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.

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.

[Effects of heat shock protein 70-2 gene polymorphisms on the transcription of HSP 70-2 mRNA and the translation of HSP 70 protein in lung cancer].

PubMed

Lu, Hao-quan; Wang, Yu-zhen; Sun, Peng-hui; Liang, Shou-pei; Li, Jie; Wang, Xiao-long; Xu, Dong; Yao, Wu; Wu, Yi-ming; Zhou, Fang

2012-05-01

This study aimed to investigate the effects of gene polymorphism of heat shock protein 70-2 (HSP 70-2) 1267A/G on the mRNA level HSP 70-2 mRNA and the protein level HSP 70 in human lung cancer. Forty six lung cancer patients diagnosed histopathologically between February and August 2008 from a hospital in zhengzhou were enrolled as the subjects in this study. Gene polymorphism of HSP 70-2 1276A/G in 46 patients with lung cancer was detected by PCR-RFLP. The mRNA levels of HSP 70-2 mRNA and the protein levels of HSP 70 in lung tissue and para-cancerous tissues of these subjects were determined by RT-PCR and Western blotting respectively. The expression levels of HSP 70-2 mRNA (1.02 ± 0.30) and HSP 70 protein (0.44 ± 0.12) in the lung cancer tissues was significantly higher than that in para-cancerous tissues (0.19 ± 0.04, 0.12 ± 0.02). The relative levels of HSP 70-2 mRNA in the subjects with AA genotype (1.32 ± 0.22) were significantly higher than the patients with AG genotype or GG genotype (0.95 ± 0.17, 0.70 ± 0.16) at the site of 1267 (A/G) (P < 0.01); however, the relative protein levels of HSP 70 were 0.47 ± 0.13 (AA genotype), 0.42 ± 0.11 (AG genotype), 0.45 ± 0.11 (GG genotype), respectively, which showed no statistically significant difference (P > 0.05). The polymorphism of HSP 70-2 1267 (A/G) is highly associated with the transcription level of HSP 70-2 mRNA, but not with the expression level of HSP 70 protein.

Rapid, transient, and dose-dependent expression of Hsp70 messenger RNA in the rat brain after morphine treatment

PubMed Central

Ammon-Treiber, Susanne; Grecksch, Gisela; Stumm, Ralf; Riechert, Uta; Tischmeyer, Helga; Reichenauer, Anke; Höllt, Volker

2004-01-01

Induction of Hsp70 in the brain has been reported after intake of drugs of abuse like amphetamine and lysergic acid diethylamide. In this investigation, gene expression of Hsp70 and other heat shock genes in the rat brain was studied in response to morphine. Twenty milligrams per kilogram morphine intraperitoneally resulted in a marked induction of Hsp70 messenger RNA (mRNA) expression in the frontal cortex with a maximum increase of 13.2-fold after 2 hours. A moderate increase of Hsp27 mRNA expression (6.7-fold) could be observed after 4 hours, whereas mRNA expression of Hsp90 and of the constitutive Hsc70 did not exceed a mean factor of 1.8-fold during the 24 hours interval. The increase in Hsp70 mRNA was dose dependent, showing a significant elevation after doses ranging from 10 to 50 mg/kg morphine. In situ hybridization revealed enhanced Hsp70 mRNA expression mainly in cortical areas, in the hippocampus, in the paraventricular and supraoptic nuclei of the hypothalamus, in the locus coeruleus, as well in the pineal body. The double in situ hybridization technique revealed increased Hsp70 mRNA expression mainly in VGLUT1-positive neurons and to a lesser extent in olig1-positive oligodendroglia. Immunohistochemistry revealed a marked increase of Hsp70 protein in neuronal cells and blood vessels after 12 hours. In contrast to animal experiments, morphine did not increase Hsp70 mRNA expression in vitro in μ-opioid receptor (MOR1)–expressing human embryonic kidney 293 cells, suggesting no direct MOR1-mediated cellular effect. To exclude a body temperature–related morphine effect on Hsp70 mRNA expression, the temperature was recorded. Five to 20 mg/kg resulted in hyperthermia (maximum 40.6°), whereas a high dose (50 mg/kg) that produced the highest mRNA induction, showed a clear hypothermia (minimum 37.2°C). These findings argue against the possibility that Hsp70 induction by morphine is caused by its effect on body temperature. It may be speculated that

Syntenic conservation of HSP70 genes in cattle and humans

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

Grosz, M.D.; Womack, J.E.; Skow, L.C.

