, and the +94 A/A genotype could be a potential marker available in future selection of Zhikong scallop with heat tolerance.

Identification of bilateral changes in TID1 expression in the 6-OHDA rat model of Parkinson's disease.

PubMed

Proft, Juliane; Faraji, Jamshid; Robbins, Jerrah C; Zucchi, Fabiola C R; Zhao, Xiaoxi; Metz, Gerlinde A; Braun, Janice E A

2011-01-01

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra and the aggregation of α-synuclein into Lewy bodies. Existing therapies address motor dysfunction but do not halt progression of the disease. A still unresolved question is the biochemical pathway that modulates the outcome of protein misfolding and aggregation processes in PD. The molecular chaperone network plays an important defensive role against cellular protein misfolding and has been identified as protective in experimental models of protein misfolding diseases like PD. Molecular mechanisms underlying chaperone-neuroprotection are actively under investigation. Current evidence implicates a number of molecular chaperones in PD including Hsp25, Hsp70 and Hsp90, however their precise involvement in the neurodegenerative cascade is unresolved. The J protein family (DnaJ or Hsp40 protein family) has long been known to be important in protein conformational processes.We assessed sensory and motor function of control and PD rats and then evaluated the brain region-specific expression levels of select J proteins by Western analysis. Surprisingly, we observed a widespread 26 kDa breakdown product of the J protein, TID1, (tumorous imaginal discs, mtHsp40 or DnaJ3) in a 6-hydroxydopamine (6-OHDA) rat model of PD in which food handling, gait symmetry and sensory performance were impaired. Greater behavioral deficits were associated with lower TID1 expression. Furthermore, direct application of either 6-OHDA or MPP+ (1-methyl-4-phenylpyridinum) to CAD (CNS-derived catecholinaminergic neuronal cell line) cell cultures, reduced TID1 expression levels.Our results suggest that changes in cellular TID1 are a factor in the pathogenesis of PD by impeding functional and structural compensation and exaggerating neurodegenerative processes. In contrast, no changes were observed in CSPα, Hsp40, Hsp70, Hsc70 and PrP(C) levels and no activation of caspase3 was observed. This study links TID1 to PD and provides a new target for therapeutics that halts the PD progression.

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.

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

PubMed

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

2015-08-18

Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell-derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser(1177), a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser(1177) in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells. Copyright © 2015, American Association for the Advancement of Science.

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

PubMed Central

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

2016-01-01

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

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3

PubMed Central

Olcese, Chiara; Patel, Mitali P.; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J.; Vaughan, Cara K.; Hayward, Jane; Goldenberg, Alice; Emes, Richard D.; Munye, Mustafa M.; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean- François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R.; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M. K.; Antonarakis, Stylianos E.; Loebinger, Michael R.; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Beales, Philip L.; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Allan, Daly; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; McCarthy, Shane; Muddyman, Dawn; Muntoni, Francesco; Parker, Victoria; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter J.; Schmidts, Miriam; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M.

2017-01-01

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins. PMID:28176794

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.

PubMed

Olcese, Chiara; Patel, Mitali P; Shoemark, Amelia; Kiviluoto, Santeri; Legendre, Marie; Williams, Hywel J; Vaughan, Cara K; Hayward, Jane; Goldenberg, Alice; Emes, Richard D; Munye, Mustafa M; Dyer, Laura; Cahill, Thomas; Bevillard, Jeremy; Gehrig, Corinne; Guipponi, Michel; Chantot, Sandra; Duquesnoy, Philippe; Thomas, Lucie; Jeanson, Ludovic; Copin, Bruno; Tamalet, Aline; Thauvin-Robinet, Christel; Papon, Jean-François; Garin, Antoine; Pin, Isabelle; Vera, Gabriella; Aurora, Paul; Fassad, Mahmoud R; Jenkins, Lucy; Boustred, Christopher; Cullup, Thomas; Dixon, Mellisa; Onoufriadis, Alexandros; Bush, Andrew; Chung, Eddie M K; Antonarakis, Stylianos E; Loebinger, Michael R; Wilson, Robert; Armengot, Miguel; Escudier, Estelle; Hogg, Claire; Amselem, Serge; Sun, Zhaoxia; Bartoloni, Lucia; Blouin, Jean-Louis; Mitchison, Hannah M

2017-02-08

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome.

PubMed

Grose, Julianne H; Langston, Kelsey; Wang, Xiaohui; Squires, Shayne; Mustafi, Soumyajit Banerjee; Hayes, Whitney; Neubert, Jonathan; Fischer, Susan K; Fasano, Matthew; Saunders, Gina Moore; Dai, Qiang; Christians, Elisabeth; Lewandowski, E Douglas; Ping, Peipei; Benjamin, Ivor J

2015-01-01

Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 "cardiac interactome" to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease.

Heat stress-induced nuclear transport mediated by Hikeshi confers nuclear function of Hsp70s.

PubMed

Imamoto, Naoko

2018-06-01

The prime feature of eukaryotic cells is the separation of the intracellular space into two compartments, the nucleus and the cytoplasm. Active nuclear transport is crucial for the maintenance of this separation. In this report, we focus on a nuclear transport receptor named Hikeshi, which mediates the heat stress-induced nuclear import of 70-kDa heat shock proteins (Hsp70s), and discuss how the same protein can function differently depending on the cellular compartment in which it is localized. Hsp70 is a molecular chaperone that is predominantly localized in the cytoplasm under normal conditions but is known to accumulate in the nucleus under conditions of heat stress. Although the reported function of Hsp70 is mostly attributed to its molecular function in the cytoplasm, the functions of Hsp70 may extend beyond molecular chaperone activity in the nucleus. Copyright © 2018 The Author. Published by Elsevier Ltd.. All rights reserved.

Extracts Obtained from Pterocarpus angolensis DC and Ziziphus mucronata Exhibit Antiplasmodial Activity and Inhibit Heat Shock Protein 70 (Hsp70) Function.

PubMed

Zininga, Tawanda; Anokwuru, Chinedu P; Sigidi, Muendi T; Tshisikhawe, Milingoni P; Ramaite, Isaiah I D; Traoré, Afsatou N; Hoppe, Heinrich; Shonhai, Addmore; Potgieter, Natasha

2017-07-28

Malaria parasites are increasingly becoming resistant to currently used antimalarial therapies, therefore there is an urgent need to expand the arsenal of alternative antimalarial drugs. In addition, it is also important to identify novel antimalarial drug targets. In the current study, extracts of two plants, Pterocarpus angolensis and Ziziphus mucronata were obtained and their antimalarial functions were investigated. Furthermore, we explored the capability of the extracts to inhibit Plasmodium falciparum heat shock protein 70 (Hsp70) function. Heat shock protein 70 (Hsp70) are molecular chaperones whose function is to facilitate protein folding. Plasmodium falciparum the main agent of malaria, expresses two cytosol-localized Hsp70s: PfHsp70-1 and PfHsp70-z. The PfHsp70-z has been reported to be essential for parasite survival, while inhibition of PfHsp70-1 function leads to parasite death. Hence both PfHsp70-1 and PfHsp70-z are potential antimalarial drug targets. Extracts of P. angolensis and Z. mucronata inhibited the basal ATPase and chaperone functions of the two parasite Hsp70s. Furthermore, fractions of P. angolensis and Z. mucronata inhibited P. falciparum 3D7 parasite growth in vitro. The extracts obtained in the current study exhibited antiplasmodial activity as they killed P. falciparum parasites maintained in vitro. In addition, the findings further suggest that some of the compounds in P. angolensis and Z. mucronata may target parasite Hsp70 function.

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.

Curcumin Suppresses Soluble Tau Dimers and Corrects Molecular Chaperone, Synaptic, and Behavioral Deficits in Aged Human Tau Transgenic Mice*

PubMed Central

Ma, Qiu-Lan; Zuo, Xiaohong; Yang, Fusheng; Ubeda, Oliver J.; Gant, Dana J.; Alaverdyan, Mher; Teng, Edmond; Hu, Shuxin; Chen, Ping-Ping; Maiti, Panchanan; Teter, Bruce; Cole, Greg M.; Frautschy, Sally A.

2013-01-01

The mechanisms underlying Tau-related synaptic and cognitive deficits and the interrelationships between Tau species, their clearance pathways, and synaptic impairments remain poorly understood. To gain insight into these mechanisms, we examined these interrelationships in aged non-mutant genomic human Tau mice, with established Tau pathology and neuron loss. We also examined how these interrelationships changed with an intervention by feeding mice either a control diet or one containing the brain permeable beta-amyloid and Tau aggregate binding molecule curcumin. Transgene-dependent elevations in soluble and insoluble phospho-Tau monomer and soluble Tau dimers accompanied deficits in behavior, hippocampal excitatory synaptic markers, and molecular chaperones (heat shock proteins (HSPs)) involved in Tau degradation and microtubule stability. In human Tau mice but not control mice, HSP70, HSP70/HSP72, and HSP90 were reduced in membrane-enriched fractions but not in cytosolic fractions. The synaptic proteins PSD95 and NR2B were reduced in dendritic fields and redistributed into perikarya, corresponding to changes observed by immunoblot. Curcumin selectively suppressed levels of soluble Tau dimers, but not of insoluble and monomeric phospho-Tau, while correcting behavioral, synaptic, and HSP deficits. Treatment increased PSD95 co-immunoprecipitating with NR2B and, independent of transgene, increased HSPs implicated in Tau clearance. It elevated HSP90 and HSC70 without increasing HSP mRNAs; that is, without induction of the heat shock response. Instead curcumin differentially impacted HSP90 client kinases, reducing Fyn without reducing Akt. In summary, curcumin reduced soluble Tau and elevated HSPs involved in Tau clearance, showing that even after tangles have formed, Tau-dependent behavioral and synaptic deficits can be corrected. PMID:23264626

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.

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

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

Hsp31, a member of the DJ-1 superfamily, is a multitasking stress responder with chaperone activity

PubMed Central

Aslam, Kiran; Hazbun, Tony R.

2016-01-01

ABSTRACT Among different types of protein aggregation, amyloids are a biochemically well characterized state of protein aggregation that are associated with a large number of neurodegenerative diseases including Parkinson's disease, Alzheimer and Creutzfeldt-Jakob disease. Yeast, Saccharomyces cerevisiae is an insightful model to understand the underlying mechanism of protein aggregation. Many yeast molecular chaperones can modulate aggregation and misfolding of proteins including α-Syn and the Sup35 prion. Hsp31 is a homodimeric protein structurally similar to human DJ-1, a Parkinson's disease-linked protein, and both are members of the DJ-1/ThiJ/PfpI superfamily. An emerging view is that Hsp31 and its associated superfamily members each have divergent multitasking functions that have the common theme of responding and managing various types of cellular stress. Hsp31 has several biochemical activities including chaperone and detoxifying enzyme activities that modulate at various points of a stress pathway such as toxicity associated with protein misfolding. However, we have shown the protective role of Hsp31's chaperone activity can operate independent of detoxifying enzyme activities in preventing the early stages of protein aggregate formation and associated cellular toxicities. We provide additional data that collectively supports the multiple functional roles that can be accomplished independent of each other. We present data indicating Hsp31 purified from yeast is more active compared to expression and purification from E. coli suggesting that posttranslational modifications could be important for Hsp31 to be fully active. We also compare the similarities and differences in activities among paralogs of Hsp31 supporting a model in which this protein family has overlapping but diverging roles in responding to various sources of cellular stresses. PMID:27097320

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.

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.

Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins.

PubMed

Cortajarena, Aitziber L; Kajander, Tommi; Pan, Weilan; Cocco, Melanie J; Regan, Lynne

2004-04-01

Protein design aims to understand the fundamentals of protein structure by creating novel proteins with pre-specified folds. An equally important goal is to understand protein function by creating novel proteins with pre-specified activities. Here we describe the design and characterization of a tetratricopeptide (TPR) protein, which binds to the C-terminal peptide of the eukaryotic chaperone Hsp90. The design emphasizes the importance of both direct, short-range protein-peptide interactions and of long-range electrostatic optimization. We demonstrate that the designed protein binds specifically to the desired peptide and discriminates between it and the similar C-terminal peptide of Hsp70.

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria.

PubMed

Zmijewski, Michał A; Skórko-Glonek, Joanna; Tanfani, Fabio; Banecki, Bogdan; Kotlarz, Agnieszka; Macario, Alberto J L; Lipińska, Barbara

2007-01-01

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

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

Immunodominant protein MIP_05962 from Mycobacterium indicus pranii displays chaperone activity.

PubMed

Sharma, Ashish; Equbal, Md Javed; Pandey, Saurabh; Sheikh, Javaid A; Ehtesham, Nasreen Z; Hasnain, Seyed E; Chaudhuri, Tapan K

2017-05-01

Tuberculosis, a contagious disease of infectious origin is currently a major cause of deaths worldwide. Mycobacterium indicus pranii (MIP), a saprophytic nonpathogen and a potent immunomodulator is currently being investigated as an intervention against tuberculosis along with many other diseases with positive outcome. The apparent paradox of multiple chaperones in mycobacterial species and enigma about the cellular functions of the client proteins of these chaperones need to be explored. Chaperones are the known immunomodulators; thus, there is need to exploit the proteome of MIP for identification and characterization of putative chaperones. One of the immunogenic proteins, MIP_05962 is a member of heat shock protein (HSP) 20 family due to the presence of α-crystallin domain, and has amino acid similarity with Mycobacterium lepraeHSP18 protein. The diverse functions of M. lepraeHSP18 in stress conditions implicate MIP_05962 as an important protein that needs to be explored. Biophysical and biochemical characterization of the said protein proved it to be a chaperone. The observations of aggregation prevention and refolding of substrate proteins in the presence of MIP_05962 along with interaction with non-native proteins, surface hydrophobicity, formation of large oligomers, in-vivo thermal rescue of Escherichia coli expressing MIP_05962, enhancing solubility of insoluble protein maltodextrin glucosidase (MalZ) under in-vivo conditions, and thermal stability and reversibility confirmed MIP_05962 as a molecular chaperone. © 2017 Federation of European Biochemical Societies.

Chaperoning the Cancer: The Proteostatic Functions of the Heat Shock Proteins in Cancer.

PubMed

Vahid, Sepideh; Thaper, Daksh; Zoubeidi, Amina

2017-01-01

Protein homeostasis (proteostasis) is vital for the survival of cells in physiological and pathological conditions. Particularly, cancer cells are in constant state of cellular stress due to rapid proliferation and decreased quality control in proteosynthesis and therefore, are exceedingly dependent on the homeostasis pathways. Among the complex biological mechanisms regulating proteostasis are the highly conserved molecular chaperones, heat shock proteins (HSPs). HSPs assist cell survival by catalysing the proper folding of proteins, modulation of the apoptotic machinery and finally regulating the protein degradation machinery, providing either the stability or the degradation of selected proteins under stress conditions. Inevitably, HSPs are upregulated in malignancies and participate in different hallmarks of cancer, with indispensable roles in the onset and progression of the disease. Moreover, high levels of HSPs contribute to poor prognosis and treatment resistance in various cancers. Therefore these molecular chaperones present as attractive targets for anti-cancer therapy. This review describes how HSPs regulate different hallmarks of cancer and provides an overview on the most relevant patents which have recently appeared in the literature. The patents were extracted from Google Patents (2012-2016) while the clinical trial results were mined from www.clinicaltrial.gov. Review of literature shows that the proteostatic functions of HSPs can modify different hallmarks of cancer. Moreover, targeting HSPs (notably HSP27, HSP70 and HSP90) exhibited positive results in clinical trials so far. However, more studies should be designed to optimize the efficacy of mono or combination therapy in various malignancies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Role and mechanism of the Hsp70 molecular chaperone machines in bacterial pathogens.

PubMed

Ghazaei, Ciamak

2017-03-01

Heat shock proteins are highly conserved, stress-inducible, ubiquitous proteins that maintain homeostasis in both eukaryotes and prokaryotes. Hsp70 proteins belong to the heat shock protein family and enhance bacterial survival in hostile environments. Hsp70, known as DnaK in prokaryotes, supports numerous processes such as the assembly and disassembly of protein complexes, the refolding of misfolded and clustered proteins, membrane translocation and the regulation of regulatory proteins. The chaperone-based activity of Hsp70 depends on dynamic interactions between its two domains, known as the ATPase domain and the substrate-binding domain. It also depends on interactions between these domains and other co-chaperone molecules such as the Hsp40 protein family member DnaJ and nucleotide exchange factors. DnaJ is the primary chaperone that interacts with nascent polypeptide chains and functions to prevent their premature release from the ribosome and misfolding before it is targeted by DnaK. Adhesion of bacteria to host cells is mediated by both host and bacterial Hsp70. Following infection of the host, bacterial Hsp70 (DnaK) is in a position to initiate bacterial survival processes and trigger an immune response by the host. Any mutations in the dnaK gene have been shown to decrease the viability of bacteria inside the host. This review will give insights into the structure and mechanism of Hsp70 and its role in regulating the protein activity that contributes to pathogenesis.

Antioxidant and molecular chaperone defences during estivation and arousal in the South American apple snail Pomacea canaliculata.

PubMed

Giraud-Billoud, Maximiliano; Vega, Israel A; Tosi, Martín E Rinaldi; Abud, María A; Calderón, María L; Castro-Vazquez, Alfredo

2013-02-15

The invasive Pomacea canaliculata estivates during periods of drought and should cope with harmful effects of reoxygenation during arousal. We studied thiobarbituric acid reactive substances (TBARS), enzymatic (superoxide dismutase, SOD and catalase, CAT) and non-enzymatic antioxidants (uric acid and reduced glutathione), and heat shock protein expression (Hsc70, Hsp70 and Hsp90) in (1) active control snails, (2) snails after 45 days of estivation, and (3) aroused snails 20 min and (4) 24 h after water exposure, in midgut gland, kidney and foot. Both kidney and foot (but not the midgut gland) showed a TBARS increase during estivation and a decrease after arousal. Tissue SOD and CAT did not change in any experimental groups. Uric acid increased during estivation in all tissues, and it decreased after arousal in the kidney. Allantoin, the oxidation product of uric acid, remained constant in the midgut gland but it decreased in the kidney until 20 min after arousal; however, allantoin levels rose in both kidney and foot 24 h after arousal. Reduced glutathione decreased during estivation and arousal, in both midgut gland and kidney, and it remained constant in the foot. Hsc70 and Hsp70 kidney levels were stable during the activity-estivation cycle and Hsp90 expression decreases during estivation and recovers in the early arousal. In foot, the expression of Hsp70 and Hsp90 was high during activity and estivation periods and disminished after arousal. Results indicate that a panoply of antioxidant and molecular chaperone defences may be involved during the activity-estivation cycle in this freshwater gastropod.

Metabolic energy sensors (AMPK and SIRT1), protein carbonylation and cardiac failure as biomarkers of thermal stress in an intertidal limpet: linking energetic allocation with environmental temperature during aerial emersion.

PubMed

Han, Guo-dong; Zhang, Shu; Marshall, David J; Ke, Cai-huan; Dong, Yun-wei

2013-09-01

The effects of heat stress on organisms are manifested at the levels of organ function, metabolic activity, protein stability and gene expression. Here, we examined effects of high temperature on the intertidal limpet Cellana toreuma to determine how the temperatures at which (1) organ failure (cardiac function), (2) irreversible protein damage (carbonylation) and (3) expression of genes encoding proteins involved in molecular chaperoning (hsp70 and hsp90) and metabolic regulation (ampk and sirt1) occur compare with field temperatures, which commonly exceed 30°C and can reach 46°C. Heart failure, indexed by the Arrhenius break temperature, occurred at 34.3°C. Protein carbonylation rose significantly at 38°C. Genes for heat shock proteins HSP70 (hsp70) and HSP90 (hsp90), for two subunits of AMP-activated protein kinase (AMPK) (ampkα and ampkβ) and for histone/protein deacetylase SIRT1 (sirt1) all showed increased expression at 30°C. Temperatures of maximal expression differed among genes, as did temperatures at which upregulation ceased. Expression patterns for ampk and sirt1 indicate that heat stress influenced cellular energy homeostasis; above ~30°C, upregulation of ATP-generating pathways is suggested by elevated expression of genes for ampk; an altered balance between reliance on carbohydrate and lipid fuels is indicated by changes in expression of sirt1. These results show that C. toreuma commonly experiences temperatures that induce expression of genes associated with the stress response (hsp70 and hsp90) and regulation of energy metabolism (ampk and sirt1). At high temperatures, there is likely to be a shift away from anabolic processes such as growth to catabolic processes, to provide energy for coping with stress-induced damage, notably to proteins.

Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles

PubMed Central

Rosenzweig, Rina; Sekhar, Ashok; Nagesh, Jayashree; Kay, Lewis E

2017-01-01

The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state. DOI: http://dx.doi.org/10.7554/eLife.28030.001 PMID:28708484

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas.

PubMed

Ochoa, Gina H; Clark, Ying Mei; Matsumoto, Brian; Torres-Ruiz, Jose A; Robles, Laura J

2002-02-01

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.

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.

Multiple functions of the E3 ubiquitin ligase CHIP in immunity.

PubMed

Zhan, Shaohua; Wang, Tianxiao; Ge, Wei

2017-09-03

The carboxyl terminal of Hsp70-interacting protein (CHIP) is an E3 ubiquitin ligase that plays a pivotal role in the protein quality control system by shifting the balance of the folding-refolding machinery toward the degradative pathway. However, the precise mechanisms by which nonnative proteins are selected for degradation by CHIP either directly or indirectly via chaperone Hsp70 or Hsp90 are still not clear. In this review, we aim to provide a comprehensive model of the mechanism by which CHIP degrades its substrate in a chaperone-dependent or direct manner. In addition, through tight regulation of the protein level of its substrates, CHIP plays important roles in many physiological and pathological conditions, including cancers, neurological disorders, cardiac diseases, bone metabolism, immunity, and so on. Nonetheless, the precise mechanisms underlying the regulation of the immune system by CHIP are still poorly understood despite accumulating developments in our understanding of the regulatory roles of CHIP in both innate and adaptive immune responses. In this review, we also aim to provide a view of CHIP-mediated regulation of immune responses and the signaling pathways involved in the model described. Finally, we discuss the roles of CHIP in immune-related diseases.

Sequence analyses reveal that a TPR-DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR-DP domains and prokaryotic GerD proteins.

PubMed

Hernández Torres, Jorge; Papandreou, Nikolaos; Chomilier, Jacques

2009-05-01

The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR-DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR-DP domains.

Heat shock proteins on the human sperm surface.