1992-12-01

A phage library of bovine genomic DNA was screened for hybridization with a human HSP70 cDNA probe, and 21 positive plaques were identified and isolated. Restriction mapping and blot hybridization analysis of DNA from the recombinant plaques demonstrated that the cloned DNAs were derived from three different regions of the bovine genome. Ore region contains two tandemly arrayed HSP70 sequences, designated HSP70-1 and HSP70-2, separated by approximately 8 kb of DNA. Single HSP70 sequences, designated HSP70-3 and HSP70-4, were found in two other genomic regions. Locus-specific probes of unique flanking sequences from representative HSP70 clones were hybridized to restriction endonuclease-digestedmore » DNA from bovine-hamster and bovine-mouse somatic cell hybrid panels to determine the chromosomal location of the HSP70 sequences. The probe for the tandemly arrayed HSP70-1 and HSP70-2 sequences mapped to bovine chromosome 23, syntenic with glyoxalase 1, 21 steroid hydroxylase, and major histocompatibility class I loci. HSP70-3 sequences mapped to bovine chromosome 10, syntenic with nucleoside phosphorylase and murine osteosarcoma viral oncogene (v-fos), and HSP70-4 mapped to bovine syntenic group U6, syntenic with amylase 1 and phosphoglucomutase 1. On the basis of these data, the authors propose that bovine HSP70-1,2 are homologous to human HSPA1 and HSPA1L on chromosome 6p21.3, bovine HSP70-3 is the homolog of an unnamed human HSP70 gene on chromosome 14q22-q24, and bovine HSP70-4 is homologous to one of the human HSPA-6,-7 genes on chromosome 1. 34 refs., 2 figs., 1 tab.« less

Imbalance of Hsp70 family variants fosters tau accumulation.

PubMed

Jinwal, Umesh K; Akoury, Elias; Abisambra, Jose F; O'Leary, John C; Thompson, Andrea D; Blair, Laura J; Jin, Ying; Bacon, Justin; Nordhues, Bryce A; Cockman, Matthew; Zhang, Juan; Li, Pengfei; Zhang, Bo; Borysov, Sergiy; Uversky, Vladimir N; Biernat, Jacek; Mandelkow, Eckhard; Gestwicki, Jason E; Zweckstetter, Markus; Dickey, Chad A

2013-04-01

Dysfunctional tau accumulation is a major contributing factor in tauopathies, and the heat-shock protein 70 (Hsp70) seems to play an important role in this accumulation. Several reports suggest that Hsp70 proteins can cause tau degradation to be accelerated or slowed, but how these opposing activities are controlled is unclear. Here we demonstrate that highly homologous variants in the Hsp70 family can have opposing effects on tau clearance kinetics. When overexpressed in a tetracycline (Tet)-based protein chase model, constitutive heat shock cognate 70 (Hsc70) and inducible Hsp72 slowed or accelerated tau clearance, respectively. Tau synergized with Hsc70, but not Hsp72, to promote microtubule assembly at nearly twice the rate of either Hsp70 homologue in reconstituted, ATP-regenerating Xenopus extracts supplemented with rhodamine-labeled tubulin and human recombinant Hsp72 and Hsc70. Nuclear magnetic resonance spectroscopy with human recombinant protein revealed that Hsp72 had greater affinity for tau than Hsc70 (I/I0 ratio difference of 0.3), but Hsc70 was 30 times more abundant than Hsp72 in human and mouse brain tissue. This indicates that the predominant Hsp70 variant in the brain is Hsc70, suggesting that the brain environment primarily supports slower tau clearance. Despite its capacity to clear tau, Hsp72 was not induced in the Alzheimer's disease brain, suggesting a mechanism for age-associated onset of the disease. Through the use of chimeras that blended the domains of Hsp72 and Hsc70, we determined that the reason for these differences between Hsc70 and Hsp72 with regard to tau clearance kinetics lies within their C-terminal domains, which are essential for their interactions with substrates and cochaperones. Hsp72 but not Hsc70 in the presence of tau was able to recruit the cochaperone ubiquitin ligase CHIP, which is known to facilitate the ubiquitination of tau, describing a possible mechanism of how the C-termini of these homologous Hsp70 variants

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.

Suppression of Melanin Production by Expression of HSP70*

PubMed Central

Hoshino, Tatsuya; Matsuda, Minoru; Yamashita, Yasuhiro; Takehara, Masaya; Fukuya, Masayo; Mineda, Kazutaka; Maji, Daisuke; Ihn, Hironobu; Adachi, Hiroaki; Sobue, Gen; Funasaka, Yoko; Mizushima, Tohru

2010-01-01

Skin hyperpigmentation disorders due to abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and cosmetic problem. UV irradiation stimulates melanin production in melanocytes by increasing intracellular cAMP. Expression of heat shock proteins (HSPs), especially HSP70, is induced by various stressors, including UV irradiation, to provide cellular resistance to such stressors. In this study we examined the effect of expression of HSP70 on melanin production both in vitro and in vivo. 3-Isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, stimulated melanin production in cultured mouse melanoma cells, and this stimulation was suppressed in cells overexpressing HSP70. IBMX-dependent transcriptional activation of the tyrosinase gene was also suppressed in HSP70-overexpressing cells. Expression of microphthalmia-associated transcription factor (MITF), which positively regulates transcription of the tyrosinase gene, was up-regulated by IBMX; however, this up-regulation was not suppressed in HSP70-overexpressing cells. On the other hand, immunoprecipitation and immunostaining analyses revealed a physical interaction between and co-localization of MITF and HSP70, respectively. Furthermore, the transcription of tyrosinase gene in nuclear extract was inhibited by HSP70. In vivo, UV irradiation of wild-type mice increased the amount of melanin in the basal layer of the epidermis, and this increase was suppressed in transgenic mice expressing HSP70. This study provides the first evidence of an inhibitory effect of HSP70 on melanin production both in vitro and in vivo. This effect seems to be mediated by modulation of MITF activity through a direct interaction between HSP70 and MITF. PMID:20177067

Carboxy terminus of heat shock protein (HSP) 70-interacting protein (CHIP) inhibits HSP70 in the heart.