PubMed

Naaby-Hansen, Soren; Herr, John C

2010-01-01

The sperm plasma membrane is known to be critical to fertilization and to be highly regionalized into domains of head, mid- and principal pieces. However, the molecular composition of the sperm plasma membrane and its alterations during genital tract passage, capacitation and the acrosome reaction remains to be fully dissected. A two-dimensional gel-based proteomic study previously identified 98 human sperm proteins which were accessible for surface labelling with both biotin and radioiodine. In this report twelve dually labelled protein spots were excised from stained gels or PDVF membranes and analysed by mass spectrometry (MS) and Edman degradation. Seven members from four different heat shock protein (HSP) families were identified including HYOU1 (ORP150), HSPC1 (HSP86), HSPA5 (Bip), HSPD1 (HSP60), and several isoforms of the two testis-specific HSP70 chaperones HSPA2 and HSPA1L. An antiserum raised against the testis-specific HSPA2 chaperone reacted with three 65kDa HSPA2 isoforms and three high molecular weight surface proteins (78-79kDa, 84kDa and 90-93kDa). These proteins, together with seven 65kDa HSP70 forms, reacted with human anti-sperm IgG antibodies that blocked in vitro fertilization in humans. Three of these surface biotinylated human sperm antigens were immunoprecipitated with a rabbit antiserum raised against a linear peptide epitope in Chlamydia trachomatis HSP70. The results indicate diverse HSP chaperones are accessible for surface labelling on human sperm. Some of these share epitopes with C. trachomatis HSP70, suggesting an association between genital tract infection, immunity to HSP70 and reproductive failure. 2009 Elsevier Ireland Ltd. All rights reserved.

Tight Control of Trehalose Content Is Required for Efficient Heat-induced Cell Elongation in Candida albicans*

PubMed Central

Serneels, Joke; Tournu, Hélène; Van Dijck, Patrick

2012-01-01

The ability to form hyphae in the human pathogenic fungus Candida albicans is a prerequisite for virulence. It contributes to tissue infection, biofilm formation, as well as escape from phagocytes. Cell elongation triggered by human body temperature involves the essential heat shock protein Hsp90, which negatively governs a filamentation program dependent upon the Ras-protein kinase A (PKA) pathway. Tight regulation of Hsp90 function is required to ensure fast appropriate response and maintenance of a wide range of regulatory and signaling proteins. Client protein activation by Hsp90 relies on a conformational change of the chaperone, whose ATPase activity is competitively inhibited by geldanamycin. We demonstrate a novel regulatory mechanism of heat- and Hsp90-dependent induced morphogenesis, whereby the nonreducing disaccharide trehalose acts as a negative regulator of Hsp90 release. By means of a mutant strain deleted for Gpr1, the G protein-coupled receptor upstream of PKA, we demonstrate that elevated trehalose content in that strain, resulting from misregulation of enzymatic activities involved in trehalose metabolism, disrupts the filamentation program in response to heat. Addition of geldanamycin does not result in hyphal extensions at 30 °C in the gpr1Δ/gpr1Δ mutant as it does in wild type cells. In addition, validamycin, a specific inhibitor of trehalase, the trehalose-degrading enzyme, inhibits cell elongation in response to heat and geldanamycin. These results place Gpr1 as a regulator of trehalose metabolism in C. albicans and illustrate that trehalose modulates Hsp90-dependent activation of client proteins and signaling pathways leading to filamentation in the human fungal pathogen. PMID:22952228

Cloning, expression and crystallisation of SGT1 co-chaperone protein from Glaciozyma antarctica

NASA Astrophysics Data System (ADS)

Yusof, Nur Athirah; Bakar, Farah Diba Abu; Beddoe, Travis; Murad, Abdul Munir Abdul

2013-11-01

Studies on psycrophiles are now in the limelight of today's post genomic era as they fascinate the research and development industries. The discovery from Glaciozyma antarctica, an extreme cold adapted yeast from Antarctica shows promising future to provide cost effective natural sustainable energy and create wider understanding of the property that permits this organisms to adapt to extreme temperature downshift. In plants and yeast, studies show the interaction between SGT1 and HSP90 are essential for disease resistance and heat stress by activating a number of resistance proteins. Here we report for the first time cloning, expression and crystallization of the recombinant SGT1 protein of G. antarctica (rGa_SGT1), a highly conserved eukaryotic protein that interacts with the molecular chaperones HSP90 (heat shock protein 90) apparently associated in a role of co-chaperone that may play important role in cold adaptation. The sequence analysis of rGa_SGT1 revealed the presence of all the characteristic features of SGT1 protein. In this study, we present the outlines and results of protein structural study of G. antarctica SGT1 protein. We validate this approach by starting with cloning the target insert into Ligation Independent Cloning system proceeded with expression using E. coli system, and crystallisation of the target rGA_SGT1 protein. The work is still on going with the target subunit of the complex proteins yielded crystals. These results, still ongoing, open a platform for better understanding of the uniqueness of this crucial molecular machine function in cold adaptation.

Interplay between Heat Shock Proteins HSP101 and HSA32 Prolongs Heat Acclimation Memory Posttranscriptionally in Arabidopsis1[W][OA

PubMed Central

Wu, Ting-ying; Juan, Yu-ting; Hsu, Yang-hsin; Wu, Sze-hsien; Liao, Hsiu-ting; Fung, Raymond W.M.; Charng, Yee-yung

2013-01-01

Heat acclimation improves the tolerance of organisms to severe heat stress. Our previous work showed that in Arabidopsis (Arabidopsis thaliana), the “memory” of heat acclimation treatment decayed faster in the absence of the heat-stress-associated 32-kD protein HSA32, a heat-induced protein predominantly found in plants. The HSA32 null mutant attains normal short-term acquired thermotolerance but is defective in long-term acquired thermotolerance. To further explore this phenomenon, we isolated Arabidopsis defective in long-term acquired thermotolerance (dlt) mutants using a forward genetic screen. Two recessive missense alleles, dlt1-1 and dlt1-2, encode the molecular chaperone heat shock protein101 (HSP101). Results of immunoblot analyses suggest that HSP101 enhances the translation of HSA32 during recovery after heat treatment, and in turn, HSA32 retards the decay of HSP101. The dlt1-1 mutation has little effect on HSP101 chaperone activity and thermotolerance function but compromises the regulation of HSA32. In contrast, dlt1-2 impairs the chaperone activity and thermotolerance function of HSP101 but not the regulation of HSA32. These results suggest that HSP101 has a dual function, which could be decoupled by the mutations. Pulse-chase analysis showed that HSP101 degraded faster in the absence of HSA32. The autophagic proteolysis inhibitor E-64d, but not the proteasome inhibitor MG132, inhibited the degradation of HSP101. Ectopic expression of HSA32 confirmed its effect on the decay of HSP101 at the posttranscriptional level and showed that HSA32 was not sufficient to confer long-term acquired thermotolerance when the HSP101 level was low. Taken together, we propose that a positive feedback loop between HSP101 and HSA32 at the protein level is a novel mechanism for prolonging the memory of heat acclimation. PMID:23439916

Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity.

PubMed

Rutsdottir, Gudrun; Härmark, Johan; Weide, Yoran; Hebert, Hans; Rasmussen, Morten I; Wernersson, Sven; Respondek, Michal; Akke, Mikael; Højrup, Peter; Koeck, Philip J B; Söderberg, Christopher A G; Emanuelsson, Cecilia

2017-05-12

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended I X V X I motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the I X V X I motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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.

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

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

Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

PubMed

Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

1997-03-15

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes in protein structure as a result of calcium binding may facilitate phosphorylation. A small, but significant, movement of metal ions and sidechains could position catalytically important threonine residues for phosphorylation. The second calcium site represents a new calcium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in the active site could be transmitted to the peptide-binding domain.

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.

The heat-shock protein/chaperone network and multiple stress resistance.

PubMed

Jacob, Pierre; Hirt, Heribert; Bendahmane, Abdelhafid

2017-04-01

Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multistress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat-shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone 'client proteins', many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat-shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signalling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/chaperone network. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

1H, 15N and 13C resonance assignments of the J-domain of co-chaperone Sis1 from Saccharomyces cerevisiae.

PubMed

Pinheiro, Glaucia M S; Amorim, Gisele C; Iqbal, Anwar; Ramos, C H I; Almeida, Fabio C L

2018-04-30

Protein folding in the cell is usually aided by molecular chaperones, from which the Hsp70 (Hsp = heat shock protein) family has many important roles, such as aiding nascent folding and participating in translocation. Hsp70 has ATPase activity which is stimulated by binding to the J-domain present in co-chaperones from the Hsp40 family. Hsp40s have many functions, as for instance the binding to partially folded proteins to be delivered to Hsp70. However, the presence of the J-domain characterizes Hsp40s or, by this reason, as J-proteins. The J-domain alone can stimulate Hsp70 ATPase activity. Apparently, it also maintains the same conformation as in the whole protein although structural information on full J-proteins is still missing. This work reports the 1 H, 15 N and 13 C resonance assignments of the J-domain of a Hsp40 from Saccharomyces cerevisiae, named Sis1. Secondary structure and order parameter prediction from chemical shifts are also reported. Altogether, the data show that Sis1 J-domain is highly structured and predominantly formed by α-helices, results that are in very good agreement with those previously reported for the crystallographic structure.

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.

The exported chaperone Hsp70-x supports virulence functions for Plasmodium falciparum blood stage parasites

PubMed Central

Charnaud, Sarah C.; Dixon, Matthew W. A.; Nie, Catherine Q.; Chappell, Lia; Sanders, Paul R.; Nebl, Thomas; Hanssen, Eric; Berriman, Matthew; Chan, Jo-Anne; Blanch, Adam J.; Beeson, James G.; Rayner, Julian C.; Przyborski, Jude M.; Tilley, Leann; Crabb, Brendan S.

2017-01-01

Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins. PMID:28732045

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 Hsp40 proteins of Plasmodium falciparum and other apicomplexa: regulating chaperone power in the parasite and the host.

PubMed

Botha, M; Pesce, E-R; Blatch, G L

2007-01-01

Extensive structural and functional remodelling of Plasmodium falciparum (malaria)-infected erythrocytes follows the export of a range of proteins of parasite origin (exportome) across the parasitophorous vacuole into the host erythrocyte. The genome of P. falciparum encodes a diverse chaperone complement including at least 43 members of the heat shock protein 40kDa (Hsp40) family, and six members of the heat shock protein 70kDa (Hsp70) family. Nearly half of the Hsp40 proteins of P. falciparum are predicted to contain a PEXEL/HT (Plasmodium export element/host targeting signal) sequence motif, and hence are likely to be part of the exportome. In this review we critically evaluate the classification, sequence similarity and clustering, and possible interactors of the P. falciparum Hsp40 chaperone machinery. In addition to the types I, II and III Hsp40 proteins all exhibiting the signature J-domain, the P. falciparum genome also encodes a number of specialized Hsp40 proteins with a J-like domain, which we have categorized as type IV Hsp40 proteins. Analysis of the potential P. falciparum Hsp40 protein interaction network revealed connections predominantly with cytoskeletal and membrane proteins, transcriptional machinery, DNA repair and replication machinery, translational machinery, the proteasome and proteolytic enzymes, and enzymes involved in cellular physiology. Comparison of the Hsp40 proteins of P. falciparum to those of other apicomplexa reveals that most of the proteins (especially the PEXEL/HT-containing proteins) are unique to P. falciparum. Furthermore, very few of the P. falciparum Hsp40 proteins have human homologs, except for those proteins implicated in fundamental biological processes. Our analysis suggests that P. falciparum has evolved an expanded and specialized Hsp40 protein machinery to enable it successfully to invade and remodel the human erythrocyte, and we propose a model in which these proteins are involved in chaperone-mediated translocation, folding, assembly and regulation of parasite and host proteins.

Heat shock proteins and proteasomal degradation in normal and tumor cells.

PubMed

Karademir, Betul; Bozaykut, Perinur; Kartal Ozer, Nesrin

2014-10-01

Proteasomal degradation of oxidized proteins is a crucial mechanism to prevent the accumulation of cellular damage. The removal of the damage is generally a required process for healthy organisms to keep the integrity while in cancer cells the situation may be different. In normal conditions, cancer cells have higher proteasome activity compared to normal cells. During cancer treatment, cellular damage by chemotherapy is an expected process to be able to kill the tumor cells. And the accumulation of this damage accompanied by the decrease in protein repair and removal systems may increase the efficacy of the cancer therapy. Heat shock proteins (Hsp) as molecular chaperones are involved in the folding, activation and assembly of a variety of proteins. Among these Hsp40, Hsp70 and Hsp90 are believed to act as a chaperone system to regulate the proteasomal degradation. In this study, we tested the role of heat stress response on the proteasomal degradation of oxidized proteins. We used two different cell lines to observe the difference in normal and tumor cells. First the effect of heat stress (42°C, 1h) were tested in terms of protein oxidation tested by protein carbonyl formation and proteasomal degradation. The results were extremely different in normal fibroblast cells and hippocampal tumor cells. In the same direction, the expressions of Hsp40, Hsp70 and Hsp90 were affected in a different manner in two cell lines, will be discussed in detail. Supported by TUBITAK COST-CM1001-110S281. Copyright © 2014. Published by Elsevier Inc.

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.

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

Sporadic renal angiomyolipoma in a patient with Birt-Hogg-Dubé: chaperones in pathogenesis.

PubMed

Sager, Rebecca A; Woodford, Mark R; Shapiro, Oleg; Mollapour, Mehdi; Bratslavsky, Gennady

2018-04-24

Birt-Hogg-Dubé (BHD) is an autosomal dominant genetic syndrome caused by germline mutations in the FLCN gene that predisposes patients to develop renal tumors. Renal angiomyolipoma (AML) is not a renal tumor sub-type associated with BHD. AML is, however, a common phenotypic manifestation of Tuberous Sclerosis Complex (TSC) syndrome caused by mutations in either the TSC1 or TSC2 tumor suppressor genes. Previous case reports of renal AML in patients with BHD have speculated on the molecular and clinical overlap of these two syndromes as a result of described involvement of the gene products in the mTOR pathway. Our recent work provided a new molecular link between these two syndromes by identifying FLCN and Tsc2 as clients of the molecular chaperone Hsp90. Folliculin interacting proteins FNIP1/2 and Tsc1 are important for FLCN and Tsc2 stability as new Hsp90 co-chaperones. Here we present a case of sporadic AML as a result of somatic Tsc1/2 loss in a patient with BHD. We further demonstrate that FNIP1 and Tsc1 are capable of compensating for each other in the chaperoning of mutated FLCN tumor suppressor. Our findings demonstrate interconnectivity and compensatory mechanisms between the BHD and TSC pathways.

Insights into Hsp70 Chaperone Activity from a Crystal Structure of the Yeast Hsp110 Sse1

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

Liu,Q.; Hendrickson, W.

2007-01-01

Classic Hsp70 chaperones assist in diverse processes of protein folding and translocation, and Hsp110s had seemed by sequence to be distant relatives within an Hsp70 superfamily. The 2.4 Angstroms resolution structure of Sse1 with ATP shows that Hsp110s are indeed Hsp70 relatives, and it provides insight into allosteric coupling between sites for ATP and polypeptide-substrate binding in Hsp70s. Subdomain structures are similar in intact Sse1(ATP) and in the separate Hsp70 domains, but conformational dispositions are radically different. Interfaces between Sse1 domains are extensive, intimate, and conservative in sequence with Hsp70s. We propose that Sse1(ATP) may be an evolutionary vestige ofmore » the Hsp70(ATP) state, and an analysis of 64 mutant variants in Sse1 and three Hsp70 homologs supports this hypothesis. An atomic-level understanding of Hsp70 communication between ATP and substrate-binding domains follows. Requirements on Sse1 for yeast viability are in keeping with the distinct function of Hsp110s as nucleotide exchange factors.« less

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.

Conserved conformational selection mechanism of Hsp70 chaperone-substrate interactions

PubMed Central

Velyvis, Algirdas; Zoltsman, Guy; Rosenzweig, Rina; Bouvignies, Guillaume

2018-01-01

Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event. PMID:29460778

Heat shock protein responses to aging and proteotoxicity in the olfactory bulb

PubMed Central

Posimo, Jessica M.; Mason, Daniel M.; Broeren, Matthew T.; Heinemann, Scott D.; Wipf, Peter; Brodsky, Jeffrey L.; Leak, Rehana K.

2015-01-01

The olfactory bulb is one of the most vulnerable brain regions in age-related proteinopathies. Proteinopathic stress is mitigated by the heat shock protein (Hsp) family of chaperones. Here we describe age-related decreases in Hsc70 in the olfactory bulb of the female rat and higher levels of Hsp70 and Hsp25 in middle and old age than at 2-4 months. In order to model proteotoxic and oxidative stress in the olfactory bulb, primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin and MG132 or the pro-oxidant paraquat. Toxin-induced increases were observed in Hsp70, Hsp25, and Hsp32. In order to determine the functional consequences of the increase in Hsp70, we attenuated Hsp70 activity with two mechanistically distinct inhibitors. The Hsp70 inhibitors greatly potentiated the toxicity of sublethal lactacystin or MG132 but not of paraquat. Although ubiquitinated protein levels were unchanged with aging in vivo or with sublethal MG132 in vitro, there was a large, synergistic increase in ubiquitinated proteins when proteasome and Hsp70 functions were simultaneously inhibited. Our study suggests that olfactory bulb cells rely heavily on Hsp70 chaperones to maintain homeostasis during mild proteotoxic, but not oxidative insults, and that Hsp70 prevents the accrual of ubiquitinated proteins in these cells. PMID:25640060

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

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation*

PubMed Central

Jakobsson, Magnus E.; Moen, Anders; Bousset, Luc; Egge-Jacobsen, Wolfgang; Kernstock, Stefan; Melki, Ronald; Falnes, Pål Ø.

2013-01-01

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein. PMID:23921388

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.

PROFILES OF GENE EXPRESSION ASSOCIATED WITH TETRACYCLINE OVER EXPRESSION OF HSP70 IN MCF-7 BREAST CANCER CELLS

EPA Science Inventory

Profiles of gene expression associated with tetracycline over expression of HSP70 in MCF-7 breast cancer cells.

Heat shock proteins (HSPs) protect cells from damage through their function as molecular chaperones. Some cancers reveal high levels of HSP70 expression in asso...

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

PubMed

Asea, A; Kraeft, S K; Kurt-Jones, E A; Stevenson, M A; Chen, L B; Finberg, R W; Koo, G C; Calderwood, S K

2000-04-01

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

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.

Protein unfolding as a switch from self-recognition to high-affinity client binding

PubMed Central

Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

2016-01-01

Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal α-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PubMed Central

Morgan, Rhodri M. L.; Hernández-Ramírez, Laura C.; Trivellin, Giampaolo; Zhou, Lihong; Roe, S. Mark; Korbonits, Márta; Prodromou, Chrisostomos

2012-01-01

Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20. PMID:23300914

Sequence analyses reveal that a TPR–DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR–DP domains and prokaryotic GerD proteins

PubMed Central

Papandreou, Nikolaos; Chomilier, Jacques

2008-01-01

The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR–DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR–DP domains. Electronic supplementary material The online version of this article (doi:10.1007/s12192-008-0083-8) contains supplementary material, which is available to authorized users. PMID:18987995

Regulation of sGC via hsp90, Cellular Heme, sGC Agonists, and NO: New Pathways and Clinical Perspectives

PubMed Central

Ghosh, Arnab

2017-01-01

Abstract Significance: Soluble guanylate cyclase (sGC) is an intracellular enzyme that plays a primary role in sensing nitric oxide (NO) and transducing its multiple signaling effects in mammals. Recent Advances: The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells, including sGC, where it helps to drive heme insertion into the sGC-β1 subunit. This allows sGC-β1 to associate with a partner sGC-α1 subunit and mature into an NO-responsive active form. Critical Issues: In this article, we review evidence to date regarding the mechanisms that modulate sGC activity by a pathway where binding of hsp90 or sGC agonist to heme-free sGC dictates the assembly and fate of an active sGC heterodimer, both by NO and heme-dependent or heme-independent pathways. Future Directions: We discuss some therapeutic implications of the NO-sGC-hsp90 nexus and its potential as a marker of inflammatory disease. Antioxid. Redox Signal. 26, 182–190. PMID:26983679

The Role of Co-chaperones in Synaptic Proteostasis and Neurodegenerative Disease

PubMed Central

Gorenberg, Erica L.; Chandra, Sreeganga S.

2017-01-01

Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs): Cysteine String Protein alpha (CSPα; DNAJC5), auxilin (DNAJC6), and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13). These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70), enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110), which interacts with Hsc70, DNAJAs, and DNAJBs to constitute a disaggregase. Hsp110-related disaggregase activity is present at the synapse and is known to protect against aggregation of proteins such as α-synuclein. Congruent with their importance in the nervous system, mutations of these co-chaperones lead to familial neurodegenerative disease. CSPα mutations cause adult neuronal ceroid lipofuscinosis, while auxilin mutations result in early-onset Parkinson's disease, demonstrating their significance in preservation of the nervous system. PMID:28579939

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

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

Molecular chaperones and protein folding as therapeutic targets in Parkinson's disease and other synucleinopathies.

PubMed

Ebrahimi-Fakhari, Darius; Saidi, Laiq-Jan; Wahlster, Lara

2013-12-05

Changes in protein metabolism are key to disease onset and progression in many neurodegenerative diseases. As a prime example, in Parkinson's disease, folding, post-translational modification and recycling of the synaptic protein α-synuclein are clearly altered, leading to a progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. Altered protein folding is one of the first steps of an increasingly understood cascade in which α-synuclein forms complex oligomers and finally distinct protein aggregates, termed Lewy bodies and Lewy neurites. In neurons, an elaborated network of chaperone and co-chaperone proteins is instrumental in mediating protein folding and re-folding. In addition to their direct influence on client proteins, chaperones interact with protein degradation pathways such as the ubiquitin-proteasome-system or autophagy in order to ensure the effective removal of irreversibly misfolded and potentially pathogenic proteins. Because of the vital role of proper protein folding for protein homeostasis, a growing number of studies have evaluated the contribution of chaperone proteins to neurodegeneration. We herein review our current understanding of the involvement of chaperones, co-chaperones and chaperone-mediated autophagy in synucleinopathies with a focus on the Hsp90 and Hsp70 chaperone system. We discuss genetic and pathological studies in Parkinson's disease as well as experimental studies in models of synucleinopathies that explore molecular chaperones and protein degradation pathways as a novel therapeutic target. To this end, we examine the capacity of chaperones to prevent or modulate neurodegeneration and summarize the current progress in models of Parkinson's disease and related neurodegenerative disorders.