PubMed

Zhao, Bijun; Sun, Guocheng; Feng, Guanli; Duan, Weixun; Zhu, Xiaoling; Chen, Shaoyang; Hou, Lichao; Jin, Zhenxiao; Yi, Dinghua

2012-12-01

Heat shock protein (HSP) 70 plays a critical role in protecting the heart from various stressor-induced cell injuries; the mechanism remains to be further understood. The present study aims to elucidate the effect of a probiotics-derived protein, LGG-derived protein p75 (LGP), in alleviating the ischemia/reperfusion (I/R)-induced heart injury. We treated rats with the I/R with or without preadministration with LGP. The levels of HSP70 and carboxy terminus of HSP70-interacting protein (CHIP) in the heart tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of CHIP on suppression of HSP70 and the effect of LGP on suppression of CHIP were investigated with an I/R rat model and a cell culture model. The results showed that I/R-induced infarction in the heart could be alleviated by pretreatment with LGP. HSP70 was detected in naïve rat heart tissue extracts. I/R treatment significantly suppressed the level of HSP70 and increased the levels of CHIP in the heart. A complex of CHIP/HSP70 was detected in heart tissue extracts. The addition of recombinant CHIP to culture inhibited HSP70 in heart cells. LGP was bound CHIP in heart cells and prevented the CHIP from binding HSP70. In summary, I/R can suppress HSP70 and increase CHIP in heart cells. CHIP can suppress HSP70 that can be prevented by pretreatment with LGP. The results imply that CHIP may be a potential target in the prevention of I/R-induced heart cell injury.

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

Induction of HSP70 promotes DeltaF508 CFTR trafficking.

PubMed

Choo-Kang, L R; Zeitlin, P L

2001-07-01

The DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is a temperature-sensitive trafficking mutant that is detected as an immature 160-kDa form (band B) in gel electrophoresis. The goal of this study was to test the hypothesis that HSP70, a member of the 70-kDa heat shock protein family, promotes DeltaF508 CFTR processing to the mature 180-kDa form (band C). Both pharmacological and genetic techniques were used to induce HSP70. IB3-1 cells were treated with sodium 4-phenylbutyrate (4PBA) to promote maturation of DeltaF508 CFTR to band C. A dose-dependent increase in band C and total cellular HSP70 was observed. Under these conditions, HSP70-CFTR complexes were increased and 70-kDa heat shock cognate protein-CFTR complexes were decreased. Increased DeltaF508 CFTR maturation was also seen after transfection with an HSP70 expression plasmid and exposure to glutamine, an inducer of HSP70. With immunofluorescence techniques, the increased appearance of CFTR band C correlated with CFTR distribution beyond the perinuclear regions. These data suggest that induction of HSP70 promotes DeltaF508 CFTR maturation and trafficking.

A Protective Hsp70-TLR4 Pathway in Lethal Oxidant Lung Injury

PubMed Central

Zhang, Yi; Zhang, Xuchen; Shan, Peiying; Hunt, Clayton R.; Pandita, Tej K.; Lee, Patty J.

2013-01-01

Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. However, prolonged exposures can exacerbate respiratory failure. Our previous study showed that toll-like receptor 4 (TLR4) confers protection against hyperoxia-induced lung injury and mortality. Hsp70 has potent cytoprotective properties and has been described as a TLR4 ligand in cell lines. We sought to elucidate the relationship between TLR4 and Hsp70 in hyperoxia-induced lung injury in vitro and in vivo and to define the signaling mechanisms involved. Wild type, TLR4−/− and Trif−/− (a TLR4 adapter protein) murine lung endothelial cells (MLEC) were exposed to hyperoxia. We found markedly elevated levels of intracellular and secreted Hsp70 from mice lung and MLEC after hyperoxia. We confirmed that Hsp70 and TLR4 co-immunoprecipitate in lung tissue and MLEC. Hsp70-mediated NFκB activation appears to depend upon TLR4. In the absence of TLR4, Hsp70 loses its protective effects in endothelial cells. Furthermore, these protective properties of Hsp70 are TLR4 adapter Trif-dependent, MyD88-independent. Hsp70-deficient mice have increased mortality during hyperoxia and lung-targeted adenoviral delivery of Hsp70 effectively rescues both Hsp70-deficient and wild type mice. Our studies are the first to define an Hsp70-TLR4-Trif cytoprotective axis in the lung and endothelial cells. This pathway is a potential therapeutic target against a range of oxidant-induced lung injuries. PMID:23817427

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

PubMed

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

2016-08-01

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

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

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

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

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.

The role of heat shock protein 70 (Hsp70) in radiation-induced immunomodulation.