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

Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1 from Saccharomyces cerevisiae.

PubMed

Hu, Wanhui; Wu, Huiwen; Zhang, Hong; Gong, Weibin; Perrett, Sarah

2015-10-01

Hsp70 chaperone proteins play crucial roles in the cell. Extensive structural and functional studies have been performed for bacterial and mammalian Hsp70s. Ssa1 from Saccharomyces cerevisiae is a member of the Hsp70 family. In vivo and biochemical studies on Ssa1 have revealed that it regulates prion propagation and the cell cycle. However, no structural data has been obtained for Ssa1 up to now. Here we report the almost complete (96 %) (1)H, (13)C, (15)N backbone and side chain NMR assignment of the 18.8 kDa Ssa1 substrate binding domain. The construct includes residues 382-554, which corresponds to the entire substrate binding domain and two following α-helices in homologous structures. The secondary structure predicted from the assigned chemical shifts is consistent with that of homologous Hsp70 substrate binding domains.

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.

Molecular chaperone Hsp27 regulates the Hippo tumor suppressor pathway in cancer

PubMed Central

Vahid, Sepideh; Thaper, Daksh; Gibson, Kate F.; Bishop, Jennifer L.; Zoubeidi, Amina

2016-01-01

Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers. PMID:27555231

Molecular chaperone Hsp27 regulates the Hippo tumor suppressor pathway in cancer.

PubMed

Vahid, Sepideh; Thaper, Daksh; Gibson, Kate F; Bishop, Jennifer L; Zoubeidi, Amina

2016-08-24

Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers.

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.

Djhsp90s are crucial regulators during planarian regeneration and tissue homeostasis.

PubMed

Dong, Zimei; Chu, Gengbo; Sima, Yingxu; Chen, Guangwen

2018-04-15

Heat shock protein 90 family members (HSP90s), as molecular chaperones, have conserved roles in the physiological processes of eukaryotes regulating cytoprotection, increasing host resistance and so on. However, whether HSP90s affect regeneration in animals is unclear. Planarians are emerging models for studying regeneration in vivo. Here, the roles of three hsp90 genes from planarian Dugesia japonica are investigated by WISH and RNAi. The results show that: (1) Djhsp90s expressions are induced by heat and cold shock, tissue damage and ionic liquid; (2) Djhsp90s mRNA are mainly distributed each side of the body in intact worms as well as blastemas in regenerative worms; (3) the worms show head regression, lysis, the body curling and the regeneration arrest or even failure after Djhsp90s RNAi; (4) Djhsp90s are involved in autophagy and locomotion of the body. The research results suggest that Djhsp90s are not only conserved in cytoprotection, but also involved in homeostasis maintenance and regeneration process by regulating different pathways in planarians. Copyright © 2018 Elsevier Inc. All rights reserved.

Inflammatory stress of pancreatic beta cells drives release of extracellular heat-shock protein 90α.

PubMed

Ocaña, Gail J; Pérez, Liliana; Guindon, Lynette; Deffit, Sarah N; Evans-Molina, Carmella; Thurmond, Debbie C; Blum, Janice S

2017-06-01

A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1β, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity. © 2017 John Wiley & Sons Ltd.

The heat shock protein 90 of Plasmodium falciparum and antimalarial activity of its inhibitor, geldanamycin

PubMed Central

Kumar, Rajinder; Musiyenko, Alla; Barik, Sailen

2003-01-01

Background The naturally occurring benzoquinone ansamycin compound, geldanamycin (GA), is a specific inhibitor of heat shock protein 90 (Hsp90) and is a potential anticancer agent. Since Plasmodium falciparum has been reported to have an Hsp90 ortholog, we tested the possibility that GA might inhibit it and thereby display antiparasitic activity. Results We provide direct recombinant DNA evidence for the Hsp90 protein of Plasmodium falciparum, the causative agent of fatal malaria. While the mRNA of Hsp90 was mainly expressed in ring and trophozoite stages, the protein was found in all stages, although schizonts contained relatively lower amounts. In vitro the parasitic Hsp90 exhibited an ATP-binding activity that could be specifically inhibited by GA. Plasmodium growth in human erythrocyte culture was strongly inhibited by GA with an IC50 of 20 nM, compared to the IC50 of 15 nM for chloroquine (CQ) under identical conditions. When used in combination, the two drugs acted synergistically. GA was equally effective against CQ-sensitive and CQ-resistant strains (3D7 and W2, respectively) and on all erythrocytic stages of the parasite. Conclusions Together, these results suggest that an active and essential Hsp90 chaperone cycle exists in Plasmodium and that the ansamycin antibiotics will be an important tool to dissect its role in the parasite. Additionally, the favorable pharmacology of GA, reported in human trials, makes it a promising antimalarial drug. PMID:14514358

The heat shock protein 90 of Plasmodium falciparum and antimalarial activity of its inhibitor, geldanamycin.

PubMed

Kumar, Rajinder; Musiyenko, Alla; Barik, Sailen

2003-09-15

The naturally occurring benzoquinone ansamycin compound, geldanamycin (GA), is a specific inhibitor of heat shock protein 90 (Hsp90) and is a potential anticancer agent. Since Plasmodium falciparum has been reported to have an Hsp90 ortholog, we tested the possibility that GA might inhibit it and thereby display antiparasitic activity. We provide direct recombinant DNA evidence for the Hsp90 protein of Plasmodium falciparum, the causative agent of fatal malaria. While the mRNA of Hsp90 was mainly expressed in ring and trophozoite stages, the protein was found in all stages, although schizonts contained relatively lower amounts. In vitro the parasitic Hsp90 exhibited an ATP-binding activity that could be specifically inhibited by GA. Plasmodium growth in human erythrocyte culture was strongly inhibited by GA with an IC50 of 20 nM, compared to the IC50 of 15 nM for chloroquine (CQ) under identical conditions. When used in combination, the two drugs acted synergistically. GA was equally effective against CQ-sensitive and CQ-resistant strains (3D7 and W2, respectively) and on all erythrocytic stages of the parasite. Together, these results suggest that an active and essential Hsp90 chaperone cycle exists in Plasmodium and that the ansamycin antibiotics will be an important tool to dissect its role in the parasite. Additionally, the favorable pharmacology of GA, reported in human trials, makes it a promising antimalarial drug.

PDE5 inhibitors enhance the lethality of pemetrexed through inhibition of multiple chaperone proteins and via the actions of cyclic GMP and nitric oxide

PubMed Central

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Gordon, Sarah; Dent, Paul

2017-01-01

Phosphodiesterase 5 (PDE5) inhibitors prevent the breakdown of cGMP that results in prolonged protein kinase G activation and the generation of nitric oxide. PDE5 inhibitors enhanced the anti-NSCLC cell effects of the NSCLC therapeutic pemetrexed. [Pemetrexed + sildenafil] activated an eIF2α – ATF4 – CHOP – Beclin1 pathway causing formation of toxic autophagosomes; activated a protective IRE1 – XBP-1 – chaperone induction pathway; and activated a toxic eIF2α – CHOP – DR4 / DR5 / CD95 induction pathway. [Pemetrexed + sildenafil] reduced the expression of c-FLIP-s, MCL-1 and BCL-XL that was blocked in a cell-type -dependent fashion by either over-expression of HSP90 / GRP78 / HSP70 / HSP27 or by blockade of eIF2α-CHOP signaling. Knock down of PKGI/II abolished the ability of sildenafil to enhance pemetrexed toxicity whereas pan-inhibition of NOS using L-NAME or knock down of [iNOS + eNOS] only partially reduced the lethal drug interaction. Pemetrexed reduced the ATPase activities of HSP90 and HSP70 in an ATM-AMPK-dependent fashion that was enhanced by sildenafil signaling via PKGI/II. The drug combination activated an ATM-AMPK-TSC2 pathway that was associated with reduced mTOR S2448 and ULK-1 S757 phosphorylation and increased ULK-1 S317 and ATG13 S318 phosphorylation. These effects were prevented by chaperone over-expression or by expression of an activated form of mTOR that prevented autophagosome formation and reduced cell killing. In two models of NSCLC, sildenafil enhanced the ability of pemetrexed to suppress tumor growth. Collectively we argue that the combination of [pemetrexed + PDE5 inhibitor] should be explored in a new NSCLC phase I trial. PMID:27903966

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

PubMed Central

Tsai, Chai-jui; Aslam, Kiran; Drendel, Holli M.; Asiago, Josephat M.; Goode, Kourtney M.; Paul, Lake N.; Rochet, Jean-Christophe; Hazbun, Tony R.

2015-01-01

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H2O2, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins. PMID:26306045

Swa2, the yeast homolog of mammalian auxilin, is specifically required for the propagation of the prion variant [URE 3‐1

PubMed Central

Troisi, Elizabeth M.; Rockman, Michael E.; Nguyen, Phil P.; Oliver, Emily E.

2015-01-01

Summary Yeast prions require a core set of chaperone proteins including Sis1, Hsp70 and Hsp104 to generate new amyloid templates for stable propagation, yet emerging studies indicate that propagation of some prions requires additional chaperone activities, demonstrating chaperone specificity beyond the common amyloid requirements. To comprehensively assess such prion‐specific requirements for the propagation of the [URE 3] prion variant [URE 3‐1], we screened 12 yeast cytosolic J‐proteins, and here we report a novel role for the J‐protein Swa2/Aux1. Swa2 is the sole yeast homolog of the mammalian protein auxilin, which, like Swa2, functions in vesicle‐mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. We found that, in addition to Sis1, [URE 3‐1] is specifically dependent upon Swa2, but not on any of the 11 other J‐proteins. Further, we show that [URE 3‐1] propagation requires both a functional J‐domain and the tetratricopeptide repeat (TPR) domain, but surprisingly does not require Swa2‐clathrin binding. Because the J‐domain of Swa2 can be replaced with the J‐domains of other proteins, our data strongly suggest that prion‐chaperone specificity arises from the Swa2 TPR domain and supports a model where Swa2 acts through Hsp70, most likely to provide additional access points for Hsp104 to promote prion template generation. PMID:26031938

Engineering and Evolution of Molecular Chaperones and Protein Disaggregases with Enhanced Activity

PubMed Central

Mack, Korrie L.; Shorter, James

2016-01-01

Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector. PMID:27014702

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

Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

PubMed Central

Chou, Ming-Lun; Fitzpatrick, Lynda M.; Tu, Shuh-Long; Budziszewski, Gregory; Potter-Lewis, Sharon; Akita, Mitsuru; Levin, Joshua Z.; Keegstra, Kenneth; Li, Hsou-min

2003-01-01

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane. PMID:12805212

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.

Small molecule sensitization to TRAIL is mediated via nuclear localization, phosphorylation and inhibition of chaperone activity of Hsp27

PubMed Central

Mellier, G; Liu, D; Bellot, G; Lisa Holme, A; Pervaiz, S

2013-01-01

The small chaperone protein Hsp27 confers resistance to apoptosis, and therefore is an attractive anticancer drug target. We report here a novel mechanism underlying the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitizing activity of the small molecule LY303511, an inactive analog of the phosphoinositide 3-kinase inhibitor inhibitor LY294002, in HeLa cells that are refractory to TRAIL-induced apoptosis. On the basis of the fact that LY303511 is derived from LY294002, itself derived from quercetin, and earlier findings indicating that quercetin and LY294002 affected Hsp27 expression, we investigated whether LY303511 sensitized cancer cells to TRAIL via a conserved inhibitory effect on Hsp27. We provide evidence that upon treatment with LY303511, Hsp27 is progressively sequestered in the nucleus, thus reducing its protective effect in the cytosol during the apoptotic process. LY303511-induced nuclear translocation of Hsp27 is linked to its sustained phosphorylation via activation of p38 kinase and MAPKAP kinase 2 and the inhibition of PP2A. Furthermore, Hsp27 phosphorylation leads to the subsequent dissociation of its large oligomers and a decrease in its chaperone activity, thereby further compromising the death inhibitory activity of Hsp27. Furthermore, genetic manipulation of Hsp27 expression significantly affected the TRAIL sensitizing activity of LY303511, which corroborated the Hsp27 targeting activity of LY303511. Taken together, these data indicate a novel mechanism of small molecule sensitization to TRAIL through targeting of Hsp27 functions, rather than its overall expression, leading to decreased cellular protection, which could have therapeutic implications for overcoming chemotherapy resistance in tumor cells. PMID:24176848

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

PubMed

Lee, Eun Jeong; Yun, Un-Jung; Koo, Kyung Hee; Sung, Jee Young; Shim, Jaegal; Ye, Sang-Kyu; Hong, Kyeong-Man; Kim, Yong-Nyun

2014-01-01

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function. © 2013.

Heat Shock Protein 70: Roles in Multiple Sclerosis

PubMed Central

Mansilla, María José; Montalban, Xavier; Espejo, Carmen

2012-01-01

Heat shock proteins (HSP) have long been considered intracellular chaperones that possess housekeeping and cytoprotective functions. Consequently, HSP overexpression was proposed as a potential therapy for neurodegenerative diseases characterized by the accumulation or aggregation of abnormal proteins. Recently, the discovery that cells release HSP with the capacity to trigger proinflammatory as well as immunoregulatory responses has focused attention on investigating the role of HSP in chronic inflammatory autoimmune diseases such as multiple sclerosis (MS). To date, the most relevant HSP is the inducible Hsp70, which exhibits both cytoprotectant and immunoregulatory functions. Several studies have presented contradictory evidence concerning the involvement of Hsp70 in MS or experimental autoimmune encephalomyelitis (EAE), the MS animal model. In this review, we dissect the functions of Hsp70 and discuss the controversial data concerning the role of Hsp70 in MS and EAE. PMID:22669475

Structural mechanisms of chaperone mediated protein disaggregation

PubMed Central

Sousa, Rui

2014-01-01

The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism—entropic pulling—may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context. PMID:25988153

Phloretin increases the anti-tumor efficacy of intratumorally delivered heat-shock protein 70 kDa (HSP70) in a murine model of melanoma.

PubMed

Abkin, Sergey V; Ostroumova, Olga S; Komarova, Elena Y; Meshalkina, Darya A; Shevtsov, Maxim A; Margulis, Boris A; Guzhova, Irina V

2016-01-01

Recombinant HSP70 chaperone exerts a profound anticancer effect when administered intratumorally. This action is based on the ability of HSP70 to penetrate tumor cells and extract its endogenous homolog. To enhance the efficacy of HSP70 cycling, we employed phloretin, a flavonoid that enhances the pore-forming activity of the chaperone on artificial membranes. Phloretin increased the efficacy of HSP70 penetration in B16 mouse melanoma cells and K-562 human erythroblasts; this was accompanied with increased transport of the endogenous HSP70 to the plasma membrane. Importantly, treatment with HSP70 combined with phloretin led to the elevation of cell sensitivity to cytotoxic lymphocytes by 16-18 % compared to treatment with the chaperone alone. The incubation of K-562 cells with biotinylated HSP70 and phloretin increased the amount of the chaperone released from cells, suggesting that chaperone cycling could trigger a specific anti-tumor response. We studied the effect of the combination of HSP70 and phloretin using B16 melanoma and a novel method of HSP70-gel application. We found that the addition of phloretin to the gel reduced tumor weight almost fivefold compared with untreated mice, while the life span of the animals extended from 25 to 39 days. The increased survival was corroborated by the activation of innate and adaptive immunity; interestingly, HSP70 was more active in induction of CD8+ cell-mediated toxicity and γIFN production while phloretin contributed largely to the CD56+ cell response. In conclusion, the combination of HSP70 with phloretin could be a novel treatment for efficient immunotherapy of intractable cancers such as skin melanoma.

Geldanamycin induces heat shock protein 70 and protects against MPTP-induced dopaminergic neurotoxicity in mice.

PubMed

Shen, Hai-Ying; He, Jin-Cai; Wang, Yumei; Huang, Qing-Yuan; Chen, Jiang-Fan

2005-12-02

As key molecular chaperone proteins, heat shock proteins (HSPs) represent an important cellular protective mechanism against neuronal cell death in various models of neurological disorders. In this study, we investigated the effect as well as the molecular mechanism of geldanamycin (GA), an inhibitor of Hsp90, on 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a mouse model of Parkinson disease. Neurochemical analysis showed that pretreatment with GA (via intracerebral ventricular injection 24 h prior to MPTP treatment) increased residual dopamine content and tyrosine hydroxylase immunoreactivity in the striatum 24 h after MPTP treatment. To dissect out the molecular mechanism underlying this neuroprotection, we showed that the GA-mediated protection against MPTP was associated with a reduction of cytosolic Hsp90 and an increase in Hsp70, with no significant changes in Hsp40 and Hsp25 levels. Furthermore, in parallel with the induction of Hsp70, striatal nuclear HSF1 levels and HSF1 binding to heat shock element sites in the Hsp70 promoter were significantly enhanced by the GA pretreatment. Together these results suggested that the molecular cascade leading to the induction of Hsp70 is critical to the neuroprotection afforded by GA against MPTP-induced neurotoxicity in the brain and that pharmacological inhibition of Hsp90 may represent a potential therapeutic strategy for Parkinson disease.

Chaperone roles for TMAO and HSP70 during hyposmotic stress in the spiny dogfish shark (Squalus acanthias).

PubMed

MacLellan, Robyn J; Tunnah, Louise; Barnett, David; Wright, Patricia A; MacCormack, Tyson; Currie, Suzanne

2015-10-01

Salinity decreases are experienced by many marine elasmobranchs. To understand how these fishes cope with hyposmotic stress on a cellular level, we used the spiny dogfish shark (Squalus acanthias) as a model to test whether a reciprocal relationship exists between the cell's two primary protein protection mechanisms, the chemical (e.g., trimethylamine oxide, TMAO) and molecular (e.g., heat shock protein 70, HSP70) chaperone systems. This relationship is interesting given that many elasmobranchs are expected to gain water and lose osmolytes, chemical chaperones, and ions as they osmoconform to new, lowered salinity. Dogfish were cannulated for repeated blood sampling and exposed to 70% seawater (SW) for 48 h. These hyposmotic conditions had no effect on red blood cell (RBC) and white muscle TMAO concentrations, and did not result in HSP70 induction or signs of protein damage (i.e., increased ubiquitin), suggesting that TMAO levels were sufficiently protective in these tissues. However, in the gill, we observed a significant decrease in TMAO concentration and a significant induction of HSP70 as well as signs of protein damage. In the face of this cellular stress response, gill Na(+)/K(+)-ATPase (NKA) activity significantly increased during hyposmotic conditions, as expected. We suggest that this functional preservation in the gill is partly the result of HSP70 induction with lowered salinity. We conclude a reciprocal relationship between TMAO and HSP70 in the gills of dogfish as a result of in vivo hyposmotic stress. When osmotically induced protein damage surpasses the protective capacity of remaining TMAO, HSP70 is induced to preserve tissue and organismal function.

Ovalbumin-derived precursor peptides are transferred sequentially from gp96 and calreticulin to MHC I in the endoplasmic reticulum

PubMed Central

Kropp, Laura E.; Garg, Manish; Binder, Robert J.

2010-01-01

Cellular peptides generated by proteasomal degradation of proteins in the cytosol and destined for presentation by MHC I are associated with several chaperones. Hsp70, hsp90 and the TCP1-ring complex have been implicated as important cytosolic players for chaperoning these peptides. In this study we report that gp96 and calreticulin are essential for chaperoning peptides in the endoplasmic reticulum. Importantly we demonstrate that cellular peptides are transferred sequentially from gp96 to calreticulin and then to MHC I forming a relay line. Disruption of this relay line by removal of gp96 or calreticulin prevents the binding of peptides by MHC I and hence presentation of the MHC I-peptide complex on the cell surface. Our results are important for understanding how peptides are processed and trafficked within the endoplasmic reticulum before exiting in association with MHC I heavy chains and β2-microglobulin as a trimolecular complex. PMID:20410492

Hsp90α forms a stable complex at the cilium neck for the interaction of signalling molecules in IGF-1 receptor signalling.

PubMed

Wang, Hongzhong; Zou, Xinle; Wei, Zhuang; Wu, Yuan; Li, Rongxia; Zeng, Rong; Chen, Zhengjun; Liao, Kan

2015-01-01

The primary cilium is composed of an axoneme that protrudes from the cell surface, a basal body beneath the membrane and a transition neck in between. It is a sensory organelle on the plasma membrane, involved in mediating extracellular signals. In the transition neck region of the cilium, the microtubules change from triplet to doublet microtubules. This region also contains the transition fibres that crosslink the axoneme with the membrane and the necklace proteins that regulate molecules being transported into and out of the cilium. In this protein-enriched, complex area it is important to maintain the correct assembly of all of these proteins. Here, through immunofluorescent staining and protein isolation, we identify the molecular chaperone Hsp90α clustered at the periciliary base. At the transition neck region, phosphorylated Hsp90α forms a stable ring around the axoneme. Heat shock treatment causes Hsp90α to dissipate and induces resorption of cilia. We further identify that Hsp90α at the transition neck region represents a signalling platform on which IRS-1 interacts with intracellular downstream signalling molecules involved in IGF-1 receptor signalling. © 2015. Published by The Company of Biologists Ltd.

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding.

PubMed

Żwirowski, Szymon; Kłosowska, Agnieszka; Obuchowski, Igor; Nillegoda, Nadinath B; Piróg, Artur; Ziętkiewicz, Szymon; Bukau, Bernd; Mogk, Axel; Liberek, Krzysztof

2017-03-15

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp-substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp-substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions. © 2017 The Authors.

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.

Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities.

PubMed

Nahomi, Rooban B; DiMauro, Michael A; Wang, Benlian; Nagaraj, Ram H

2015-01-01

Previous studies have identified peptides in the 'crystallin-domain' of the small heat-shock protein (sHSP) α-crystallin with chaperone and anti-apoptotic activities. We found that peptides in heat-shock protein Hsp20 (G71HFSVLLDVKHFSPEEIAVK91) and Hsp27 (D93RWRVSLDVNHFAPDELTVK113) with sequence homology to α-crystallin also have robust chaperone and anti-apoptotic activities. Both peptides inhibited hyperthermic and chemically induced aggregation of client proteins. The scrambled peptides of Hsp20 and Hsp27 showed no such effects. The chaperone activities of the peptides were better than those from αA- and αB-crystallin. HeLa cells took up the FITC-conjugated Hsp20 peptide and, when the cells were thermally stressed, the peptide was translocated from the cytoplasm to the nucleus. The two peptides inhibited apoptosis in HeLa cells by blocking cytochrome c release from the mitochondria and caspase-3 activation. We found that scrambling the last four amino acids in the two peptides (KAIV in Hsp20 and KTLV in Hsp27) made them unable to enter cells and ineffective against stress-induced apoptosis. Intraperitoneal injection of the peptides prevented sodium-selenite-induced cataract formation in rats by inhibiting protein aggregation and oxidative stress. Our study has identified peptides from Hsp20 and Hsp27 that may have therapeutic benefit in diseases where protein aggregation and apoptosis are contributing factors.