PubMed

Multhoff, Gabriele; Pockley, Alan G; Schmid, Thomas E; Schilling, Daniela

2015-11-28

Despite enormous progress in radiation technologies (high precision image-guided irradiation, proton irradiation, heavy ion irradiation) and radiotherapeutic concepts (hypofractionated irradiation schemes), the clinical outcome of radiotherapy in locally advanced and metastasized tumors and in hypoxic tumors which are radiation-resistant remains unsatisfactory. Given their key influence on a number of biological and immunological parameters, this article considers the influence of irradiation-induced stress proteins on radiation-induced immunomodulation. Depending on its location, the major stress-inducible Heat shock protein 70 (Hsp70) has been found to fulfill multiple roles. On the one hand, increased intracellular Hsp70 levels have been found to play a key role in the recovery from stress such as radio(chemo)therapy, and on the other hand extracellular Hsp70 proteins are potent stimulators of the innate immune system and mediators of anti-tumor immunity. Furthermore, if loaded with tumor-derived peptides, members of the Heat Shock Protein 70 (HSP70) and 90 (HSP90) families can stimulate the adaptive immune system via antigen cross-presentation. An irradiation-induced enhancement of the selective expression of a membrane form of Hsp70 on the surface of tumor cells which can act as a recognition structure for activated NK cells might have significant clinical relevance, in that the outcome of irradiation therapy for advanced tumors could be improved by combining it with cell-based and other immunotherapies that target this membrane form of Hsp70. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Influence of Hsp70 and HLA-E on the killing of leukemic blasts by cytokine/Hsp70 peptide-activated human natural killer (NK) cells.

PubMed

Stangl, Stefan; Gross, Catharina; Pockley, Alan G; Asea, Alexzander A; Multhoff, Gabriele

2008-01-01

This study compared the effects of the human 70-kDa stress protein (Hsp70) peptide, TKDNNLLGRFELSG (TKD), proinflammatory cytokines, or a combination of both on the repertoire of receptors expressed by human natural killer (NK) cells and their capacity to kill human CX colon carcinoma cells, K562 erythroleukemic cells, and leukemic blasts from two patients with acute myelogenous leukemia. Low-dose interleukin (IL) 2/IL-15 and TKD increase the expression density of activatory (NKG2D, NKp30, NKp44, NKp46, CD94/NKG2C) and inhibitory (CD94/NKG2A) receptors on NK cells. Concomitantly, IL-2/TKD treatment enhances the cytotoxicity of NK cells (as reflected by their secretion of granzyme B) against Hsp70 membrane-positive and human leukocyte antigen (HLA)-E membrane-negative (Hsp70(+)/HLA-E(-)) CX(+) and K562 cells. However, it had no effect on the responsiveness to Hsp70(-)/HLA-E(-) CX(-) cells over that induced by IL-2 alone. The cytotoxicity of IL-2/TKD-activated, purified NK cells and peripheral blood mononuclear cells against Hsp70(+)/HLA-E(+) leukemic blasts was weaker than that against Hsp70(+)/HLA-E(-) K562 cells. Hsp70-blocking and HLA-E transfection experiments confirmed membrane-bound Hsp70 as being a recognition/activatory ligand for NK cells, as cytotoxicity was reduced by the presence of the anti-Hsp70 monoclonal antibody cmHsp70.2 and by inhibiting Hsp70 synthesis using short interference ribonucleic acid. HLA-E was confirmed as an inhibitory ligand, as the extent of NK cell-mediated lysis of K562 cell populations that had been transfected with HLA-E(R) or HLA-E(G) alleles was dependent on the proportion of HLA-E-expressing cells. These findings indicate that Hsp70 (as an activatory molecule) and HLA-E (as an inhibitory ligand) expression influence the susceptibility of leukemic cells to the cytolytic activities of cytokine/TKD-activated NK cells.

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.

The role of heat shock protein 70 in mediating age-dependent mortality in sepsis.

PubMed

McConnell, Kevin W; Fox, Amy C; Clark, Andrew T; Chang, Nai-Yuan Nicholas; Dominguez, Jessica A; Farris, Alton B; Buchman, Timothy G; Hunt, Clayton R; Coopersmith, Craig M

2011-03-15

Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6- to 12-wk-old) and aged (16- to 17-mo-old) HSP70(-/-) and wild-type (WT) mice to determine whether HSP70 modulated outcome in an age-dependent fashion. Young HSP70(-/-) and WT mice subjected to cecal ligation and puncture, Pseudomonas aeruginosa pneumonia, or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70(-/-) mice than aged WT mice subjected to cecal ligation and puncture (p = 0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared with WT mice, aged septic HSP70(-/-) mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70(-/-) mice had increased systemic levels of TNF-α, IL-6, IL-10, and IL-1β compared with WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged, but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation.

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

PubMed Central

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

1996-01-01

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

Comparative baseline levels of mercury, Hsp 70 and Hsp 60 in subsistence fish from the Yukon-Kuskokwim delta region of Alaska.

PubMed

Duffy, L K; Scofield, E; Rodgers, T; Patton, M; Bowyer, R T

1999-10-01

In subsistence fish; northern pike (Esox lucius), burbot (Lota lota), whitefish (Coregonus nelsoni), grayling (Thymallus arcticus) and sheefish (Stenodus lencichthys), we determined the Hsp 60 and Hsp 70 levels in 31 samples from adult fish gills. A dot-blot analysis using antibodies to either Hsp 70 or Hsp 60 showed the average Hsp 70 concentration was 9.1 microg/mg protein, while the average Hsp 60 concentration was 147.4 microg/mg protein. Mercury levels in muscle tissue in these fish averaged 0.382 ppm. Using a subset of samples (n = 24), we determined that the major component in the muscle of Alaskan subsistence fish was methyl mercury. No correlation was observed between Hsp 60 or Hsp 70 expression in gill tissue and mercury concentrations in muscle tissue. Hsp 60 and Hsp 70 protein levels in the gills were correlated.