Allostery in the Hsp70 chaperone proteins

PubMed Central

Zuiderweg, Erik R.P.; Bertelsen, Eric B.; Rousaki, Aikaterini; Mayer, Matthias P.; Gestwicki, Jason E.; Ahmad, Atta

2013-01-01

Heat shock 70 kDa (Hsp70) chaperones are essential to in-vivo protein folding, protein transport and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter, these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimer's and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed. PMID:22576356

Hsp70 Isoforms Are Essential for the Formation of Kaposi’s Sarcoma-Associated Herpesvirus Replication and Transcription Compartments

PubMed Central

Baquero-Pérez, Belinda; Whitehouse, Adrian

2015-01-01

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with various AIDS-related malignancies. Like other herpesviruses, multiple processes required for KSHV lytic replication, including viral transcription, viral DNA synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (RTCs). Here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to KSHV-induced RTCs and thus play a key role in KSHV lytic replication. We show that several isoforms of the HSP70 chaperone family, Hsc70 and iHsp70, are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of KSHV-induced RTCs. We demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed KSHV RTCs, however during later time points the chaperones move within KSHV RTCs and completely co-localise with actively replicating viral DNA. The functional significance of Hsp70 isoforms recruitment into KSHV RTCs was also examined using the specific Hsp70 isoform small molecule inhibitor, VER-155008. Intriguingly, results highlight an essential role of Hsp70 isoforms in the KSHV replication cycle independent of protein stability and maturation. Notably, inhibition of Hsp70 isoforms precluded KSHV RTC formation and RNA polymerase II (RNAPII) relocalisation to the viral genome leading to the abolishment of global KSHV transcription and subsequent viral protein synthesis and DNA replication. These new findings have revealed novel mechanisms that regulate KSHV lytic replication and highlight the potential of HSP70 inhibitors as novel antiviral agents. PMID:26587836

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.

Functional state of the Hsp27 chaperone as a molecular marker of an unfavorable course of larynx cancer.

PubMed

Kaigorodova, Evgeniya V; Zavyalova, Marina V; Bychkov, Vyacheslav A; Perelmuter, Vladimir M; Choynzonov, Evgenii L

2016-06-24

The small heat shock protein 27 kDA (Hsp27) acts as an ATP-independent chaperone in protein folding, but is also implicated in architecture of the cytoskeleton, cell migration, metabolism, cell survival, growth/differentiation, mRNA stabilization, and tumor progression. To study the intracellular localization of phosphorylated and non-phosphorylated forms of Hsp27 in squamous cell carcinoma of the larynx (SCCL) and to evaluate their relationship with regional lymphatic metastasis and overall five-year survival. Tumor biopsies of larynx tissue were collected from 50 patients who were between the ages of 30 to 80 years and had a confirmed diagnosis of squamous cell carcinoma of the larynx. Immunohistochemistry was used to determine the intracellular localization of the phosphorylated and non-phosphorylated forms of Hsp27. The study revealed that the Hsp27 chaperone was expressed in both the cytoplasm and the nucleus of tumor cells in SCCL. The biopsies of patients with lymph node metastases showed significantly higher expression of the phosphorylated and unphosphorylated forms of Hsp27 in the nucleus compared to those of patients without lymph node metastases. At the same time, the cytoplasmic expression of Hsp27 in these patients did not differ statistically. Analysis of the overall five-year survival rates showed that negative Hsp27 expression in the nucleus of tumor cells is associated with the survival rate of patients with SCCL. The nuclear expression of phosphorylated and unphosphorylated forms of Hsp27 is a molecular marker of unfavorable squamous cell carcinoma of the larynx associated with lymphogenous metastasis and decreased total five-year survival.

Synthesis and Anticancer Mechanism Investigation of Dual Hsp27 and Tubulin Inhibitors

PubMed Central

Zhong, Bo; Chennamaneni, Snigdha; Lama, Rati; Yi, Xin; Geldenhuys, Werner J.; Pink, John J.; Dowlati, Afshin; Xu, Yan; Zhou, Aimin; Su, Bin

2013-01-01

Heat shock protein 27 (Hsp27) is a chaperone protein, and its expression is increased in response to various stress stimuli including anticancer chemotherapy, which allows the cells to survive and causes drug resistance. We previously identified lead compounds that bound to Hsp27 and tubulin via proteomic approaches. Systematic ligand based optimization in the current study significantly increased the cell growth inhibition and apoptosis inducing activities of the compounds. Compared to the lead compounds, one of the new derivatives exhibited much better potency to inhibit tubulin polymerization but a decreased activity to inhibit Hsp27 chaperone function, suggesting that the structural modification dissected the dual targeting effects of the compound. The most potent compounds 20 and 22 exhibited strong cell proliferation inhibitory activities at subnanomolar concentration against 60 human cancer cell lines conducted by Developmental Therapeutic Program at the National Cancer Institute and represented promising candidates for anticancer drug development. PMID:23767669

Conformational changes in human Hsp70 induced by high hydrostatic pressure produce oligomers with ATPase activity but without chaperone activity.

PubMed

Araujo, Thaís L S; Borges, Julio Cesar; Ramos, Carlos H; Meyer-Fernandes, José Roberto; Oliveira Júnior, Reinaldo S; Pascutti, Pedro G; Foguel, Debora; Palhano, Fernando L

2014-05-13

We investigated the folding of the 70 kDa human cytosolic inducible protein (Hsp70) in vitro using high hydrostatic pressure as a denaturing agent. We followed the structural changes in Hsp70 induced by high hydrostatic pressure using tryptophan fluorescence, molecular dynamics, circular dichroism, high-performance liquid chromatography gel filtration, dynamic light scattering, ATPase activity, and chaperone activity. Although monomeric, Hsp70 is very sensitive to hydrostatic pressure; after pressure had been removed, the protein did not return to its native sate but instead formed oligomeric species that lost chaperone activity but retained ATPase activity.

HSP70 and heat shock factor 1 cooperate to repress Ras-induced transcriptional activation of the c-fos gene.

PubMed

He, H; Chen, C; Xie, Y; Asea, A; Calderwood, S K

2000-11-01

Heat shock protein 70 (HSP70) is a molecular chaperone involved in protein folding and resistance to the deleterious effects of stress. Here we show that HSP70 suppresses transcription of c-fos, an early response gene that is a key component of the ubiquitous AP-1 transcription factor complex. HSP70 repressed Ras-induced c-fos transcription only in the presence of functional heat shock factor1 (HSF1). This suggests that HSP70 functions as a corepressor with HSF1 to inhibit c-fos gene transcription. Therefore, besides its known function in the stress response, HSP70 also has the property of a corepressor and combines with HSF1 to antagonize Fos expression and may thus impact multiple aspects of cell regulation.

Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis.

PubMed

Chung-Davidson, Yu-Wen; Yeh, Chu-Yin; Bussy, Ugo; Li, Ke; Davidson, Peter J; Nanlohy, Kaben G; Brown, C Titus; Whyard, Steven; Li, Weiming

2015-12-01

Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics. We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection. HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.

Arginine methylation of HSP70 regulates retinoid acid-mediated RARβ2 gene activation

PubMed Central

Gao, Wei-wei; Xiao, Rong-quan; Peng, Bing-ling; Xu, Huan-teng; Shen, Hai-feng; Huang, Ming-feng; Shi, Tao-tao; Yi, Jia; Zhang, Wen-juan; Wu, Xiao-nan; Gao, Xiang; Lin, Xiang-zhi; Dorrestein, Pieter C.; Rosenfeld, Michael G.; Liu, Wen

2015-01-01

Although “histone” methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain–containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor β2 (RARβ2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70’s function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control. PMID:26080448

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

PubMed Central

Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

2015-01-01

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

Thermally induced disintegration of the oligomeric structure of alphaB-crystallin mutant F28S is associated with diminished chaperone activity.

PubMed

Kelley, Patrick B; Abraham, Edathara C

2003-10-01

alphaB-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins. The present work has investigated the role of phenylalanine-28 (F28) of the 22RLFDQFF28 region of alphaB-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress. Bovine alphaB-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse alphaB-crystallins. F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type alphaB (alphaB-wt) in E. coli and subsequent purification of the protein by size-exclusion chromatography. Secondary and tertiary structure analyses showed some structural changes in the mutant. Chaperone activity and oligomeric size of the mutant was unchanged at 37 degrees C whereas at 58 degrees C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure. The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.

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

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.

Application of SGT1-Hsp90 chaperone complex for soluble expression of NOD1 LRR domain in E. coli

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

Hong, Tae-Joon; Hahn, Ji-Sook

NOD1 is an intracellular sensor of innate immunity which is related to a number of inflammatory diseases. NOD1 is known to be difficult to express and purify for structural and biochemical studies. Based on the fact that Hsp90 and its cochaperone SGT1 are necessary for the stabilization and activation of NOD1 in mammals, SGT1 was chosen as a fusion partner of the leucine-rich repeat (LRR) domain of NOD1 for its soluble expression in Escherichia coli. Fusion of human SGT1 (hSGT1) to NOD1 LRR significantly enhanced the solubility, and the fusion protein was stabilized by coexpression of mouse Hsp90α. The expressionmore » level of hSGT1-NOD1 LRR was further enhanced by supplementation of rare codon tRNAs and exchange of antibiotic marker genes. - Highlights: • The NOD1 LRR domain was solubilized by SGT1 fusion in E. coli. • The coexpression of HSP90 stabilized the SGT1-NOD1 LRR fusion protein. • Several optimizations could enhance the expression level of the fusion protein.« less

The novel HSP90 inhibitor NVP-AUY922 shows synergistic anti-leukemic activity with cytarabine in vivo.

PubMed

Wendel, Torunn; Zhen, Yan; Suo, Zenhe; Bruheim, Skjalg; Wiedlocha, Antoni

2016-01-15

HSP90 is a molecular chaperone essential for stability, activity and intracellular sorting of many proteins, including oncoproteins, such as tyrosine kinases, transcription factors and cell cycle regulatory proteins. Therefore, inhibitors of HSP90 are being investigated for their potential as anti-cancer drugs. Here we show that the HSP90 inhibitor NVP-AUY922 induced degradation of the fusion oncoprotein FOP2-FGFR1 in a human acute myeloid leukemia (AML) cell line, KG-1a. Concordantly, downstream signaling cascades, such as STAT1, STAT3 and PLCγ were abrogated. At concentrations that caused FOP2-FGFR1 degradation and signaling abrogation, NVP-AUY922 treatment caused significant cell death and inhibition of proliferation of KG-1a cells in vitro. In an animal model for AML, NVP-AUY922 administrated alone showed no anti-leukemic activity. However, when NVP-AUY922 was administered in combination with cytarabine, the two compounds showed significant synergistic anti-leukemic activity in vivo. Thus NVP-AUY922 and cytarabine combination therapy might be a prospective strategy for AML treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Specific cerebral heat shock proteins and histamine receptor cross-talking mechanisms promote distinct lead-dependent neurotoxic responses in teleosts

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

Giusi, Giuseppina; Alo, Raffaella; Crudo, Michele

Recent interests are beginning to be directed towards toxic neurobiological dysfunctions caused by lead (Pb) in aquatic vertebrates. In the present work, treatment with a maximum acceptable toxic concentration of this heavy metal was responsible for highly significant (p < 0.01) abnormal motor behaviors such as hyperactive movements in the teleost Thalassoma pavo and the same treatment accounted for significantly (p < 0.05) enhanced hyperventilating states. On the other hand, greater abnormal motor behaviors were detected in the presence of the histamine (HA) receptor subtype 2 (H{sub 2}R) antagonist cimetidine (Cim), as shown by the very robust (p < 0.001)more » increases of the two behavioral states. Interestingly, elevated expression levels of stress-related factors, i.e. heat shock protein70/90 (HSP90/70) orthologs were reported for the first time in hypothalamic and mesencephalic areas of Pb-treated teleosts. In particular, an up-regulation of HSP70 was readily detected when this heavy metal was given concomitantly with Cim, while the histamine subtype 3 antagonist (H{sub 3}R) thioperamide (Thio), instead, blocked Pb-dependent up-regulatory trends of both chaperones in mostly hypothalamic areas. Moreover, intense neuronal damages of the above brain regions coincided with altered expressions of HSP70 and HSP90 when treated only with Cim. Overall these first results show that distinct H{sub n}R are able to exert a net neuroprotective role arising from their interaction with chaperones in fish exposed to Pb-dependent stressful conditions making this a potentially key interaction especially for T. pavo, aquatic species which plays an important ecological role towards the survival of other commercially vital fishes.« less

EXPRESSION OF INDUCIBLE HSP70 ENHANCES THE PROLIFERATION OF MCF-7 BREAST CANCER CELLS AND PROTECTS AGAINST THE CYTOTOXIC EFFECTS OF HYPERTHERMIA

EPA Science Inventory

Heat shock proteins (HSPs) are ubiquitous proteins that are induced following exposure to sub-lethal heat shock, are highly conserved during evolution and protect cells from damage through their function as molecular chaperones. Some cancers demonstrate elevated levels of Hsp70 ...

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

PubMed

Cappelletti, Pamela; Binda, Elisa; Tunesi, Marta; Albani, Diego; Giordano, Carmen; Molla, Gianluca; Pollegioni, Loredano

2017-10-01

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence.

PubMed

Workman, Paul; van Montfort, Rob

2014-06-01

The crystal structure of a conserved tubulin-binding region of the EML1 protein reveals a highly atypical fold in one of its β-propeller domains. Disruption of the EML1 core region domain in many of the oncogenic EML4-ALK fusion protein variants that drive non-small cell lung cancer explains their dependence on the HSP90 molecular chaperone, provides a basis to allow more precise patient stratification for therapy, and suggests a more general model for other oncogenic fusion proteins. ©2014 American Association for Cancer Research.

Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins

PubMed Central

Gibert, Benjamin; Eckel, Bénédicte; Fasquelle, Lydie; Moulin, Maryline; Bouhallier, Frantz; Gonin, Vincent; Mellier, Gregory; Simon, Stéphanie; Kretz-Remy, Carole; Arrigo, André-Patrick; Diaz-Latoud, Chantal

2012-01-01

Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions. PMID:22238643

The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers.

PubMed

Newman, Joseph; Asfor, Amin S; Berryman, Stephen; Jackson, Terry; Curry, Stephen; Tuthill, Tobias J

2018-03-01

Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug. IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity involved for this process to occur, which could be the basis for a novel antiviral control mechanism for FMDV. Copyright © 2018 Newman et al.

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor σ32 of Escherichia coli

PubMed Central

Perrody, Elsa; Cirinesi, Anne-Marie; Desplats, Carine; Keppel, France; Schwager, Françoise; Tranier, Samuel; Georgopoulos, Costa; Genevaux, Pierre

2012-01-01

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ32 facilitates RB43 bacteriophage proliferation is discussed. PMID:23133404

Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.

PubMed

Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid

2006-01-01

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

Differential gene expressions in testes of L2 strain Taiwan country chicken in response to acute heat stress.

PubMed

Wang, Shih-Han; Cheng, Chuen-Yu; Tang, Pin-Chi; Chen, Chih-Feng; Chen, Hsin-Hsin; Lee, Yen-Pai; Huang, San-Yuan

2013-01-15

Acute heat stress affects genes involved in spermatogenesis in mammals. However, there is apparently no elaborate research on the effects of acute heat stress on gene expression in avian testes. The purpose of this study was to investigate global gene expression in testes of the L2 strain of Taiwan country chicken after acute heat stress. Twelve roosters, 45 weeks old, were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2-hour recovery, and with 6-hour recovery, respectively. Testis samples were collected for RNA isolation and microarray analysis. Based on gene expression profiles, 169 genes were upregulated and 140 genes were downregulated after heat stress using a cutoff value of twofold or greater change. Based on gene ontology analysis, differentially expressed genes were mainly related to response to stress, transport, signal transduction, and metabolism. A functional network analysis displayed that heat shock protein genes and related chaperones were the major upregulated groups in chicken testes after acute heat stress. A quantitative real-time polymerase chain reaction analysis of mRNA expressions of HSP70, HSP90AA1, BAG3, SERPINB2, HSP25, DNAJA4, CYP3A80, CIRBP, and TAGLN confirmed the results of the microarray analysis. Because the HSP genes (HSP25, HSP70, and HSP90AA1) and the antiapoptotic BAG3 gene were dramatically altered in heat-stressed chicken testes, we concluded that these genes were important factors in the avian testes under acute heat stress. Whether these genes could be candidate genes for thermotolerance in roosters requires further investigation. Copyright © 2013 Elsevier Inc. All rights reserved.

HSP70 and heat shock factor 1 cooperate to repress Ras-induced transcriptional activation of the c-fos gene

PubMed Central

He, Haiying; Chen, Changmin; Xie, Yue; Asea, Alexzander; Calderwood, Stuart K.

2000-01-01

Heat shock protein 70 (HSP70) is a molecular chaperone involved in protein folding and resistance to the deleterious effects of stress. Here we show that HSP70 suppresses transcription of c-fos, an early response gene that is a key component of the ubiquitous AP-1 transcription factor complex. HSP70 repressed Ras-induced c-fos transcription only in the presence of functional heat shock factor1 (HSF1). This suggests that HSP70 functions as a corepressor with HSF1 to inhibit c-fos gene transcription. Therefore, besides its known function in the stress response, HSP70 also has the property of a corepressor and combines with HSF1 to antagonize Fos expression and may thus impact multiple aspects of cell regulation. PMID:11189444

Stress-induced interaction between p38 MAPK and HSP70

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

Gong, Xiaowei, E-mail: gongxw@fimmu.com; Luo, Tingting; Deng, Peng

2012-08-24

Highlights: Black-Right-Pointing-Pointer HSP70 interacts to p38 MAPK in vitro and in vivo. Black-Right-Pointing-Pointer HSP70 co-localizes with p38 MAPK in the nucleus upon stress. Black-Right-Pointing-Pointer HSP70 is involved in the nuclear phosphorylation of MK2 by p38 MAPK. -- Abstract: p38 MAPK, one of the four MAPK subfamilies in mammalian cells, is activated by environmental stresses and pro-inflammatory cytokines, playing fundamental roles in many biological processes. Despite all that is known on the structure and functions of p38, many questions still exist. The coupling of activation and nuclear translocation represents an important aspect of p38 signaling. In our effort in exploring themore » potential chaperone for p38 translocation, we performed an endogenous pull-down assay and identified HSP70 as a potential interacting protein of p38. We confirmed the interaction between p38 and HSP70 in vitro and in vivo, and identified their interaction domains. We also showed stress-induced nuclear co-localization of these two proteins. Our preliminary result indicated that HSP70 was related to the phosphorylation of MK2, a specific nuclear downstream target of p38, suggesting HSP70 is a potential chaperone for the nuclear translocation of p38.« less

Inducible HSP70 Is Critical in Preventing the Aggregation and Enhancing the Processing of PMP22

PubMed Central

Chittoor-Vinod, Vinita G.; Lee, Sooyeon; Judge, Sarah M.

2015-01-01

Chaperones, also called heat shock proteins (HSPs), transiently interact with proteins to aid their folding, trafficking, and degradation, thereby directly influencing the transport of newly synthesized molecules. Induction of chaperones provides a potential therapeutic approach for protein misfolding disorders, such as peripheral myelin protein 22 (PMP22)-associated peripheral neuropathies. Cytosolic aggregates of PMP22, linked with a demyelinating Schwann cell phenotype, result in suppression of proteasome activity and activation of proteostatic mechanisms, including the heat shock pathway. Although the beneficial effects of chaperones in preventing the aggregation and improving the trafficking of PMP22 have been repeatedly observed, the requirement for HSP70 in events remains elusive. In this study, we show that activation of the chaperone pathway in fibroblasts from PMP22 duplication-associated Charcot–Marie–Tooth disease type 1A patient with an FDA-approved small molecule increases HSP70 expression and attenuates proteasome dysfunction. Using cells from an HSP70.1/3−/− (inducible HSP70) mouse model, we demonstrate that under proteotoxic stress, this chaperone is critical in preventing the aggregation of PMP22, and this effect is aided by macroautophagy. When examined at steady-state, HSP70 appears to play a minor role in the trafficking of wild-type-PMP22, while it is crucial for preventing the buildup of the aggregation-prone Trembler-J-PMP22. HSP70 aids the processing of Trembler-J-PMP22 through the Golgi and its delivery to lysosomes via Rab7-positive vesicles. Together, these results demonstrate a key role for inducible HSP70 in aiding the processing and hindering the accumulation of misfolded PMP22, which in turn alleviates proteotoxicity within the cells. PMID:25694550

Molecular Cloning and mRNA Expression of Heat Shock Protein Genes and Their Response to Cadmium Stress in the Grasshopper Oxya chinensis.

PubMed

Zhang, Yuping; Liu, Yaoming; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

2015-01-01

Heat shock proteins (Hsps) are highly conserved molecular chaperones that are synthesized in response to stress. In this study, we cloned the full-length sequences of the Grp78 (glucose-regulated protein 78), Hsp70, Hsp90, and Hsp40 genes from the Chinese rice grasshopper Oxya chinensis. The full-length cDNA sequences of OcGrp78, OcHsp70, OcHsp90, and OcHsp40 contain open reading frames of 1947, 1920, 2172, and 1042 bp that encode proteins of 649, 640, 724, and 347 amino acids, respectively. Fluorescent real-time quantitative PCR (RT-qPCR) was performed to quantify the relative transcript levels of these Hsp genes in different tissues and developmental stages. The mRNAs encoding these four Hsp genes were present at all developmental stages and in all tissues examined but were expressed at varying levels. Additionally, we investigated the mRNA expression profiles of these four Hsps in O. chinensis subjected to Cadmium (Cd) stress. OcGrp78, OcHsp70, OcHsp90, and OcHsp40 mRNA expression was induced under acute Cd stress; the levels reached a maximum within a short time (6 h), were reduced significantly at 12 h, and were lowered to or below control levels by 48 h. Regarding induction efficiency, OcHsp70 was the most sensitive gene to acute Cd stress. Chronic Cd exposure showed that dietary Cd treatment induced increased OcGrp78, OcHsp90, and OcHsp40 expression. However, dietary Cd induced a significant reduction of OcHsp70 expression. In the period tested, no significant difference in the mortality of the grasshoppers was observed. Our results suggest that these four Hsps genes, especially OcHsp70, are sensitive to acute Cd stress and could be used as molecular markers for toxicology studies. However, our results also indicate that OcHsp70 is not suitable for use as a molecular marker of chronic Cd contamination.