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

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.

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

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

PubMed

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

2017-09-19

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

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

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.

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

Inner ear supporting cells protect hair cells by secreting HSP70

PubMed Central

May, Lindsey A.; Kramarenko, Inga I.; Brandon, Carlene S.; Voelkel-Johnson, Christina; Roy, Soumen; Truong, Kristy; Francis, Shimon P.; Monzack, Elyssa L.; Lee, Fu-Shing; Cunningham, Lisa L.

2013-01-01

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug–induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70–/– mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress. PMID:23863716

Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

PubMed Central

Wisniewska, Magdalena; Karlberg, Tobias; Lehtiö, Lari; Johansson, Ida; Kotenyova, Tetyana; Moche, Martin; Schüler, Herwig

2010-01-01

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. PMID:20072699

Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

PubMed

Wisniewska, Magdalena; Karlberg, Tobias; Lehtiö, Lari; Johansson, Ida; Kotenyova, Tetyana; Moche, Martin; Schüler, Herwig

2010-01-11

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Therapeutic inducers of the HSP70/HSP110 protect mice against traumatic brain injury.

PubMed

Eroglu, Binnur; Kimbler, Donald E; Pang, Junfeng; Choi, Justin; Moskophidis, Demetrius; Yanasak, Nathan; Dhandapani, Krishnan M; Mivechi, Nahid F

2014-09-01

Traumatic brain injury (TBI) induces severe harm and disability in many accident victims and combat-related activities. The heat-shock proteins Hsp70/Hsp110 protect cells against death and ischemic damage. In this study, we used mice deficient in Hsp110 or Hsp70 to examine their potential requirement following TBI. Data indicate that loss of Hsp110 or Hsp70 increases brain injury and death of neurons. One of the mechanisms underlying the increased cell death observed in the absence of Hsp110 and Hsp70 following TBI is the increased expression of reactive oxygen species-induced p53 target genes Pig1, Pig8, and Pig12. To examine whether drugs that increase the levels of Hsp70/Hsp110 can protect cells against TBI, we subjected mice to TBI and administered Celastrol or BGP-15. In contrast to Hsp110- or Hsp70i-deficient mice that were not protected following TBI and Celastrol treatment, there was a significant improvement of wild-type mice following administration of these drugs during the first week following TBI. In addition, assessment of neurological injury shows significant improvement in contextual and cued fear conditioning tests and beam balance in wild-type mice that were treated with Celastrol or BGP-15 following TBI compared to TBI-treated mice. These studies indicate a significant role of Hsp70/Hsp110 in neuronal survival following TBI and the beneficial effects of Hsp70/Hsp110 inducers toward reducing the pathological consequences of TBI. Our data indicate that loss of Hsp110 or Hsp70 in mice increases brain injury following TBI. (a) One of the mechanisms underlying the increased cell death observed in the absence of these Hsps following TBI is the increased expression of ROS-induced p53 target genes known as Pigs. In addition, (b) using drugs (Celastrol or BGP-15) to increase Hsp70/Hsp110 levels protect cells against TBI, suggesting the beneficial effects of Hsp70/Hsp110 inducers to reduce the pathological consequences of TBI. © 2014 International Society

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.

Leptin downregulates heat shock protein-70 (HSP-70) gene expression in chicken liver and hypothalamus.

PubMed

Figueiredo, Denise; Gertler, Arieh; Cabello, Gérard; Decuypere, Eddy; Buyse, Johan; Dridi, Sami

2007-07-01

Heat shock protein (HSP)-70 is expressed in normal and stressed cells but is highly stress-inducible. Although leptin has long been suggested to be involved in the regulation of stress response, its interaction with the HSP-70 gene is still unknown, under both unstressed and stressed conditions. The present study has aimed to investigate the effect of leptin on HSP-70 gene expression in normal chicken liver, hypothalamus, and muscle. Continuous infusion of recombinant chicken leptin (8 mug/kg per hour) at a constant rate of 3 ml/h for 6 h in 3-week-old broiler chickens significantly (P < 0.05) decreased food intake and HSP-70 mRNA levels in liver and hypothalamus, but not in muscle. In an attempt to discriminate between the effect of leptin and of leptin-reduced food intake on HSP-70 gene expression, we also evaluated the effect of food deprivation on the same cellular responses in two broiler chicken lines genetically selected for low (LL) or high (FL) abdominal fat pad size. Food deprivation for 16 h did not affect HSP-70 gene expression in any of the studied tissues indicating that the effect of leptin was independent of the inhibition of food intake. Regardless of the nutritional status, HSP-70 mRNA levels were significantly (P < 0.05) higher in the hypothalamus of FL compared with LL chickens consistent with higher mRNA levels for hypothalamic corticotropin-releasing factor. To assess, whether the effects of leptin were direct or indirect, we carried out in vitro studies. Leptin treatments did not affect HSP-70 mRNA levels in a leghorn male hepatoma cell line or quail myoblast cell line suggesting that the effect of leptin on HSP-70 gene expression is mediated through the central nervous system. Furthermore, HSP-70 gene expression was gender-dependent with significantly (P < 0.05) higher levels in male than in female chickens.

Induction of hsp70, hsp60, hsp83 and hsp26 and oxidative stress markers in benzene, toluene and xylene exposed Drosophila melanogaster: Role of ROS generation

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

Singh, Mahendra Pratap; Reddy, M.M. Krishna.; Mathur, N.

2009-03-01

Exposure to benzene, toluene and xylene in the human population may pose a health risk. We tested a working hypothesis that these test chemicals cause cellular toxicity to a non-target organism, Drosophila melanogaster. Third instar larvae of D. melanogaster transgenic for hsp70, hsp83 and hsp26 and Oregon R{sup +} strain were exposed to 1.0-100.0 mM benzene, toluene and xylene for 2-48 h to examine the heat shock proteins (hsps), ROS generation, anti-oxidant stress markers and developmental end points. The test chemicals elicited a concentration- and time-dependent significant (p < 0.01) induction of the hsps in the exposed organism in themore » order of hsp70 > hsp83 {>=} hsp26 as evident by {beta}-galactosidase activity after 24 h. RT-PCR amplification studies in Oregon R{sup +} larvae revealed a similar induction pattern of these genes along with hsp60 in the order of hsp70 > hsp60 > hsp26 {>=} hsp83. Under similar experimental conditions, a significant induction of ROS generation and oxidative stress markers viz. superoxide dismutase, catalase, glutathione S-transferase, thioredoxin reductase, glutathione, malondialdehyde and protein carbonyl content was observed. Sub-organismal response was propagated towards organismal response i.e., a delay in the emergence of flies and their reproductive performance. While hsp70 was predominantly induced in the organism till 24 h of treatment with the test chemicals, a significant or insignificant regression of Hsp70 after 48 h was concurrent with a significant induction (p < 0.01) of hsp60 > hsp83 {>=} hsp26 in comparison to the former. A significant positive correlation was observed between ROS generation and these hsps in the exposed organism till 24 h and a negative correlation between ROS generation and hsp70 in them after 48 h indicating a modulatory role of ROS in the induction of hsps. The study suggests that among the tested hsps, hsp70 may be used as an early bioindicator of cellular toxicity against benzene

The study of the relationship between aberrant expression of hot shock protein 70 (HSP70) and spontaneous abortion.

PubMed

Peng, Y-B; Liu, H; Huang, S-H; Lai, H; Zhou, Q; Luo, Y; Zhang, Z-Y; Xi, B-R; Ouyang, X

2017-02-01

The present study is aimed to explore the relationship between aberrant expression of heat shock protein 70 (HSP) and spontaneous abortion. 50 patients with spontaneous abortion and 50 patients with induced abortion were continuously selected based on the nearest matching principle, and the proportion of age and gestational age was 1:1. The decidual tissues were obtained, and the cell apoptosis was determined by TUNEL assay. Further, the expression of HSP70 was assayed by immune-histochemical staining, and the expression of HSP70 mRNA was detected by the RT-PCR approach. Apoptosis rate, HSP70 expression and HSP70 mRNA expression in the observation group were significantly higher than the control group. HSP70 might induce apoptosis so as to cause spontaneous abortion.

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

Heat shock protein 70.1 (Hsp70.1) affects neuronal cell fate by regulating lysosomal acid sphingomyelinase.

PubMed

Zhu, Hong; Yoshimoto, Tanihiro; Yamashima, Tetsumori

2014-10-03

The inducible expression of heat shock protein 70.1 (Hsp70.1) plays cytoprotective roles in its molecular chaperone function. Binding of Hsp70 to an endolysosomal phospholipid, bis(monoacylglycero)phosphate (BMP), has been recently shown to stabilize lysosomal membranes by enhancing acid sphingomyelinase (ASM) activity in cancer cells. Using the monkey experimental paradigm, we have reported that calpain-mediated cleavage of oxidized Hsp70.1 causes neurodegeneration in the hippocampal cornu ammonis 1 (CA1), whereas expression of Hsp70.1 in the motor cortex without calpain activation contributes to neuroprotection. However, the molecular mechanisms of the lysosomal destabilization/stabilization determining neuronal cell fate have not been elucidated. To elucidate whether regulation of lysosomal ASM could affect the neuronal fate, we analyzed Hsp70.1-BMP binding and ASM activity by comparing the motor cortex and the CA1. We show that Hsp70.1 being localized at the lysosomal membrane, lysosomal lipid BMP levels, and the lipid binding domain of Hsp70.1 are crucial for Hsp70.1-BMP binding. In the postischemic motor cortex, Hsp70.1 being localized at the lysosomal membrane could bind to BMP without calpain activation and decreased BMP levels, resulting in increasing ASM activity and lysosomal stability. However, in the postischemic CA1, calpain activation and a concomitant decrease in the lysosomal membrane localization of Hsp70.1 and BMP levels may diminish Hsp70.1-BMP binding, resulting in decreased ASM activity and lysosomal rupture with leakage of cathepsin B into the cytosol. A TUNEL assay revealed the differential neuronal vulnerability between the CA1 and the motor cortex. These results suggest that regulation of ASM activation in vivo by Hsp70.1-BMP affects lysosomal stability and neuronal survival or death after ischemia/reperfusion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

HSP-70 as a nonspecific early marker in cisplatin ototoxicity.