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

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

Hsp70 in cancer: back to the future

PubMed Central

Sherman, Michael Y.; Gabai, Vladimir L.

2014-01-01

Mechanistic studies from cell culture and animal models have revealed critical roles for the heat shock protein Hsp70 in cancer initiation and progression. Surprisingly, many effects of Hsp70 on cancer have not been related to its chaperone activity, but rather to its role(s) in regulating cell signaling. A major factor that directs Hsp70 signaling activity appears to be the co-chaperone Bag3. Here, we review these recent breakthroughs, and how these discoveries drive drug development efforts. PMID:25347739

Structural variants of yeast prions show conformer-specific requirements for chaperone activity

PubMed Central

Stein, Kevin C.; True, Heather L.

2016-01-01

Summary Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1, and its human ortholog Hdj1, had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation. PMID:25060529

AsHSP17, a creeping bentgrass small heat shock protein modulates plant photosynthesis and ABA-dependent and independent signalling to attenuate plant response to abiotic stress.

PubMed

Sun, Xinbo; Sun, Chunyu; Li, Zhigang; Hu, Qian; Han, Liebao; Luo, Hong

2016-06-01

Heat shock proteins (HSPs) are molecular chaperones that accumulate in response to heat and other abiotic stressors. Small HSPs (sHSPs) belong to the most ubiquitous HSP subgroup with molecular weights ranging from 12 to 42 kDa. We have cloned a new sHSP gene, AsHSP17 from creeping bentgrass (Agrostis stolonifera) and studied its role in plant response to environmental stress. AsHSP17 encodes a protein of 17 kDa. Its expression was strongly induced by heat in both leaf and root tissues, and by salt and abscisic acid (ABA) in roots. Transgenic Arabidopsis plants constitutively expressing AsHSP17 exhibited enhanced sensitivity to heat and salt stress accompanied by reduced leaf chlorophyll content and decreased photosynthesis under both normal and stressed conditions compared to wild type. Overexpression of AsHSP17 also led to hypersensitivity to exogenous ABA and salinity during germination and post-germinative growth. Gene expression analysis indicated that AsHSP17 modulates expression of photosynthesis-related genes and regulates ABA biosynthesis, metabolism and ABA signalling as well as ABA-independent stress signalling. Our results suggest that AsHSP17 may function as a protein chaperone to negatively regulate plant responses to adverse environmental stresses through modulating photosynthesis and ABA-dependent and independent signalling pathways. © 2015 John Wiley & Sons Ltd.

Binding of human nucleotide exchange factors to heat shock protein 70 (Hsp70) generates functionally distinct complexes in vitro.

PubMed

Rauch, Jennifer N; Gestwicki, Jason E

2014-01-17

Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone heat shock protein 70 (Hsp70). There are six BAG family NEFs in humans, and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete for binding to Hsp70 or how they might differentially shape its biochemical activities. Toward these questions, we measured the binding of human Hsp72 (HSPA1A) to BAG1, BAG2, BAG3, and the unrelated NEF Hsp105. These studies revealed a clear hierarchy of affinities: BAG3 > BAG1 > Hsp105 ≫ BAG2. All of the NEFs competed for binding to Hsp70, and their relative affinity values predicted their potency in nucleotide and peptide release assays. Finally, we combined the Hsp70-NEF pairs with cochaperones of the J protein family (DnaJA1, DnaJA2, DnaJB1, and DnaJB4) to generate 16 permutations. The activity of the combinations in ATPase and luciferase refolding assays were dependent on the identity and stoichiometry of both the J protein and NEF so that some combinations were potent chaperones, whereas others were inactive. Given the number and diversity of cochaperones in mammals, it is likely that combinatorial assembly could generate a large number of distinct permutations.

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

Pea Chaperones under Centrifugation

NASA Astrophysics Data System (ADS)

Talalaiev, Oleksandr

2008-06-01

Etiolated Pisum sativum seedlings were subjected to altered g-forces by centrifugation (3-14g). By using semiquantitative RT-PCR, we studied transcripts of pea genes coding for chaperones that are representatives of small heat shock proteins (sHsps) family. Four members from the different classes of sHsps: cytosolic Hsp17.7 and Hsp18.1 (class I and class II accordingly), chloroplast Hsp21 (class III) and endoplasmic reticulum Hsp22.7 (class IV) were investigated. We conclude that exposure to 3, 7, 10 and 14g for 1h did not affect the level of sHsp transcripts.

Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem.

PubMed

Aguado, Alejandra; Fernández-Higuero, José Angel; Moro, Fernando; Muga, Arturo

2015-08-15

The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival during severe stress. The protein disaggregase activity of these members of the Hsp100 family is linked to the activity of chaperones from the Hsp70 and Hsp40 families. The precise mechanism by which these proteins untangle protein aggregates remains unclear. Strikingly, Hsp100 proteins are not present in metazoans. This does not mean that animal cells do not have a disaggregase activity, but that this activity is performed by the Hsp70 system and a representative of the Hsp110 family instead of a Hsp100 protein. This review describes the actual view of Hsp100-mediated aggregate reactivation, including the ATP-induced conformational changes associated with their disaggregase activity, the dynamics of the oligomeric assembly that is regulated by its ATPase cycle and the DnaK system, and the tight allosteric coupling between the ATPase domains within the hexameric ring complexes. The lack of homologs of these disaggregases in metazoans has suggested that they might be used as potential targets to develop antimicrobials. The current knowledge of the human disaggregase machinery and the role of Hsp110 are also discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication

PubMed Central

Stetz, Gabrielle; Verkhivker, Gennady M.

2017-01-01

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms. PMID:28095400

Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2017-01-01

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms.

The Barley Powdery Mildew Candidate Secreted Effector Protein CSEP0105 Inhibits the Chaperone Activity of a Small Heat Shock Protein1[OPEN

PubMed Central

Ahmed, Ali Abdurehim; Pedersen, Carsten; Schultz-Larsen, Torsten; Kwaaitaal, Mark; Jørgensen, Hans Jørgen Lyngs; Thordal-Christensen, Hans

2015-01-01

Pathogens secrete effector proteins to establish a successful interaction with their host. Here, we describe two barley (Hordeum vulgare) powdery mildew candidate secreted effector proteins, CSEP0105 and CSEP0162, which contribute to pathogen success and appear to be required during or after haustorial formation. Silencing of either CSEP using host-induced gene silencing significantly reduced the fungal haustorial formation rate. Interestingly, both CSEPs interact with the barley small heat shock proteins, Hsp16.9 and Hsp17.5, in a yeast two-hybrid assay. Small heat shock proteins are known to stabilize several intracellular proteins, including defense-related signaling components, through their chaperone activity. CSEP0105 and CSEP0162 localized to the cytosol and the nucleus of barley epidermal cells, whereas Hsp16.9 and Hsp17.5 are cytosolic. Intriguingly, only those specific CSEPs changed localization and became restricted to the cytosol when coexpressed with Hsp16.9 and Hsp17.5, confirming the CSEP-small heat shock protein interaction. As predicted, Hsp16.9 showed chaperone activity, as it could prevent the aggregation of Escherichia coli proteins during thermal stress. Remarkably, CSEP0105 compromised this activity. These data suggest that CSEP0105 promotes virulence by interfering with the chaperone activity of a barley small heat shock protein essential for defense and stress responses. PMID:25770154

Oral administration of an HSP90 inhibitor, 17-DMAG, intervenes tumor-cell infiltration into multiple organs and improves survival period for ATL model mice

PubMed Central

Ikebe, E; Kawaguchi, A; Tezuka, K; Taguchi, S; Hirose, S; Matsumoto, T; Mitsui, T; Senba, K; Nishizono, A; Hori, M; Hasegawa, H; Yamada, Y; Ueno, T; Tanaka, Y; Sawa, H; Hall, W; Minami, Y; Jeang, K T; Ogata, M; Morishita, K; Hasegawa, H; Fujisawa, J; Iha, H

2013-01-01

In the peripheral blood leukocytes (PBLs) from the carriers of the human T-lymphotropic virus type-1 (HTLV-1) or the patients with adult T-cell leukemia (ATL), nuclear factor kappaB (NF-κB)-mediated antiapoptotic signals are constitutively activated primarily by the HTLV-1-encoded oncoprotein Tax. Tax interacts with the I κB kinase regulatory subunit NEMO (NF-κB essential modulator) to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, heat-shock protein 90 (HSP90), and its co-chaperone cell division cycle 37 (CDC37). The antibiotic geldanamycin (GA) inhibits HSP90's ATP binding for its proper interaction with client proteins. Administration of a novel water-soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), to Tax-expressing ATL-transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines, although this treatment has no apparent effects on normal PBLs. 17-DMAG also downregulated Tax-mediated intracellular signals including the activation of NF-κB, activator protein 1 or HTLV-1 long terminal repeat in Tax-transfected HEK293 cells. Oral administration of 17-DMAG to ATL model mice xenografted with lymphomatous transgenic Lck-Tax (Lck proximal promoter-driven Tax transgene) cells or HTLV-1-producing tumor cells dramatically attenuated aggressive infiltration into multiple organs, inhibited de novo viral production and improved survival period. These observations identified 17-DMAG as a promising candidate for the prevention of ATL progression. PMID:23955587

The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.

PubMed

Heikkila, John J

2017-01-01

Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of an Atriplex nummularia gene encoding a protein homologous to the bacterial molecular chaperone DnaJ.

PubMed

Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M

1993-03-01

DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress.

Crystal structures of Hsp104 N-terminal domains from Saccharomyces cerevisiae and Candida albicans suggest the mechanism for the function of Hsp104 in dissolving prions

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

Wang, Peng; Li, Jingzhi; Weaver, Clarissa

Hsp104 is a yeast member of the Hsp100 family which functions as a molecular chaperone to disaggregate misfolded polypeptides. To understand the mechanism by which the Hsp104 N-terminal domain (NTD) interacts with its peptide substrates, crystal structures of the Hsp104 NTDs fromSaccharomyces cerevisiae(ScHsp104NTD) andCandida albicans(CaHsp104NTD) have been determined at high resolution. The structures of ScHsp104NTD and CaHsp104NTD reveal that the yeast Hsp104 NTD may utilize a conserved putative peptide-binding groove to interact with misfolded polypeptides. In the crystal structures ScHsp104NTD forms a homodimer, while CaHsp104NTD exists as a monomer. The consecutive residues Gln105, Gln106 and Lys107, and Lys141 around themore » putative peptide-binding groove mediate the monomer–monomer interactions within the ScHsp104NTD homodimer. Dimer formation by ScHsp104NTD suggests that the Hsp104 NTD may specifically interact with polyQ regions of prion-prone proteins. The data may reveal the mechanism by which Hsp104 NTD functions to suppress and/or dissolve prions.« less

Genome-wide analysis of the Hsp70 family genes in pepper (Capsicum annuum L.) and functional identification of CaHsp70-2 involvement in heat stress.

PubMed

Guo, Meng; Liu, Jin-Hong; Ma, Xiao; Zhai, Yu-Fei; Gong, Zhen-Hui; Lu, Ming-Hui

2016-11-01

Hsp70s function as molecular chaperones and are encoded by a multi-gene family whose members play a crucial role in plant response to stress conditions, and in plant growth and development. Pepper (Capsicum annuum L.) is an important vegetable crop whose genome has been sequenced. Nonetheless, no overall analysis of the Hsp70 gene family is reported in this crop plant to date. To assess the functionality of Capsicum annuum Hsp70 (CaHsp70) genes, pepper genome database was analyzed in this research. A total of 21 CaHsp70 genes were identified and their characteristics were also described. The promoter and transcript expression analysis revealed that CaHsp70s were involved in pepper growth and development, and heat stress response. Ectopic expression of a cytosolic gene, CaHsp70-2, regulated expression of stress-related genes and conferred increased thermotolerance in transgenic Arabidopsis. Taken together, our results provide the basis for further studied to dissect CaHsp70s' function in response to heat stress as well as other environmental stresses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai).

PubMed

Cheng, Peizhou; Liu, Xiao; Zhang, Guofan; He, Jianguo

2007-01-01

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631bp, consisting of a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 573bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response.

Advanced drug delivery of N-acetylcarnosine (N-acetyl-beta-alanyl-L-histidine), carcinine (beta-alanylhistamine) and L-carnosine (beta-alanyl-L-histidine) in targeting peptide compounds as pharmacological chaperones for use in tissue engineering, human disease management and therapy: from in vitro to the clinic.

PubMed

Babizhayev, Mark A; Yegorov, Yegor E

2010-11-01

A pharmacological chaperone is a relatively new concept in the treatment of certain chronic disabling diseases. Cells maintain a complete set of functionally competent proteins normally and in the face of injury or environmental stress with the use of various mechanisms, including systems of proteins called molecular chaperones. Proteins that are denatured by any form of proteotoxic stress are cooperatively recognized by heat shock proteins (HSP) and directed for refolding or degradation. Under non-denaturing conditions HSP have important functions in cell physiology such as in transmembrane protein transport and in enabling assembly and folding of newly synthesized polypeptides. Besides cellular molecular chaperones, which are stress-induced proteins, there have been recently reported chemical, or so-called pharmacological chaperones with demonstrated ability to be effective in preventing misfolding of different disease causing proteins, specifically in the therapeutic management of sight-threatening eye diseases, essentially reducing the severity of several neurodegenerative disorders (such as age-related macular degeneration), cataract and many other protein-misfolding diseases. This work reviews the biological and therapeutic activities protected with the patents of the family of imidazole-containing peptidomimetics Carcinine (β-alanylhistamine), N-acetylcarnosine (N-acetyl-β-alanylhistidine) and Carnosine (β-alanyl-L-histidine) which are essential constituents possessing diverse biological and pharmacological chaperone properties in human tissues.

Hsp70 Is a Novel Posttranscriptional Regulator of Gene Expression That Binds and Stabilizes Selected mRNAs Containing AU-Rich Elements

PubMed Central

Kishor, Aparna; Tandukar, Bishal; Ly, Yann V.; Toth, Eric A.; Suarez, Yvelisse; Brewer, Gary

2013-01-01

The AU-rich elements (AREs) encoded within many mRNA 3′ untranslated regions (3′UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP- and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3′UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes. PMID:23109422

Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking

PubMed Central

Bird, Jonathan E.; Takagi, Yasuharu; Billington, Neil; Strub, Marie-Paule; Sellers, James R.; Friedman, Thomas B.

2014-01-01

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin–specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end–directed motor that moves actin filaments in a gliding assay (∼430 nm·s−1 at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (kcat ∼6 s−1) was similar to the actin-detachment rate (kdet = 6.2 s−1) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells. PMID:25114250

CHIP as a membrane-shuttling proteostasis sensor

PubMed Central

Kopp, Yannick; Martínez-Limón, Adrián; Hofbauer, Harald F; Ernst, Robert; Calloni, Giulia

2017-01-01

Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function). PMID:29091030

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.

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.

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.

Tandem Duplication Events in the Expansion of the Small Heat Shock Protein Gene Family in Solanum lycopersicum (cv. Heinz 1706).

PubMed

Krsticevic, Flavia J; Arce, Débora P; Ezpeleta, Joaquín; Tapia, Elizabeth

2016-10-13

In plants, fruit maturation and oxidative stress can induce small heat shock protein (sHSP) synthesis to maintain cellular homeostasis. Although the tomato reference genome was published in 2012, the actual number and functionality of sHSP genes remain unknown. Using a transcriptomic (RNA-seq) and evolutionary genomic approach, putative sHSP genes in the Solanum lycopersicum (cv. Heinz 1706) genome were investigated. A sHSP gene family of 33 members was established. Remarkably, roughly half of the members of this family can be explained by nine independent tandem duplication events that determined, evolutionarily, their functional fates. Within a mitochondrial class subfamily, only one duplicated member, Solyc08g078700, retained its ancestral chaperone function, while the others, Solyc08g078710 and Solyc08g078720, likely degenerated under neutrality and lack ancestral chaperone function. Functional conservation occurred within a cytosolic class I subfamily, whose four members, Solyc06g076570, Solyc06g076560, Solyc06g076540, and Solyc06g076520, support ∼57% of the total sHSP RNAm in the red ripe fruit. Subfunctionalization occurred within a new subfamily, whose two members, Solyc04g082720 and Solyc04g082740, show heterogeneous differential expression profiles during fruit ripening. These findings, involving the birth/death of some genes or the preferential/plastic expression of some others during fruit ripening, highlight the importance of tandem duplication events in the expansion of the sHSP gene family in the tomato genome. Despite its evolutionary diversity, the sHSP gene family in the tomato genome seems to be endowed with a core set of four homeostasis genes: Solyc05g014280, Solyc03g082420, Solyc11g020330, and Solyc06g076560, which appear to provide a baseline protection during both fruit ripening and heat shock stress in different tomato tissues. Copyright © 2016 Krsticevic et al.

Tandem Duplication Events in the Expansion of the Small Heat Shock Protein Gene Family in Solanum lycopersicum (cv. Heinz 1706)

PubMed Central

Krsticevic, Flavia J.; Arce, Débora P.; Ezpeleta, Joaquín; Tapia, Elizabeth

2016-01-01

In plants, fruit maturation and oxidative stress can induce small heat shock protein (sHSP) synthesis to maintain cellular homeostasis. Although the tomato reference genome was published in 2012, the actual number and functionality of sHSP genes remain unknown. Using a transcriptomic (RNA-seq) and evolutionary genomic approach, putative sHSP genes in the Solanum lycopersicum (cv. Heinz 1706) genome were investigated. A sHSP gene family of 33 members was established. Remarkably, roughly half of the members of this family can be explained by nine independent tandem duplication events that determined, evolutionarily, their functional fates. Within a mitochondrial class subfamily, only one duplicated member, Solyc08g078700, retained its ancestral chaperone function, while the others, Solyc08g078710 and Solyc08g078720, likely degenerated under neutrality and lack ancestral chaperone function. Functional conservation occurred within a cytosolic class I subfamily, whose four members, Solyc06g076570, Solyc06g076560, Solyc06g076540, and Solyc06g076520, support ∼57% of the total sHSP RNAm in the red ripe fruit. Subfunctionalization occurred within a new subfamily, whose two members, Solyc04g082720 and Solyc04g082740, show heterogeneous differential expression profiles during fruit ripening. These findings, involving the birth/death of some genes or the preferential/plastic expression of some others during fruit ripening, highlight the importance of tandem duplication events in the expansion of the sHSP gene family in the tomato genome. Despite its evolutionary diversity, the sHSP gene family in the tomato genome seems to be endowed with a core set of four homeostasis genes: Solyc05g014280, Solyc03g082420, Solyc11g020330, and Solyc06g076560, which appear to provide a baseline protection during both fruit ripening and heat shock stress in different tomato tissues. PMID:27565886

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.

Secreted HSP Vaccine for Malaria Prophylaxis

DTIC Science & Technology

2016-10-26

AWARD NUMBER: W81XWH-13-2-0098 TITLE: Secreted HSP Vaccine for Malaria Prophylaxis PRINCIPAL INVESTIGATOR: Dr. Natasa Strbo CONTRACTING ORGANIZATION...Secreted HSP Vaccine for Malaria Prophylaxis 4. TITLE AND SUBTITLE NATASA STRBO, M.D., D.SC NAME(S) AND E-M tzA UNIVERS]TY OF MTAMI 1600 NW 1OTH AVENUE ROOM...Here we developed malaria vaccine that relies on secreted gp96-lg chaperon-ing Plasmodium falciparum antigenic sporozoite proteins CSP and AMA1. The

Metazoan Hsp70 machines use Hsp110 to power protein disaggregation.

PubMed

Rampelt, Heike; Kirstein-Miles, Janine; Nillegoda, Nadinath B; Chi, Kang; Scholz, Sebastian R; Morimoto, Richard I; Bukau, Bernd

2012-11-05

Accumulation of aggregation-prone misfolded proteins disrupts normal cellular function and promotes ageing and disease. Bacteria, fungi and plants counteract this by solubilizing and refolding aggregated proteins via a powerful cytosolic ATP-dependent bichaperone system, comprising the AAA+ disaggregase Hsp100 and the Hsp70-Hsp40 system. Metazoa, however, lack Hsp100 disaggregases. We show that instead the Hsp110 member of the Hsp70 superfamily remodels the human Hsp70-Hsp40 system to efficiently disaggregate and refold aggregates of heat and chemically denatured proteins in vitro and in cell extracts. This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP-driven chaperoning is not required. Knock-down of nematode Caenorhabditis elegans Hsp110, but not an unrelated nucleotide exchange factor, compromises dissolution of heat-induced protein aggregates and severely shortens lifespan after heat shock. We conclude that in metazoa, Hsp70-Hsp40 powered by Hsp110 nucleotide exchange represents the crucial disaggregation machinery that reestablishes protein homeostasis to counteract protein unfolding stress.

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

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

Barends, Thomas R. M., E-mail: thomas.barends@mpimf-heidelberg.mpg.de; Brosi, Richard W. W.; Steinmetz, Andrea

2013-08-01

The crystal structure of the N-terminal part of T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed. Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein,more » but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.« less

Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

PubMed

Smith, T; Ferreira, L R; Hebert, C; Norris, K; Sauk, J J

1995-08-04

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.

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.

Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model.