PubMed

Ramírez-Camacho, R; Citores, M J; Trinidad, A; Verdaguer, J M; García-Berrocal, J R; Marero, A Martín; Puente, A; González-García, J A; Vargas, J A

2007-06-01

The great variety of pathological entities related to the presence of circulating HSP-70 suggests a nonspecific cellular damage. As the present study shows, positive results decrease with respect to the time elapsed after the injection of the ototoxic agent. HSP-70 appears as an early and transient marker that could permit early detection of inner ear damage. The aim of this study was to determine the presence of HSP-70 at different time points by means of Western blot immunoassay in the sera of rats treated with cisplatin. Thirty-six Wistar rats were intraperitoneally injected with cisplatin at a dose of 5 mg/kg and blood samples were collected at 7 and 90 days. Determination of HSP-70 was made by means of a modified Western blot immunoassay kit originally used for human HSP-70 antigen detection. A control group of 18 animals was used for comparison. Western blot was positive in 77.8% of the animals in the 7 days group, decreasing to a 44.4% in the 90 days group. In the control group, Western blot was positive in 5.5%.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-07-01

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

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members.

PubMed

Heinen, R C; Diniz-Mendes, L; Silva, J T; Paschoalin, V M F

2006-11-01

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

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

Hsp70 Regulates Immune Response in Experimental Autoimmune Encephalomyelitis

PubMed Central

Mansilla, M. José; Costa, Carme; Eixarch, Herena; Tepavcevic, Vanja; Castillo, Mireia; Martin, Roland; Lubetzki, Catherine; Aigrot, Marie-Stéphane; Montalban, Xavier; Espejo, Carmen

2014-01-01

Heat shock protein (Hsp)70 is one of the most important stress-inducible proteins. Intracellular Hsp70 not only mediates chaperone-cytoprotective functions but can also block multiple steps in the apoptosis pathway. In addition, Hsp70 is actively released into the extracellular milieu, thereby promoting innate and adaptive immune responses. Thus, Hsp70 may be a critical molecule in multiple sclerosis (MS) pathogenesis and a potential target in this disease due to its immunological and cytoprotective functions. To investigate the role of Hsp70 in MS pathogenesis, we examined its immune and cytoprotective roles using both in vitro and in vivo experimental procedures. We found that Hsp70.1-deficient mice were more resistant to developing experimental autoimmune encephalomyelitis (EAE) compared with their wild-type (WT) littermates, suggesting that Hsp70.1 plays a critical role in promoting an effective myelin oligodendrocyte glycoprotein (MOG)-specific T cell response. Conversely, Hsp70.1-deficient mice that developed EAE showed an increased level of autoreactive T cells to achieve the same production of cytokines compared with the WT mice. Although a neuroprotective role of HSP70 has been suggested, Hsp70.1-deficient mice that developed EAE did not exhibit increased demyelination compared with the control mice. Accordingly, Hsp70 deficiency did not influence the vulnerability to apoptosis of oligodendrocyte precursor cells (OPCs) in culture. Thus, the immunological role of Hsp70 may be relevant in EAE, and specific therapies down-regulating Hsp70 expression may be a promising approach to reduce the early autoimmune response in MS patients. PMID:25153885

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.

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin

PubMed Central

Ciocca, Daniel R.; Rozados, Viviana R.; Carrión, F. Darío Cuello; Gervasoni, Silvia I.; Matar, Pablo; Scharovsky, O. Graciela

2003-01-01

Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in

HSP70 expression in Biomphalaria glabrata snails exposed to cadmium.

PubMed

da Silva Cantinha, Rebeca; Borrely, Sueli Ivone; Oguiura, Nancy; de Bragança Pereira, Carlos Alberto; Rigolon, Marcela M; Nakano, Eliana

2017-06-01

In this study, the effects of the heavy metal cadmium on the stress protein HSP70 are investigated in freshwater mollusks Biomphalaria glabrata. Adult snails were exposed for 96h to CdCl 2 at concentrations ranging from 0.09 to 0.7mgL -1 (LC 50/96h =0.34 (0.30-0.37). Time and concentration-dependent increases in the expression of HSP70 were observed at sub-lethal levels in the immunoblotting assay. Further, an increased survival to a lethal heat shock was observed in animals pre-exposed to a nonlethal concentration of cadmium, evidencing the induction of acquired tolerance. The present study demonstrated the inducibility of B. glabrata HSP70 by cadmium, a relevant environmental contaminant, at non-lethal levels, providing evidences that the assessment of HSP70 in B. glabrata can be regarded as a suitable biomarker for ecotoxicological studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Using Lymphocyte and Plasma Hsp70 as Biomarkers for Assessing Coke Oven Exposure among Steel Workers