PubMed

de la Mare, Jo-Anne; Jurgens, Tamarin; Edkins, Adrienne L

2017-03-16

Tumour metastasis remains the major cause of death in cancer patients and, to date, the mechanism and signalling pathways governing this process are not completely understood. The TGF-β pathway is the most commonly mutated pathway in cancer, however its role in cancer progression is controversial as it can function as both a promoter and a suppressor of metastasis. Although previous studies have suggested a role for the molecular chaperone Hsp90 in regulating the TGF-β pathway, the level at which this occurs as well as the consequences in terms of colon cancer metastasis are unknown. The paired SW480 and SW620 colon cancer cell lines, derived from a primary tumour and its lymph node metastasis, respectively, were used as an in vitro model to study key cellular processes required for metastasis. The status of the TGF-β pathway was examined in these cells using ELISA, flow cytometry, western blot analysis and confocal microscopy. Furthermore, the effect of addition or inhibition of the TGF-β pathway and Hsp90 on adhesion, migration and anchorage-independent growth, was determined in the cell lines. When comparing the canonical TGF-β1 pathway in the genetically paired cell lines our data suggests that this pathway may be constitutively active in the SW620 metastasis-derived cell line and not the SW480 primary tumour-derived line. In addition, we report that, when present in combination, TGF-β1 and Hsp90β stimulate anchorage-independent growth, reduce adhesion and stimulate migration. This effect is potentiated by inhibition of the TGF-β1 receptor and occurs via an alternate TGF-β1 pathway, mediated by αvβ6 integrin. Interestingly, in the SW620 cells, activation of this alternate TGF-β1 signalling machinery does not appear to require inhibition of the canonical TGF-β1 receptor, which would allow them to respond more effectively to the pro-metastasis stimulus of a combination of Hsp90β and TGF-β1 and this could account for the increased migratory capacity of these cells. In this study we report an apparent synergy between TGF-β1 and Hsp90β in stimulating migratory behaviour of colon cancer cells when signalling occurs via αvβ6 integrin as opposed to the canonical TGF-β1 pathway.

The Functional Landscape of Hsp27 Reveals New Cellular Processes such as DNA Repair and Alternative Splicing and Proposes Novel Anticancer Targets*

PubMed Central

Katsogiannou, Maria; Andrieu, Claudia; Baylot, Virginie; Baudot, Anaïs; Dusetti, Nelson J.; Gayet, Odile; Finetti, Pascal; Garrido, Carmen; Birnbaum, Daniel; Bertucci, François; Brun, Christine; Rocchi, Palma

2014-01-01

Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells. PMID:25277244

Oligomerization and chaperone-like activity of Drosophila melanogaster small heat shock protein DmHsp27 and three arginine mutants in the alpha-crystallin domain.

PubMed

Moutaoufik, Mohamed Taha; Morrow, Geneviève; Maaroufi, Halim; Férard, Céline; Finet, Stéphanie; Tanguay, Robert M

2017-07-01

The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.

Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

PubMed Central

Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

2016-01-01

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

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

The role of molecular chaperones in clathrin mediated vesicular trafficking

PubMed Central

Sousa, Rui; Lafer, Eileen M.

2015-01-01

The discovery that the 70 kD “uncoating ATPase,” which removes clathrin coats from vesicles after endocytosis, is the constitutively expressed Hsc70 chaperone was a surprise. Subsequent work, however, revealed that uncoating is an archetypal Hsp70 reaction: the cochaperone auxilin, which contains a clathrin binding domain and an Hsc70 binding J domain, recruits Hsc70*ATP to the coat and, concomitant with ATP hydrolysis, transfers it to a hydrophobic Hsc70-binding element found on a flexible tail at the C-terminus of the clathrin heavy chain. Release of clathrin in association with Hsc70*ADP follows, and the subsequent, persistent association of clathrin with Hsc70 is important to prevent aberrant clathrin polymerization. Thus, the two canonical functions of Hsp70—dissociation of existing protein complexes or aggregates, and binding to a protein to inhibit its inappropriate aggregation—are recapitulated in uncoating. Association of clathrin with Hsc70 in vivo is regulated by Hsp110, an Hsp70 NEF that is itself a member of the Hsp70 family. How Hsp110 activity is itself regulated to make Hsc70-free clathrin available for endocytosis is unclear, though at synapses it's possible that the influx of calcium that accompanies depolarization activates the Ca++/calmodulin dependent calcineurin phosphatase which then dephosphorylates and activates Hsp110 to stimulate ADP/ATP exchange and release clathrin from Hsc70*ADP:clathrin complexes. PMID:26042225

Elevated CO2 increases R gene-dependent resistance of Medicago truncatula against the pea aphid by up-regulating a heat shock gene.

PubMed

Sun, Yucheng; Guo, Huijuan; Yuan, Erliang; Ge, Feng

2018-03-01

Resistance against pathogens and herbivorous insects in many plant results from the expression of resistance (R) genes. Few reports, however, have considered the effects of elevated CO 2 on R gene-based resistance in plants. The current study determined the responses of two near isogenic Medicago truncatula genotypes (Jester has an R gene and A17 does not) to the pea aphid and elevated CO 2 in open-top chambers in the field. Aphid abundance, mean relative growth rate and feeding efficiency were increased by elevated CO 2 on A17 plants but were reduced on Jester plants. According to proteomic and gene expression data, elevated CO 2 enhanced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) but decreased the effector-triggered immunity (ETI) in aphid-infested A17 plants. For aphid-infested Jester plants, by contrast, elevated CO 2 enhanced the ETI-related heat shock protein (HSP) 90 and its co-chaperones, the jasmonic acid (JA) signaling pathway, and ubiquitin-mediated proteolysis. In a loss-of-function experiment, silencing of the HSP90 gene in Jester plants impaired the JA signaling pathway and ubiquitin-mediated proteolysis against the aphid under ambient CO 2 , and negated the increased resistance against the aphid under elevated CO 2 . Our results suggest that increases in expression of HSP90 are responsible for the enhanced resistance against the aphid under elevated CO 2 . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Isoflavone-deprived soy peptide suppresses mammary tumorigenesis by inducing apoptosis

PubMed Central

Park, Kyoungsook; Choi, Kyusam; Kim, Hyemee; Kim, Kwangbae; Lee, Mi Hee; Kim Rim, Jean Chinock

2009-01-01

During carcinogenesis, NF-κB mediates processes associated with deregulation of the normal control of proliferation, angiogenesis, and metastasis. Thus, suppression of NF-κB has been linked with chemoprevention of cancer. Accumulating findings reveal that heat shock protein 90 (HSP90) is a molecular chaperone and a component of the IκB kinase (IKK) complex that plays a central role in NF-κB activation. HSP90 also stabilizes key proteins involved in cell cycle control and apoptosis signaling. We have determined whether the exogenous administration of isoflavone-deprived soy peptide prevents 7,12-dimethylbenz[α]anthracene (DMBA)-induced rat mammary tumorigenesis and investigated the mechanism of action. Dietary administration of soy peptide (3.3 g/rat/day) significantly reduced the incidence of ductal carcinomas (50%), the number of tumors per multiple tumor-bearing rats (49%; P < 0.05), and extended the latency period of tumor development (8.07 ± 0.92 weeks) compared to control diet animals (10.80 ± 1.30; P < 0.05). Our results have further demonstrated that soy peptide (1) dramatically inhibits the expression of HSP90, thereby suppressing signaling pathway leading to NF-κB activation; (2) induces expression of p21, p53, and caspase-3 proteins; and (3) inhibits expression of VEGF. In agreement with our in vivo data, soy peptide treatment inhibited the growth of human breast MCF-7 tumor cells in a dose-dependent manner and induced apoptosis. Taken together, our in vivo and in vitro results suggest chemopreventive and tumor suppressive functions of isoflavone-deprived soy peptide by inducing growth arrest and apoptosis. PMID:19322027

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.

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70.

PubMed

Centrone, Mariangela; Ranieri, Marianna; Di Mise, Annarita; Berlingerio, Sante Princiero; Russo, Annamaria; Deen, Peter M T; Staub, Olivier; Valenti, Giovanna; Tamma, Grazia

2017-01-01

AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation. © 2017 The Author(s). Published by S. Karger AG, Basel.

Chaperone Hsp27 Modulates AUF1 Proteolysis and AU-Rich Element-Mediated mRNA Degradation▿

PubMed Central

Knapinska, Anna M.; Gratacós, Frances M.; Krause, Christopher D.; Hernandez, Kristina; Jensen, Amber G.; Bradley, Jacquelyn J.; Wu, Xiangyue; Pestka, Sidney; Brewer, Gary

2011-01-01

AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues—Ser15, Ser78, and Ser82—by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2–Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization. PMID:21245386

Nitric oxide acts as a positive regulator to induce metamorphosis of the ascidian Herdmania momus.

PubMed

Ueda, Nobuo; Degnan, Sandie M

2013-01-01

Marine invertebrates commonly have a biphasic life cycle in which the metamorphic transition from a pelagic larva to a benthic post-larva is mediated by the nitric oxide signalling pathway. Nitric oxide (NO) is synthesised by nitric oxide synthase (NOS), which is a client protein of the molecular chaperon heat shock protein 90 (HSP90). It is notable, then, that both NO and HSP90 have been implicated in regulating metamorphosis in marine invertebrates as diverse as urochordates, echinoderms, molluscs, annelids, and crustaceans. Specifically, the suppression of NOS activity by the application of either NOS- or HSP90-inhibiting pharmacological agents has been shown consistently to induce the initiation of metamorphosis, leading to the hypothesis that a negative regulatory role of NO is widely conserved in biphasic life cycles. Further, the induction of metamorphosis by heat-shock has been demonstrated for multiple species. Here, we investigate the regulatory role of NO in induction of metamorphosis of the solitary tropical ascidian, Herdmania momus. By coupling pharmacological treatments with analysis of HmNOS and HmHSP90 gene expression, we present compelling evidence of a positive regulatory role for NO in metamorphosis of this species, in contrast to all existing ascidian data that supports the hypothesis of NO as a conserved negative regulator of metamorphosis. The exposure of competent H. momus larvae to a NOS inhibitor or an NO donor results in an up-regulation of NOS and HSP90 genes. Heat shock of competent larvae induces metamorphosis in a temperature dependent manner, up to a thermal tolerance that approaches 35°C. Both larval/post-larval survival and the appearance of abnormal morphologies in H. momus post-larvae reflect the magnitude of up-regulation of the HSP90 gene in response to heat-shock. The demonstrated role of NO as a positive metamorphic regulator in H. momus suggests the existence of inter-specific adaptations of NO regulation in ascidian metamorphosis.

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation

PubMed Central

Lawag, Abdalla A.; Napper, Jennifer M.; Hunter, Caroline A.; Bacon, Nickolas A.; Deskins, Seth; El-hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C.

2017-01-01

Abstract Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%–90% of the cells die when placed in medium where the major growth factor is granulocyte–macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer. PMID:28910138

Nitric Oxide Acts as a Positive Regulator to Induce Metamorphosis of the Ascidian Herdmania momus

PubMed Central

Ueda, Nobuo; Degnan, Sandie M.

2013-01-01

Marine invertebrates commonly have a biphasic life cycle in which the metamorphic transition from a pelagic larva to a benthic post-larva is mediated by the nitric oxide signalling pathway. Nitric oxide (NO) is synthesised by nitric oxide synthase (NOS), which is a client protein of the molecular chaperon heat shock protein 90 (HSP90). It is notable, then, that both NO and HSP90 have been implicated in regulating metamorphosis in marine invertebrates as diverse as urochordates, echinoderms, molluscs, annelids, and crustaceans. Specifically, the suppression of NOS activity by the application of either NOS- or HSP90-inhibiting pharmacological agents has been shown consistently to induce the initiation of metamorphosis, leading to the hypothesis that a negative regulatory role of NO is widely conserved in biphasic life cycles. Further, the induction of metamorphosis by heat-shock has been demonstrated for multiple species. Here, we investigate the regulatory role of NO in induction of metamorphosis of the solitary tropical ascidian, Herdmania momus. By coupling pharmacological treatments with analysis of HmNOS and HmHSP90 gene expression, we present compelling evidence of a positive regulatory role for NO in metamorphosis of this species, in contrast to all existing ascidian data that supports the hypothesis of NO as a conserved negative regulator of metamorphosis. The exposure of competent H. momus larvae to a NOS inhibitor or an NO donor results in an up-regulation of NOS and HSP90 genes. Heat shock of competent larvae induces metamorphosis in a temperature dependent manner, up to a thermal tolerance that approaches 35°C. Both larval/post-larval survival and the appearance of abnormal morphologies in H. momus post-larvae reflect the magnitude of up-regulation of the HSP90 gene in response to heat-shock. The demonstrated role of NO as a positive metamorphic regulator in H. momus suggests the existence of inter-specific adaptations of NO regulation in ascidian metamorphosis. PMID:24019877

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.

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation.

PubMed

Lawag, Abdalla A; Napper, Jennifer M; Hunter, Caroline A; Bacon, Nickolas A; Deskins, Seth; El-Hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C; Sollars, Vincent E

2017-10-01

Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%-90% of the cells die when placed in medium where the major growth factor is granulocyte-macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.

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.

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

Optimizing fluorescently tethered Hsp90 inhibitor dose for maximal specific uptake by breast tumors

NASA Astrophysics Data System (ADS)

Crouch, Brian T.; Duer, Joy; Wang, Roujia; Gallagher, Jennifer; Hall, Allison; Soo, Mary Scott; Hughes, Philip; Haystead, Timothy A. J.; Ramanujam, Nirmala

2018-03-01

Despite improvements in surgical resection, 20-40% of patients undergoing breast conserving surgery require at least one additional re-excision. Leveraging the unique surface expression of heat shock protein 90 (Hsp90), a chaperone protein involved in several key hallmarks of cancer, in breast cancer provides an exciting opportunity to identify residual disease during surgery. We developed a completely non-destructive strategy using HS-27, a fluorescently-tethered Hsp90 inhibitor, to assay surface Hsp90 expression on intact tissue specimens using a fluorescence microendoscope with a field of view of 750 μm and subcellular resolution of 4 μm. HS-27 consists of an FDA approved Hsp90 inhibitor tethered to fluorescein isothiocyanate (EX 488nm, EM 525nm). Here, we optimized ex vivo HS-27 administration in pre-clinical breast cancer models and validated our approach on 21 patients undergoing standard of care ultrasound guided core needle biopsy. HS-27 administration time was fixed at 1- minute to minimize imaging impact on clinical workflow. HS-27 and HS-217 (non-specific control) doses were modulated from 1 μM up to 100 μM to identify the dose maximizing the ratio of specific uptake (HS-27 fluorescence) to non-specific uptake (HS-217 fluorescence). The specificity ratio was maximized at 100 μM and was significantly greater than all other doses (p<0.05). We applied our optimized imaging protocol to clinical samples and demonstrated significantly greater uptake of HS-27 by tumor than non-tumor tissue (p<0.05). The ubiquitous nature of HS-27 binding to all subtypes of breast cancer makes this technology attractive for assessing tumor margins, as one agent can be used for all subtypes.

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.

A systematic atlas of chaperome deregulation topologies across the human cancer landscape

PubMed Central

Sverchkova, Angelina

2018-01-01

Proteome balance is safeguarded by the proteostasis network (PN), an intricately regulated network of conserved processes that evolved to maintain native function of the diverse ensemble of protein species, ensuring cellular and organismal health. Proteostasis imbalances and collapse are implicated in a spectrum of human diseases, from neurodegeneration to cancer. The characteristics of PN disease alterations however have not been assessed in a systematic way. Since the chaperome is among the central components of the PN, we focused on the chaperome in our study by utilizing a curated functional ontology of the human chaperome that we connect in a high-confidence physical protein-protein interaction network. Challenged by the lack of a systems-level understanding of proteostasis alterations in the heterogeneous spectrum of human cancers, we assessed gene expression across more than 10,000 patient biopsies covering 22 solid cancers. We derived a novel customized Meta-PCA dimension reduction approach yielding M-scores as quantitative indicators of disease expression changes to condense the complexity of cancer transcriptomics datasets into quantitative functional network topographies. We confirm upregulation of the HSP90 family and also highlight HSP60s, Prefoldins, HSP100s, ER- and mitochondria-specific chaperones as pan-cancer enriched. Our analysis also reveals a surprisingly consistent strong downregulation of small heat shock proteins (sHSPs) and we stratify two cancer groups based on the preferential upregulation of ATP-dependent chaperones. Strikingly, our analyses highlight similarities between stem cell and cancer proteostasis, and diametrically opposed chaperome deregulation between cancers and neurodegenerative diseases. We developed a web-based Proteostasis Profiler tool (Pro2) enabling intuitive analysis and visual exploration of proteostasis disease alterations using gene expression data. Our study showcases a comprehensive profiling of chaperome shifts in human cancers and sets the stage for a systematic global analysis of PN alterations across the human diseasome towards novel hypotheses for therapeutic network re-adjustment in proteostasis disorders. PMID:29293508

A systematic atlas of chaperome deregulation topologies across the human cancer landscape.

PubMed

Hadizadeh Esfahani, Ali; Sverchkova, Angelina; Saez-Rodriguez, Julio; Schuppert, Andreas A; Brehme, Marc

2018-01-01

Proteome balance is safeguarded by the proteostasis network (PN), an intricately regulated network of conserved processes that evolved to maintain native function of the diverse ensemble of protein species, ensuring cellular and organismal health. Proteostasis imbalances and collapse are implicated in a spectrum of human diseases, from neurodegeneration to cancer. The characteristics of PN disease alterations however have not been assessed in a systematic way. Since the chaperome is among the central components of the PN, we focused on the chaperome in our study by utilizing a curated functional ontology of the human chaperome that we connect in a high-confidence physical protein-protein interaction network. Challenged by the lack of a systems-level understanding of proteostasis alterations in the heterogeneous spectrum of human cancers, we assessed gene expression across more than 10,000 patient biopsies covering 22 solid cancers. We derived a novel customized Meta-PCA dimension reduction approach yielding M-scores as quantitative indicators of disease expression changes to condense the complexity of cancer transcriptomics datasets into quantitative functional network topographies. We confirm upregulation of the HSP90 family and also highlight HSP60s, Prefoldins, HSP100s, ER- and mitochondria-specific chaperones as pan-cancer enriched. Our analysis also reveals a surprisingly consistent strong downregulation of small heat shock proteins (sHSPs) and we stratify two cancer groups based on the preferential upregulation of ATP-dependent chaperones. Strikingly, our analyses highlight similarities between stem cell and cancer proteostasis, and diametrically opposed chaperome deregulation between cancers and neurodegenerative diseases. We developed a web-based Proteostasis Profiler tool (Pro2) enabling intuitive analysis and visual exploration of proteostasis disease alterations using gene expression data. Our study showcases a comprehensive profiling of chaperome shifts in human cancers and sets the stage for a systematic global analysis of PN alterations across the human diseasome towards novel hypotheses for therapeutic network re-adjustment in proteostasis disorders.

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

Crystal structure and function of an unusual dimeric Hsp20.1 provide insight into the thermal protection mechanism of small heat shock proteins.

PubMed

Liu, Liang; Chen, Jiyun; Yang, Bo; Wang, Yonghua

2015-03-06

Small heat shock proteins (sHSPs) are ubiquitous chaperones that play a vital role in protein homeostasis. sHSPs are characterized by oligomeric architectures and dynamic exchange of subunits. The flexible oligomeric assembling associating with function remains poorly understood. Based on the structural data, it is certainly agreed that two dimerization models depend on the presence or absence of a β6 strand to differentiate nonmetazoan sHSPs from metazoan sHSPs. Here, we report the Sulfolobus solfataricus Hsp20.1 ACD dimer structure, which shows a distinct dimeric interface. We observed that, in the absence of β6, Hsp20.1 dimer does not depend on β7 strand for forming dimer interface as metazoan sHSPs, nor dissociates to monomers. This is in contrast to other published sHSPs. Our structure reveals a variable, highly polar dimer interface that has advantages for rapid subunits exchange and substrate binding. Remarkably, we find that the C-terminal truncation variant has chaperone activity comparable to that of wild-type despite lack of the oligomer structure. Our further study indicates that the N-terminal region is essential for the oligomer and dimer binding to the target protein. Together, the structure and function of Hsp20.1 give more insight into the thermal protection mechanism of sHSPs. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of an Atriplex nummularia gene encoding a protein homologous to the bacterial molecular chaperone DnaJ.

PubMed Central

Zhu, J K; Shi, J; Bressan, R A; Hasegawa, P M

1993-01-01

DnaJ is a 36-kD heat shock protein that functions together with Dnak (Hsp70) as a molecular chaperone in Escherichia coli. We have obtained a cDNA clone from the higher plant Atriplex nummularia that encodes a 46.6-kD polypeptide (ANJ1) with an overall 35.2% amino acid sequence identity with the E. coli DnaJ. ANJ1 has 43.4% overall sequence identity with the Saccharomyces cerevisiae cytoplasmic DnaJ homolog YDJ1/MAS5. Complementation of the yeast mas5 mutation indicated that ANJ1 is a functional homolog of YDJ1/MAS5. The presence of other DnaJ homologs in A. nummularia was demonstrated by the detection of proteins that are antigenically related to the yeast mitochondrial DnaJ homolog SCJ1 and the yeast DnaJ-related protein Sec63. Expression of the ANJ1 gene was compared with that of an A. nummularia Hsp70 gene. Expression of both ANJ1 and Hsp70 transcripts was coordinately induced by heat shock. However, noncoordinate accumulation of ANJ1 and Hsp70 mRNAs occurred during the cell growth cycle and in response to NaCl stress. PMID:8467224

Extracellular Release and Signaling by Heat Shock Protein 27: Role in Modifying Vascular Inflammation

PubMed Central

Batulan, Zarah; Pulakazhi Venu, Vivek Krishna; Li, Yumei; Koumbadinga, Geremy; Alvarez-Olmedo, Daiana Gisela; Shi, Chunhua; O’Brien, Edward R.

2016-01-01

Heat shock protein 27 (HSP27) is traditionally viewed as an intracellular chaperone protein with anti-apoptotic properties. However, recent data indicate that a number of heat shock proteins, including HSP27, are also found in the extracellular space where they may signal via membrane receptors to alter gene transcription and cellular function. Therefore, there is increasing interest in better understanding how HSP27 is released from cells, its levels and composition in the extracellular space, and the cognate cell membrane receptors involved in effecting cell signaling. In this paper, the knowledge to date, as well as some emerging paradigms about the extracellular function of HSP27 is presented. Of particular interest is the role of HSP27 in attenuating atherogenesis by modifying lipid uptake and inflammation in the plaque. Moreover, the abundance of HSP27 in serum is an emerging new biomarker for ischemic events. Finally, HSP27 replacement therapy may represent a novel therapeutic opportunity for chronic inflammatory disorders, such as atherosclerosis. PMID:27507972

A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

PubMed Central

Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

2016-01-01

Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

Improving survival by exploiting tumor dependence on stabilized mutant p53 for treatment

PubMed Central

Alexandrova, EM; Yallowitz, AR; Li, D; Xu, S; Schulz, R; Proia, DA; Lozano, G; Dobbelstein, M; Moll, UM

2015-01-01

SUMMARY Missense mutations in p53 generate aberrant proteins with abrogated tumor suppressor functions that can also acquire oncogenic gain-of-functions (GOF) that promote malignant progression, invasion, metastasis and chemoresistance1–5. Mutant p53 (mutp53) proteins undergo massive constitutive stabilization specifically in tumors, which is the key requisite for GOF6–8. Although currently 11 million patients worldwide live with tumors expressing highly stabilized mutp53, it is unknown whether mutp53 is a therapeutic target in vivo. Here we use a novel mutp53 mouse model expressing an inactivatible R248Q hotspot mutation (floxQ) to show that tumors depend on sustained mutp53 expression. Upon Tamoxifen-induced mutp53 ablation, allo-transplanted and autochthonous tumors curb their growth, thus extending animal survival by 37%, and advanced tumors undergo apoptosis and tumor regression or stagnation. The HSP90/HDAC6 chaperone machinery, which is significantly upregulated in cancer compared to normal tissues, is a major determinant of mutp53 stabilization9–12. We show that long-term HSP90 inhibition significantly extends the survival of mutp53 Q/−2 and H/H (R172H allele3) mice by 59% and 48%, respectively, but not their respective p53−/− littermates. This mutp53-dependent drug effect occurs in H/H mice treated with 17DMAG+SAHA and in H/H and Q/− mice treated with the potent Hsp90 inhibitor ganetespib. Notably, drug activity correlates with induction of mutp53 degradation, tumor apoptosis and prevention of T-lymphomagenesis. These proof-of-principle data identify mutp53 as an actionable cancer-specific drug target. PMID:26009011

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy

PubMed Central

Fang, Xi; Wu, Tongbin; Liu, Canzhao; Veevers, Jennifer; Stroud, Matthew J.; Zhang, Zhiyuan; Ma, Xiaolong; Mu, Yongxin; Lao, Dieu-Hung; Dalton, Nancy D.; Gu, Yusu; Wang, Celine; Wang, Michael; Liang, Yan; Ouyang, Kunfu; Peterson, Kirk L.; Evans, Sylvia M.

2017-01-01

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2–associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure. PMID:28737513

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.

PubMed

Fang, Xi; Bogomolovas, Julius; Wu, Tongbin; Zhang, Wei; Liu, Canzhao; Veevers, Jennifer; Stroud, Matthew J; Zhang, Zhiyuan; Ma, Xiaolong; Mu, Yongxin; Lao, Dieu-Hung; Dalton, Nancy D; Gu, Yusu; Wang, Celine; Wang, Michael; Liang, Yan; Lange, Stephan; Ouyang, Kunfu; Peterson, Kirk L; Evans, Sylvia M; Chen, Ju

2017-08-01

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

HSP70 peptidembearing and peptide-negative preparations act as chaperokines.

PubMed

Asea, A; Kabingu, E; Stevenson, M A; Calderwood, S K

2000-11-01

We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-kappaB activity and subsequent augmentation of proinflammatory cytokine TNF-alpha, IL-1beta, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

HSP70 peptide-bearing and peptide-negative preparations act as chaperokines

PubMed Central

Asea, Alexzander; Kabingu, Edith; Stevenson, Mary Ann; Calderwood, Stuart K.

2000-01-01

We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-κB activity and subsequent augmentation of proinflammatory cytokine TNF-α, IL-1β, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system. PMID:11189447

A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

PubMed

Kumar, C Sudheer; Swamy, Musti J

2016-07-05

HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch.

Action of the Hsp70 chaperone system observed with single proteins

NASA Astrophysics Data System (ADS)

Nunes, João M.; Mayer-Hartl, Manajit; Hartl, F. Ulrich; Müller, Daniel J.

2015-02-01

In Escherichia coli, the binding of non-native protein substrates to the Hsp70 chaperone DnaK is mediated by the co-chaperone DnaJ. DnaJ accelerates ATP hydrolysis on DnaK, by closing the peptide-binding cleft of DnaK. GrpE catalysed nucleotide exchange and ATP re-binding then lead to substrate release from DnaK, allowing folding. Here we refold immunoglobulin 27 (I27) to better understand how DnaJ-DnaK-GrpE chaperones cooperate. When DnaJ is present, I27 is less likely to misfold and more likely to fold, whereas the unfolded state remains unaffected. Thus, the ‘holdase’ DnaJ shows foldase behaviour. Misfolding of I27 is fully abrogated when DnaJ cooperates with DnaK, which stabilizes the unfolded state and increases the probability of folding. Addition of GrpE shifts the unfolded fraction of I27 to pre-chaperone levels. These insights reveal synergistic mechanisms within the evolutionary highly conserved Hsp70 system that prevent substrates from misfolding and promote their productive transition to the native state.

Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation

PubMed Central

Zheng, Xu; Krakowiak, Joanna; Patel, Nikit; Beyzavi, Ali; Ezike, Jideofor; Khalil, Ahmad S; Pincus, David

2016-01-01

Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.18638.001 PMID:27831465

A Targetable Molecular Chaperone Hsp27 Confers Aggressiveness in Hepatocellular Carcinoma.

PubMed

Zhang, Yurong; Tao, Xuemei; Jin, Guangzhi; Jin, Haojie; Wang, Ning; Hu, Fangyuan; Luo, Qin; Shu, Huiqun; Zhao, Fangyu; Yao, Ming; Fang, Jingyuan; Cong, Wenming; Qin, Wenxin; Wang, Cun

2016-01-01

Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis.

A Targetable Molecular Chaperone Hsp27 Confers Aggressiveness in Hepatocellular Carcinoma

PubMed Central

Zhang, Yurong; Tao, Xuemei; Jin, Guangzhi; Jin, Haojie; Wang, Ning; Hu, Fangyuan; Luo, Qin; Shu, Huiqun; Zhao, Fangyu; Yao, Ming; Fang, Jingyuan; Cong, Wenming; Qin, Wenxin; Wang, Cun

2016-01-01

Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis. PMID:26941848

PREFERENTIAL SECRETION OF INDUCIBLE HSP70 BY VITILIGO MELANOCYTES UNDER STRESS

PubMed Central

Mosenson, Jeffrey A.; Flood, Kelsey; Klarquist, Jared; Eby, Jonathan M.; Koshoffer, Amy; Boissy, Raymond E.; Overbeck, Andreas; C.Tung, Rebecca; Poole, I. Caroline Le

2014-01-01

SUMMARY Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes. PMID:24354861

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

The functional landscape of Hsp27 reveals new cellular processes such as DNA repair and alternative splicing and proposes novel anticancer targets.

PubMed

Katsogiannou, Maria; Andrieu, Claudia; Baylot, Virginie; Baudot, Anaïs; Dusetti, Nelson J; Gayet, Odile; Finetti, Pascal; Garrido, Carmen; Birnbaum, Daniel; Bertucci, François; Brun, Christine; Rocchi, Palma

2014-12-01

Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Biochemical assessment of the hibernator skeletal muscle properties in search of a potential countermeasure against muscle atrophy in space microgravity

NASA Astrophysics Data System (ADS)

Lee, K.; Park, J. Y.; Gwag, T.; Yoo, W.; Choi, I.

Mammalian skeletal muscle undergoes significant loss of mass and tension capacity during spaceflight or hindlimb suspension This is contrasted by observed features of hibernators in that muscle mass and contractility remain fairly unchanged during a prolonged period of dormancy In an effort of finding potential countermeasure against muscle atrophy in space microgravity we thereby investigated the biochemical properties of the pectoral muscle in a winter-hibernating bat Murina leucogaster Two-dimensional electrophoresis on overall muscle proteins and western blot analysis on heat shock proteins HSP 60 kD 70 kD and 90 kD were conducted to compare levels of myofiber proteins and the stress responsive chaperone molecules in winter-hibernation WH versus summer-active bats SA No seasonal difference was found in the ratio of muscle mass to body mass for the pectoral muscles confirming similar results in previous reports Among more than thirty proteins identified only 14 of the proteins showed significant reduction in the level for WH compared to SA The level of HSP60 and HSP90 in WH were 63 and 71 that in SA respectively P quad 0 05 whereas that of HSP70 was not different between the two groups However when the WH were forced to arouse for 40 min from hibernation the level of HSP70 increased 1 4-fold and 1 51-fold that of WH and SA respectively while the level of HSP90 increased 1 57-fold that of WH These results suggest that the levels of many key contractile and regulatory proteins were retained during

Characterizing functional differences in sea anemone Hsp70 isoforms using budding yeast.

PubMed

Waller, Shawn J; Knighton, Laura E; Crabtree, Lenora M; Perkins, Abigail L; Reitzel, Adam M; Truman, Andrew W

2018-04-25

Marine organisms experience abiotic stressors such as fluctuations in temperature, UV radiation, salinity, and oxygen concentration. Heat shock proteins (HSPs) assist in the response of cells to these stressors by refolding and maintaining the activity of damaged proteins. The well-conserved Hsp70 chaperone family is essential for cell viability as well as the response to stress. Organisms possess a variety of Hsp70 isoforms that differ slightly in amino acid sequence, yet very little is known about their functional relevance. In this study, we undertook analysis of three principal Hsp70 isoforms NvHsp70A, B, and D from the starlet sea anemone Nematostella vectensis. The functionality of Hsp70 isoforms in the starlet sea anemone was assessed through transcriptional analysis and by heterologous expression in budding yeast Saccharomyces cerevisiae. Interestingly, these isoforms were found to not only differ in expression under stress but also appear to have functional differences in their ability to mediate the cellular stress program. These results contribute to an understanding of Hsp70 isoform specificity, their shared and unique roles in response to acute and chronic environmental stress, and the potential basis of local adaptation in populations of N. vectensis.

The C. elegans UNC-23 protein, a member of the BCL-2-associated athanogene (BAG) family of chaperone regulators, interacts with HSP-1 to regulate cell attachment and maintain hypodermal integrity.

PubMed

Rahmani, Poupak; Rogalski, Teresa; Moerman, Donald G

2015-01-01

Mutations in the unc-23 gene in the free-living nematode, Caenorhabditis elegans result in detachment and dystrophy of the anterior body wall musculature and a bent-head phenotype when grown on solid substrate. We have determined that the unc-23 gene product is the nematode ortholog of the human BAG-2 protein, a member of the Bcl-2 associated athanogene (BAG) family of molecular chaperone regulators. We show that a functional GFP-tagged UNC-23 protein is expressed throughout development in several tissues of the animal, including body wall muscle and hypodermis, and associates with adhesion complexes and attachment structures within these 2 tissues. In humans, the BAG protein family consists of 6 members that all contain a conserved 45 amino acid BAG domain near their C-termini. These proteins bind to and modulate the activity of the ATPase domain of the heat shock cognate protein 70, Hsc70. We have isolated missense mutations in the ATPase domain of the C. elegans heat shock 70 protein, HSP-1 that suppress the phenotype exhibited by unc-23(e25) mutant hermaphrodites and we show that UNC-23 and HSP-1 interact in a yeast-2-hybrid system. The interaction of UNC-23 with HSP-1 defines a role for HSP-1 function in the maintenance of muscle attachment during development.

The C. elegans UNC-23 protein, a member of the BCL-2-associated athanogene (BAG) family of chaperone regulators, interacts with HSP-1 to regulate cell attachment and maintain hypodermal integrity

PubMed Central

Rahmani, Poupak; Rogalski, Teresa; Moerman, Donald G

2015-01-01

Mutations in the unc-23 gene in the free-living nematode, Caenorhabditis elegans result in detachment and dystrophy of the anterior body wall musculature and a bent-head phenotype when grown on solid substrate. We have determined that the unc-23 gene product is the nematode ortholog of the human BAG-2 protein, a member of the Bcl-2 associated athanogene (BAG) family of molecular chaperone regulators. We show that a functional GFP-tagged UNC-23 protein is expressed throughout development in several tissues of the animal, including body wall muscle and hypodermis, and associates with adhesion complexes and attachment structures within these 2 tissues. In humans, the BAG protein family consists of 6 members that all contain a conserved 45 amino acid BAG domain near their C-termini. These proteins bind to and modulate the activity of the ATPase domain of the heat shock cognate protein 70, Hsc70. We have isolated missense mutations in the ATPase domain of the C. elegans heat shock 70 protein, HSP-1 that suppress the phenotype exhibited by unc-23(e25) mutant hermaphrodites and we show that UNC-23 and HSP-1 interact in a yeast-2-hybrid system. The interaction of UNC-23 with HSP-1 defines a role for HSP-1 function in the maintenance of muscle attachment during development. PMID:26435886

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.

A small-molecule compound inhibits a collagen-specific molecular chaperone and could represent a potential remedy for fibrosis.

PubMed

Ito, Shinya; Ogawa, Koji; Takeuchi, Koh; Takagi, Motoki; Yoshida, Masahito; Hirokawa, Takatsugu; Hirayama, Shoshiro; Shin-Ya, Kazuo; Shimada, Ichio; Doi, Takayuki; Goshima, Naoki; Natsume, Tohru; Nagata, Kazuhiro

2017-12-08

Fibrosis can disrupt tissue structure and integrity and impair organ function. Fibrosis is characterized by abnormal collagen accumulation in the extracellular matrix. Pharmacological inhibition of collagen secretion therefore represents a promising strategy for the management of fibrotic disorders, such as liver and lung fibrosis. Hsp47 is an endoplasmic reticulum (ER)-resident collagen-specific molecular chaperone essential for correct folding of procollagen in the ER. Genetic deletion of Hsp47 or inhibition of its interaction with procollagen interferes with procollagen triple helix production, which vastly reduces procollagen secretion from fibroblasts. Thus, Hsp47 could be a potential and promising target for the management of fibrosis. In this study, we screened small-molecule compounds that inhibit the interaction of Hsp47 with collagen from chemical libraries using surface plasmon resonance (BIAcore), and we found a molecule AK778 and its cleavage product Col003 competitively inhibited the interaction and caused the inhibition of collagen secretion by destabilizing the collagen triple helix. Structural information obtained with NMR analysis revealed that Col003 competitively binds to the collagen-binding site on Hsp47. We propose that these structural insights could provide a basis for designing more effective therapeutic drugs for managing fibrosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Small heat shock proteins HSP27 (HspB1), αB-crystallin (HspB5) and HSP22 (HspB8) as regulators of cell death.

PubMed

Acunzo, Julie; Katsogiannou, Maria; Rocchi, Palma

2012-10-01

Hsp27, αB-crystallin and HSP22 are ubiquitous small heat shock proteins (sHsp) whose expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. These sHsp protect cells from otherwise lethal conditions mainly by their involvement in cell death pathways such as necrosis, apoptosis or autophagy. At a molecular level, the mechanisms accounting for sHsp functions in cell death are (1) prevention of denatured proteins aggregation, (2) regulation of caspase activity, (3) regulation of the intracellular redox state, (4) function in actin polymerization and cytoskeleton integrity and (5) proteasome-mediated degradation of selected proteins. In cancer cells, these sHsp are often overexpressed and associated with increased tumorigenicity, cancer cells metastatic potential and resistance to chemotherapy. Altogether, these properties suggest that Hsp27, αB-crystallin and Hsp22 are appropriate targets for modulating cell death pathways. In the present, we briefly review recent reports showing molecular evidence of cell death regulation by these sHsp and co-chaperones. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

Structural Basis of Interdomain Communication in the Hsc70 Chaperone

PubMed Central

Jiang, Jianwen; Prasad, Kondury; Lafer, Eileen M.; Sousa, Rui

2015-01-01

Summary Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. Were-port here the 2.6 Å structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds. PMID:16307916

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

PubMed Central

Bender, Tom; Lewrenz, Ilka; Franken, Sebastian; Baitzel, Catherina; Voos, Wolfgang

2011-01-01

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria. PMID:21209324

Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline

PubMed Central

Pulvino, Mary; Chen, Luojing; Oleksyn, David; Li, Jing; Compitello, George; Rossi, Randy; Spence, Stephen; Balakrishnan, Vijaya; Jordan, Craig; Poligone, Brian; Casulo, Carla; Burack, Richard; Shapiro, Joel L.; Bernstein, Steven; Friedberg, Jonathan W.; Deshaies, Raymond J.; Land, Hartmut; Zhao, Jiyong

2015-01-01

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes. PMID:26142707

CK2 phospho-dependent binding of R2TP complex to TEL2 is essential for mTOR and SMG1 stability.

PubMed

Horejsí, Zuzana; Takai, Hiroyuki; Adelman, Carrie A; Collis, Spencer J; Flynn, Helen; Maslen, Sarah; Skehel, J Mark; de Lange, Titia; Boulton, Simon J

2010-09-24

TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone complexes during the assembly of the PIKKs. Copyright © 2010 Elsevier Inc. All rights reserved.

Structural and functional aspects of hetero-oligomers formed by the small heat shock proteins αB-crystallin and HSP27.

PubMed

Aquilina, J Andrew; Shrestha, Sudichhya; Morris, Amie M; Ecroyd, Heath

2013-05-10

αB-crystallin and HSP27 are mammalian intracellular small heat shock proteins. These proteins exchange subunits in a rapid and temperature-dependent manner. This facile subunit exchange suggests that differential expression could be used by the cell to regulate the response to stress. A robust technique defines parameters for the dynamic interaction between the major mammalian small heat shock proteins. Small heat shock proteins (sHSPs) exist as large polydisperse species in which there is constant dynamic subunit exchange between oligomeric and dissociated forms. Their primary role in vivo is to bind destabilized proteins and prevent their misfolding and aggregation. αB-Crystallin (αB) and HSP27 are the two most widely distributed and most studied sHSPs in the human body. They are coexpressed in different tissues, where they are known to associate with each other to form hetero-oligomeric complexes. In this study, we aimed to determine how these two sHSPs interact to form hetero-oligomers in vitro and whether, by doing so, there is an increase in their chaperone activity and stability compared with their homo-oligomeric forms. Our results demonstrate that HSP27 and αB formed polydisperse hetero-oligomers in vitro, which had an average molecular mass that was intermediate of each of the homo-oligomers and which were more thermostable than αB, but less so than HSP27. The hetero-oligomer chaperone function was found to be equivalent to that of αB, with each being significantly better in preventing the amorphous aggregation of α-lactalbumin and the amyloid fibril formation of α-synuclein in comparison with HSP27. Using mass spectrometry to monitor subunit exchange over time, we found that HSP27 and αB exchanged subunits 23% faster than the reported rate for HSP27 and αA and almost twice that for αA and αB. This represents the first quantitative evaluation of αB/HSP27 subunit exchange, and the results are discussed in the broader context of regulation of function and cellular proteostasis.

The force-dependent mechanism of DnaK-mediated mechanical folding

PubMed Central

Perales-Calvo, Judit; Giganti, David; Stirnemann, Guillaume; Garcia-Manyes, Sergi

2018-01-01

It is well established that chaperones modulate the protein folding free-energy landscape. However, the molecular determinants underlying chaperone-mediated mechanical folding remain largely elusive, primarily because the force-extended unfolded conformation fundamentally differs from that characterized in biochemistry experiments. We use single-molecule force-clamp spectroscopy, combined with molecular dynamics simulations, to study the effect that the Hsp70 system has on the mechanical folding of three mechanically stiff model proteins. Our results demonstrate that, when working independently, DnaJ (Hsp40) and DnaK (Hsp70) work as holdases, blocking refolding by binding to distinct substrate conformations. Whereas DnaK binds to molten globule–like forms, DnaJ recognizes a cryptic sequence in the extended state in an unanticipated force-dependent manner. By contrast, the synergetic coupling of the Hsp70 system exhibits a marked foldase behavior. Our results offer unprecedented molecular and kinetic insights into the mechanisms by which mechanical force finely regulates chaperone binding, directly affecting protein elasticity. PMID:29487911

Heat shock protein 47 expression in aged normal human fibroblasts: modulation by Salix alba extract.

PubMed

Nizard, Carine; Noblesse, Emmanuelle; Boisdé, Cécille; Moreau, Marielle; Faussat, Anne-Marie; Schnebert, Sylvianne; Mahé, Christian

2004-06-01

Heat shock protein (HSP) 47 is a specific chaperone of procollagen. This heat shock protein is responsible for the correct three-dimensional organization of procollagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP 47 in aged, photoaged, and senescent fibroblasts and its modulation by a plant extract (Salix alba). The level of HSP 47 and/or procollagen expression in fibroblasts was measured by real-time RT-PCR (mRNA transcripts) and by flow cytometry (immunochemistry technique for measurement of arbitrary fluorescence intensity). Immunochemistry techniques and confocal microscopy were used to visualize the cellular localization of HSP 47 and procollagen. These parameters were compared with different age donors, nonsenescent, and senescent fibroblasts. Fibroblasts were irradiated by a noncytotoxic dose of UVA (6 J/cm(2)), and HSP 47 level was evaluated. S. alba extract was tested for its capacity to modulate HSP 47 expression. Colocalization of HSP 47 and procollagen was shown by confocal microscopy, indicating that HSP 47 could play a role of procollagen molecular chaperone in the cellular model. It was also shown that the HSP 47 level is decreased in old-donor cells, senescent, and irradiated cells. This decrease can be modulated by a S. alba extract (polyphenols rich) in a dose-dependent manner. The evaluation of HSP 47 expression in the experimental conditions can lead to a new approach of aging and photoaging, pointing out the implication of this chaperone in these pathophysiologic phenomena. Modulation of HSP 47 expression by this family of molecules could be of cosmetic and/or dermatologic interest.

Expression of small heat shock proteins from pea seedlings under gravity altered conditions

NASA Astrophysics Data System (ADS)

Talalaev, Alexandr S.

2005-08-01

A goal of our study was to evaluate the stress gene expression in Pisum sativum seedlings exposed to altered gravity and temperature elevation. We investigate message for the two inducible forms of the cytosolic small heat shock proteins (sHsp), sHsp 17.7 and sHsp 18.1. Both proteins are able to enhance the refolding of chemically denatured proteins in an ATP- independent manner, in other words they can function as molecular chaperones. We studied sHsps expression in pea seedlings cells by Western blotting. Temperature elevation, as the positive control, significantly increased PsHsp 17.7 and PsHsp 18.1 expression. Expression of the housekeeping protein, actin was constant and comparable to unstressed controls for all treatments. We concluded that gravitational perturbations incurred by clinorotation did not change sHsp genes expression.

Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle.

PubMed

Surtees, Rebecca; Dowall, Stuart D; Shaw, Amelia; Armstrong, Stuart; Hewson, Roger; Carroll, Miles W; Mankouri, Jamel; Edwards, Thomas A; Hiscox, Julian A; Barr, John N

2016-10-15

The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease. Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is responsible for fatal human disease and is recognized as an emerging threat within Europe in response to climate change. No preventative or therapeutic strategies against nairovirus-mediated disease are currently available. Here we show that the N protein of CCHFV and the related Hazara virus interact with a cellular protein, HSP70, during both the intracellular and extracellular stages of the virus life cycle. The use of inhibitors that block HSP70 function reduces virus titers by up to 1,000-fold, suggesting that this interaction is important within the context of the nairovirus life cycle and may represent a potent target for antinairovirus therapies against which the virus cannot easily develop resistance. Copyright © 2016 Surtees et al.

Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae

PubMed Central

Jarnuczak, Andrew F.; Eyers, Claire E.; Schwartz, Jean‐Marc; Grant, Christopher M.

2015-01-01

Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC‐based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. PMID:25689132

Inhibition of HSP70 and a Collagen-Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

NASA Technical Reports Server (NTRS)

Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Kei'ichi; Akiyama, Hideo; Hirano, Masahiko; Sams, Clarence F.; Whitson, Peggy A.

2003-01-01

Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

The small heat shock protein Hsp31 cooperates with Hsp104 to modulate Sup35 prion aggregation.

PubMed

Aslam, Kiran; Tsai, Chai-Jui; Hazbun, Tony R

2016-11-01

The yeast homolog of DJ-1, Hsp31, is a multifunctional protein that is involved in several cellular pathways including detoxification of the toxic metabolite methylglyoxal and as a protein deglycase. Prior studies ascribed Hsp31 as a molecular chaperone that can inhibit α-Syn aggregation in vitro and alleviate its toxicity in vivo. It was also shown that Hsp31 inhibits Sup35 aggregate formation in yeast, however, it is unknown if Hsp31 can modulate [PSI + ] phenotype and Sup35 prionogenesis. Other small heat shock proteins, Hsp26 and Hsp42 are known to be a part of a synergistic proteostasis network that inhibits Sup35 prion formation and promotes its disaggregation. Here, we establish that Hsp31 inhibits Sup35 [PSI + ] prion formation in collaboration with a well-known disaggregase, Hsp104. Hsp31 transiently prevents prion induction but does not suppress induction upon prolonged expression of Sup35 indicating that Hsp31 can be overcome by larger aggregates. In addition, elevated levels of Hsp31 do not cure [PSI + ] strains indicating that Hsp31 cannot intervene in a pre-existing prion oligomerization cycle. However, Hsp31 can modulate prion status in cooperation with Hsp104 because it inhibits Sup35 aggregate formation and potentiates [PSI + ] prion curing upon overexpression of Hsp104. The absence of Hsp31 reduces [PSI + ] prion curing by Hsp104 without influencing its ability to rescue cellular thermotolerance. Hsp31 did not synergize with Hsp42 to modulate the [PSI + ] phenotype suggesting that both proteins act on similar stages of the prion cycle. We also showed that Hsp31 physically interacts with Hsp104 and together they prevent Sup35 prion toxicity to greater extent than if they were expressed individually. These results elucidate a mechanism for Hsp31 on prion modulation that suggest it acts at a distinct step early in the Sup35 aggregation process that is different from Hsp104. This is the first demonstration of the modulation of [PSI + ] status by the chaperone action of Hsp31. The delineation of Hsp31's role in the chaperone cycle has implications for understanding the role of the DJ-1 superfamily in controlling misfolded proteins in neurodegenerative disease and cancer.

Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae

PubMed Central

Wholey, Wei-Yun; Jakob, Ursula

2012-01-01

Summary The redox-regulated chaperone Hsp33 protects bacteria specifically against stress conditions that cause oxidative protein unfolding, such as treatment with bleach or exposure to peroxide at elevated temperatures. To gain insight into the mechanism by which expression of Hsp33 confers resistance to oxidative protein unfolding conditions, we made use of V. cholerae strain O395 lacking the Hsp33 gene hslO. We found that this strain, which is exquisitely bleach-sensitive, displays a temperature-sensitive (ts) phenotype during aerobic growth, implying that V. cholerae suffers from oxidative heat stress when cultivated at 43°C. We utilized this phenotype to select for E. coli genes that rescue the ts phenotype of V. cholerae ΔhslO when overexpressed. We discovered that expression of a single protein, the elongation factor EF-Tu, was sufficient to rescue both the ts and bleach-sensitive phenotypes of V. cholerae ΔhslO. In vivo studies revealed that V. cholerae EF-Tu is highly sensitive to oxidative protein degradation in the absence of Hsp33, indicating that EF-Tu is a vital chaperone substrate of Hsp33 in V. cholerae. These results suggest an “essential client protein” model for Hsp33’s chaperone action in Vibrio in which stabilization of a single oxidative stress-sensitive protein is sufficient to enhance the oxidative stress resistance of the whole organism. PMID:22296329

Bayesian refinement of protein structures and ensembles against SAXS data using molecular dynamics

PubMed Central

Shevchuk, Roman; Hub, Jochen S.

2017-01-01

Small-angle X-ray scattering is an increasingly popular technique used to detect protein structures and ensembles in solution. However, the refinement of structures and ensembles against SAXS data is often ambiguous due to the low information content of SAXS data, unknown systematic errors, and unknown scattering contributions from the solvent. We offer a solution to such problems by combining Bayesian inference with all-atom molecular dynamics simulations and explicit-solvent SAXS calculations. The Bayesian formulation correctly weights the SAXS data versus prior physical knowledge, it quantifies the precision or ambiguity of fitted structures and ensembles, and it accounts for unknown systematic errors due to poor buffer matching. The method further provides a probabilistic criterion for identifying the number of states required to explain the SAXS data. The method is validated by refining ensembles of a periplasmic binding protein against calculated SAXS curves. Subsequently, we derive the solution ensembles of the eukaryotic chaperone heat shock protein 90 (Hsp90) against experimental SAXS data. We find that the SAXS data of the apo state of Hsp90 is compatible with a single wide-open conformation, whereas the SAXS data of Hsp90 bound to ATP or to an ATP-analogue strongly suggest heterogenous ensembles of a closed and a wide-open state. PMID:29045407

Temperature-dependent subunit exchange and chaperone-like activities of Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis.

PubMed

Fu, Xinmiao; Chang, Zengyi

2004-04-02

Small heat shock proteins (sHsps) usually exist as oligomers that undergo dynamic oligomeric dissociation/re-association, with the dissociated oligomers as active forms to bind substrate proteins under heat shock conditions. In this study, however, we found that Hsp16.3, one sHsp from Mycobacterium tuberculosis, is able to sensitively modulate its chaperone-like activity in a range of physiological temperatures (from 25 to 37.5 degrees C) while its native oligomeric size is still maintained. Further analysis demonstrated that Hsp16.3 exposes higher hydrophobic surfaces upon temperatures increasing and that a large soluble complex between Hsp16.3 and substrate is formed only in the condition of heating temperature up to 35 and 37.5 degrees C. Structural analysis by fluorescence anisotropy showed that Hsp16.3 nonameric structure becomes more dynamic and variable at elevated temperatures. Moreover, subunit exchange between Hsp16.3 oligomers was found to occur faster upon temperatures increasing as revealed by fluorescence energy resonance transfer. These observations indicate that Hsp16.3 is able to modulate its chaperone activity by adjusting the dynamics of oligomeric dissociation/re-association process while maintaining its static oligomeric size unchangeable. A kinetic model is therefore proposed to explain the mechanism of sHsps-binding substrate proteins through oligomeric dissociation. The present study also implied that Hsp16.3 is at least capable of binding non-native proteins in vivo while expressing in the host organism that survives at 37 degrees C.

Molecular cloning and expression analysis of five heat shock protein 70 (HSP70) family members in Lateolabrax maculatus with Vibrio harveyi infection.

PubMed

Han, Ying-Li; Hou, Cong-Cong; Du, Chen; Zhu, Jun-Quan

2017-01-01

Heat shock proteins 70 (HSP70s) are molecular chaperones that aid in protection against environmental stress. In this study, we cloned and characterized five members of the HSP70 family (designated as HSPa1a, HSC70-1, HSC70-2, HSPa4 and HSPa14) from Lateolabrax maculatus using rapid amplification cDNA ends (RACE). Multiple sequence alignment and structural analysis revealed that all members of the HSP70 family had a conserved domain architecture, with some distinguishing features unique to each HSP70. Quantitative real-time (qPCR) analysis revealed that all members of the HSP70 family were ubiquitously and differentially expressed in all major types of tissues, including testicular tissue. This indicated that HSP70s have vital and conserved biological functions, and may also function in the development of germinal cells. The expression of mRNA of the five HSP70 family members mRNA expression was significantly increased in the head kidney, intestine and gill after Vibrio harveyi challenge, suggesting that HSP70s play an important role in the immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heat Shock Proteins In The Retina: Focus On Hsp70 and Alpha Crystallins In Ganglion Cell Survival

PubMed Central

Piri, Natik; Kwong, Jacky MK; Gu, Lei; Caprioli, Joseph

2016-01-01

Heat shock proteins (HSPs) belong to a superfamily of stress proteins that are critical constituents of a complex defense mechanism that enhances cell survival under adverse environmental conditions. Cell protective roles of HSPs are related to their chaperone functions, antiapoptotic and antinecrotic effects. HSPs' antiapoptotic and cytoprotective characteristics, their ability to protect cells from a variety of stressful stimuli, and the possibility of their pharmacological induction in cells under pathological stress make these proteins an attractive therapeutic target for various neurodegenerative diseases; these include Alzheimer's, Parkinson's, Huntington's, prion disease, and others. This review discusses the possible roles of HSPs, particularly HSP70 and small HSPs (alpha A and alpha B crystallins) in enhancing the survival of retinal ganglion cells (RGCs) in optic neuropathies such as glaucoma, which is characterized by progressive loss of vision caused by degeneration of RGCs and their axons in the optic nerve. Studies in animal models of RGC degeneration induced by ocular hypertension, optic nerve crush and axotomy show that upregulation of HSP70 expression by hyperthermia, zinc, geranyl-geranyl acetone, 17-AAG (a HSP90 inhibitor), or through transfection of retinal cells with AAV2-HSP70 effectively supports the survival of injured RGCs. RGCs survival was also stimulated by overexpression of alpha A and alpha B crystallins. These findings provide support for translating the HSP70- and alpha crystallin-based cell survival strategy into therapy to protect and rescue injured RGCs from degeneration associated with glaucomatous and other optic neuropathies. PMID:27017896

α-Crystallins Are Small Heat Shock Proteins: Functional and Structural Properties.

PubMed

Tikhomirova, T S; Selivanova, O M; Galzitskaya, O V

2017-02-01

During its life cycle, a cell can be subjected to various external negative effects. Many proteins provide cell protection, including small heat shock proteins (sHsp) that have chaperone-like activity. These proteins have several important functions involving prevention of apoptosis and retention of cytoskeletal integrity; also, sHsp take part in the recovery of enzyme activity. The action mechanism of sHsp is based on the binding of hydrophobic regions exposed to the surface of a molten globule. α-Crystallins presented in chordate cells as two αA- and αB-isoforms are the most studied small heat shock proteins. In this review, we describe the main functions of α-crystallins, features of their secondary and tertiary structures, and examples of their partners in protein-protein interactions.

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

Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding.

PubMed

Hageman, Jurre; Vos, Michel J; van Waarde, Maria A W H; Kampinga, Harm H

2007-11-23

Molecular chaperones are essential for cells to prevent that partially unfolded proteins form non-functional, toxic aggregates. This requirement is increased when cells experience protein unfolding stresses and such could affect all compartments in the eukaryotic cell. Whether all organelles are equipped with comparable chaperone capacities is largely unknown, mainly due to the lack of suitable reporters that allow such a comparison. Here we describe the development of fluorescent luciferase reporters that are sorted to various cellular locations (nucleus, cytoplasm, endoplasmic reticulum, and peroxisomes) and that differ minimally in their intrinsic thermal stability properties. When heating living cells, the rate of inactivation was most rapid for the nuclear-targeted luciferase, indicating that the nucleus is the most sensitive organelle toward heat-induced denaturing stress. Post-heat re-activation, however, occurred at equal kinetics irrespective of luciferase localization. Also, induction of thermotolerance by a priming heat treatment, that coordinately up-regulates all heat-inducible chaperones, resulted in a transient heat resistance of the luciferase in all organelles in a comparable manner. Overexpression of the main heat-inducible Hsp70 family member, HspA1A, protected only the cytosolic and nuclear, but not the other luciferases. Together, our data suggest that in each compartment investigated, including the peroxisome in which so far no chaperones could be detected, chaperone machines are present and can be induced with activities similar to those present in the cytosolic/nuclear compartment.

Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis.

PubMed Central

Ding, D; Parkhurst, S M; Halsell, S R; Lipshitz, H D

1993-01-01

Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. We show that maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 RNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity. In contrast, Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules--cappuccino, oskar, spire, staufen, tudor, valois, and vasa. Mislocalization of oskar RNA to the anterior pole, which has been shown to result in induction of germ cells at the anterior, leads to anterior protection of maternal Hsp83 RNA. These results suggest that Hsp83 RNA is a component of the posterior polar plasm that might be associated with polar granules. In addition, we show that zygotic expression of Hsp83 commences in the anterior third of the embryo at the syncytial blastoderm stage and is regulated by the anterior morphogen, bicoid. We consider the possible developmental significance of this complex control of Hsp83 transcript distribution. Images PMID:7684502

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.

Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

NASA Astrophysics Data System (ADS)

Schmid, Sonja; Hugel, Thorsten

2018-03-01

Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings—such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

Heat shock proteins and toll-like receptors.

PubMed

Asea, Alexzander

2008-01-01

Researchers have only just begun to elucidate the relationship between heat shock proteins (HSP) and Toll-like receptors (TLR). HSP were originally described as an intracellular molecular chaperone of naïve, aberrantly folded, or mutated proteins and primarily implicated as a cytoprotective protein when cells are exposed to stressful stimuli. However, recent studies have ascribed novel functions to the Hsp70 protein depending on its localization: Surface-bound Hsp70 specifically activate natural killer (NK) cells, while Hsp70 released into the extracellular milieu specifically bind to Toll-like receptors (TLR) 2 and 4 on antigen-presenting cells (APC) and exerts immunoregulatory effects, including upregulation of adhesion molecules, co-stimulatory molecule expression, and cytokine and chemokine release-a process known as the chaperokine activity of Hsp70. This chapter discusses the most recent advances in the understanding of heat shock protein (HSP) and TLR interactions in general and highlights recent findings that demonstrate Hsp70 is a ligand for TLR and its biological significance.

Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α.

PubMed

Sepulveda, Denisse; Rojas-Rivera, Diego; Rodríguez, Diego A; Groenendyk, Jody; Köhler, Andres; Lebeaupin, Cynthia; Ito, Shinya; Urra, Hery; Carreras-Sureda, Amado; Hazari, Younis; Vasseur-Cognet, Mireille; Ali, Maruf M U; Chevet, Eric; Campos, Gisela; Godoy, Patricio; Vaisar, Tomas; Bailly-Maitre, Béatrice; Nagata, Kazuhiro; Michalak, Marek; Sierralta, Jimena; Hetz, Claudio

2018-01-18

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR. Copyright © 2018 Elsevier Inc. All rights reserved.

A Toxoplasma gondii Class XIV Myosin, Expressed in Sf9 Cells with a Parasite Co-chaperone, Requires Two Light Chains for Fast Motility*

PubMed Central

Bookwalter, Carol S.; Kelsen, Anne; Leung, Jacqueline M.; Ward, Gary E.; Trybus, Kathleen M.

2014-01-01

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors. PMID:25231988

[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.

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

Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae.

PubMed

Jarnuczak, Andrew F; Eyers, Claire E; Schwartz, Jean-Marc; Grant, Christopher M; Hubbard, Simon J

2015-09-01

Molecular chaperones play an important role in protein homeostasis and the cellular response to stress. In particular, the HSP70 chaperones in yeast mediate a large volume of protein folding through transient associations with their substrates. This chaperone interaction network can be disturbed by various perturbations, such as environmental stress or a gene deletion. Here, we consider deletions of two major chaperone proteins, SSA1 and SSB1, from the chaperone network in Sacchromyces cerevisiae. We employ a SILAC-based approach to examine changes in global and local protein abundance and rationalise our results via network analysis and graph theoretical approaches. Although the deletions result in an overall increase in intracellular protein content, correlated with an increase in cell size, this is not matched by substantial changes in individual protein concentrations. Despite the phenotypic robustness to deletion of these major hub proteins, it cannot be simply explained by the presence of paralogues. Instead, network analysis and a theoretical consideration of folding workload suggest that the robustness to perturbation is a product of the overall network structure. This highlights how quantitative proteomics and systems modelling can be used to rationalise emergent network properties, and how the HSP70 system can accommodate the loss of major hubs. © 2015 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli.

PubMed

Cho, Younhee; Zhang, Xin; Pobre, Kristine Faye R; Liu, Yu; Powers, David L; Kelly, Jeffery W; Gierasch, Lila M; Powers, Evan T

2015-04-14

The folding fate of a protein in vivo is determined by the interplay between a protein's folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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.

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.

Breaking BAG: The Co-Chaperone BAG3 in Health and Disease.

PubMed

Behl, Christian

2016-08-01

Human BAG (Bcl-2-associated athanogene) proteins form a family of antiapoptotic proteins that currently consists of six members (BAG1-6) all sharing the BAG protein domain from which the name arises. Via this domain, BAG proteins bind to the heat shock protein 70 (Hsp70), thereby acting as a co-chaperone regulating the activity of Hsp70. In addition to their antiapoptotic activity, all human BAG proteins have distinct functions in health and disease, and BAG3 in particular is the focus of many investigations. BAG3 has a modular protein domain composition offering the possibility for manifold interactions with other proteins. Various BAG3 functions are implicated in disorders including cancer, myopathies, and neurodegeneration. The discovery of its role in selective autophagy and the description of BAG3-mediated selective macroautophagy as an adaptive mechanism to maintain cellular homeostasis, under stress as well as during aging, make BAG3 a highly interesting target for future pharmacological interventions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preliminary X-Ray Crystallographic Studies of the N-Terminal Domains of Hsp104 from Yeast Candida albicans and Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Wang, P.; Li, J.; Sha, B.

2017-12-01

Yeast Hsp104 is an ATP-dependent molecular chaperone, which can solublize and rescue denatured proteins from aggregates into active form by cooperating with Hsp70 and Hsp40 chaperones. Moreover, overexpression of Hsp104 of Saccharomyces cerevisiae (ScHsp104) cures the yeast [ PSI +] prion due to the completely dissolution of the prion seeds, demonstrating ScHsp104's potential to clear amyloid-like protein aggregates, thus making ScHsp104 a promising medication approach for human amyloidogenic neurodegenerative diseases. Because the working mechanisms for ScHsp104's activities have not been clearly elucidated yet, crystallographic determination of ScHsp104 stands for great significance. Here, the expression, purification and crystallization of the N-terminal domains of Hsp104 from yeast Candida albicans (CaHsp104N) and S. cerevisiae (ScHsp104N) are described. The CaHsp104N crystals diffracted to 1.54 Å and belonged to the sp. gr. P3221 or P3121, with unit cell parameters of a = 55.213 Å, c = 109.451 Å. The data of the ScHsp104N crystals were collected to the resolution of 2.53 Å in the sp. gr. C2, with unit cell parameters a = 148.587 Å, b = 66.255 Å, c = 74.577 Å, β = 107.369°. The phase of ScHsp104N is determined by the molecular replacement method using CaHsp104N as the search model.

The DNLZ/HEP zinc-binding subdomain is critical for regulation of the mitochondrial chaperone HSPA9

PubMed Central

Vu, Michael T; Zhai, Peng; Lee, Juhye; Guerra, Cecilia; Liu, Shirley; Gustin, Michael C; Silberg, Jonathan J

2012-01-01

Human mitochondrial DNLZ/HEP regulates the catalytic activity and solubility of the mitochondrial hsp70 chaperone HSPA9. Here, we investigate the role that the DNLZ zinc-binding and C-terminal subdomains play in regulating HSPA9. We show that truncations lacking portions of the zinc-binding subdomain (ZBS) do not affect the solubility of HSPA9 or its ATPase domain, whereas those containing the ZBS and at least 10 residues following this subdomain enhance chaperone solubility. Binding measurements further show that DNLZ requires its ZBS to form a stable complex with the HSPA9 ATPase domain, and ATP hydrolysis measurements reveal that the ZBS is critical for full stimulation of HSPA9 catalytic activity. We also examined if DNLZ is active in vivo. We found that DNLZ partially complements the growth of Δzim17Saccharomyces cerevisiae, and we discovered that a Zim17 truncation lacking a majority of the C-terminal subdomain strongly complements growth like full-length Zim17. These findings provide direct evidence that human DNLZ is a functional ortholog of Zim17. In addition, they implicate the pair of antiparallel β-strands that coordinate zinc in Zim17/DNLZ-type proteins as critical for binding and regulating hsp70 chaperones. PMID:22162012

The DNLZ/HEP zinc-binding subdomain is critical for regulation of the mitochondrial chaperone HSPA9.

PubMed

Vu, Michael T; Zhai, Peng; Lee, Juhye; Guerra, Cecilia; Liu, Shirley; Gustin, Michael C; Silberg, Jonathan J

2012-02-01

Human mitochondrial DNLZ/HEP regulates the catalytic activity and solubility of the mitochondrial hsp70 chaperone HSPA9. Here, we investigate the role that the DNLZ zinc-binding and C-terminal subdomains play in regulating HSPA9. We show that truncations lacking portions of the zinc-binding subdomain (ZBS) do not affect the solubility of HSPA9 or its ATPase domain, whereas those containing the ZBS and at least 10 residues following this subdomain enhance chaperone solubility. Binding measurements further show that DNLZ requires its ZBS to form a stable complex with the HSPA9 ATPase domain, and ATP hydrolysis measurements reveal that the ZBS is critical for full stimulation of HSPA9 catalytic activity. We also examined if DNLZ is active in vivo. We found that DNLZ partially complements the growth of Δzim17 Saccharomyces cerevisiae, and we discovered that a Zim17 truncation lacking a majority of the C-terminal subdomain strongly complements growth like full-length Zim17. These findings provide direct evidence that human DNLZ is a functional ortholog of Zim17. In addition, they implicate the pair of antiparallel β-strands that coordinate zinc in Zim17/DNLZ-type proteins as critical for binding and regulating hsp70 chaperones. Copyright © 2011 The Protein Society.