PubMed Central

Yang, Xiaobo; Zheng, Jinping; Bai, Yun; Tian, Fengjie; Yuan, Jing; Sun, Jianya; Liang, Huashan; Guo, Liang; Tan, Hao; Chen, Weihong; Tanguay, Robert M.; Wu, Tangchun

2007-01-01

Background Hsp70, an early-response protein induced when organisms are confronted with simple or complicated environmental stresses, can act as either a cellular protector or a danger signal. Objectives The goal of this study was to evaluate levels of lymphocyte and/or plasma Hsp70 as biomarkers for assessing exposure response to complex coke oven emissions (COEs). Methods We recruited 101 coke oven workers and determined levels of polycyclic aromatic hydrocarbon (PAH) exposure, urinary 1-hydroxypyrene (1-OHP), genotoxic damage by comet assay and micronuclei test, and other markers of damage, including plasma malondialdehyde (MDA) and lactate dehydrogenase (LDH). These were compared to levels of lymphocyte (intra-cellular) and plasma (extracellular) Hsp70 using Western blots and enzyme-linked immunosorbent assays (ELISA), respectively. Results We observed a COEs-related dose-dependent increase in levels of DNA damage, micronuclei rate, MDA concentration, and LDH activity. Lymphocyte Hsp70 levels increased in the intermediate-exposure group (1.39 ± 0.88) but decreased in the high-exposure group (1.10 ± 0.55), compared with the low-exposure group. In contrast, plasma Hsp70 levels progressively increased as the dose of exposure increased. Negative correlations were seen between lymphocyte Hsp70 levels and olive tail moment and LDH activity in the intermediate- and high-exposure groups. However, we observed positive correlations between plasma Hsp70 levels and LDH activity in the low and intermediate groups. Conclusions In workers exposed to COEs, high lymphocyte Hsp70 levels may provide protection and high plasma Hsp70 levels may serve as a danger marker. Larger validation studies are needed to establish the utility of Hsp70 as a response marker. PMID:18007987

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.

Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

PubMed Central

Beck, Ilse M.; Drebert, Zuzanna J.; Hoya-Arias, Ruben; Bahar, Ali A.; Devos, Michael; Clarisse, Dorien; Desmet, Sofie; Bougarne, Nadia; Ruttens, Bart; Gossye, Valerie; Denecker, Geertrui; Lievens, Sam; Bracke, Marc; Tavernier, Jan; Declercq, Wim; Gevaert, Kris; Berghe, Wim Vanden; Haegeman, Guy; De Bosscher, Karolien

2013-01-01

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA’s anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells. PMID:23935933

Western blot immunoassay for HSP-70 antibodies in idiopathic tinnitus: a preliminary report.

PubMed

Savastano, Marina; Celadin, Marilena; Pittoni, Marina; Plebani, Mario; Marioni, Gino

2006-03-01

Our preliminary study investigated the role of nonspecific immunologic tests and immunoassay for heat shock protein 70 (HSP-70) in supporting the possibility of an autoimmune inner ear process determining idiopathic tinnitus. Thirty-six consecutive patients with idiopathic tinnitus without other otologic or autoimmune diseases and 20 healthy blood donor subjects underwent determinations of circulating immune complexes (CICs) and other nonspecific immunologic factors and immunoassay for HSP-70. The mean CIC values were 4.2 microg/mL in the tinnitus patients and 0.9 microg/mL in the control group (p = .012). Thirteen of the 36 tinnitus patients and none of the control group were HSP-70-positive. Ten of the 13 HSP-70-positive patients had CIC values higher than normal. In the tinnitus group, the mean CIC values were 6.9 microg/mL and 2.6 microg/mL in the HSP-70-positive and -negative subgroups, respectively (p = .024). It may be hypothesized that in a significant number of cases, idiopathic tinnitus could be induced by immune response to inner ear-specific HSP-70.

Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant's schizothoracin (Schizothorax prenanti).

PubMed

Li, Jiuxuan; Zhang, Haibin; Zhang, Xiuyue; Yang, Shiyong; Yan, Taiming; Song, Zhaobin

2015-04-01

Through the RT-PCR and rapid amplification of cDNA ends, two complementary deoxyribonucleic acid (cDNA) clones encoding heat-shock cognate 70 (HSC70, designated Sp-HSC70) and inducible heat-shock protein 70 (HSP70, designated Sp-HSP70) were isolated from the liver of Prenant's schizothoracin (Schizothorax prenanti). The cDNAs were 2344- and 2292-bp in length and contained 1950- and 1932-bp open reading frames, encoded proteins of 649 and 643 amino acids, respectively. Amino acid sequence analysis indicated that both Sp-HSC70 and Sp-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (an ATP-binding site motif, a bipartite nuclear targeting signal), and a cytoplasmic characteristic motif EEVD. Homology analysis revealed that Sp-HSC70 and Sp-HSP70 shared 77.5% identity and Sp-HSC70 shared more than 81.1% identity with the known HSC70s of other vertebrates, while Sp-HSP70 shared more than 77.5 % identity with the known HSP70s of other vertebrates. Fluorescent real-time quantitative RT-PCR showed that Sp-HSC70 and Sp-HSP70 mRNAs were found in all tested tissues, including blood, brain, heart, liver, spleen, head kidney, white muscle, skin, gonad, hypophysis, red muscle, and gill. The Sp-HSC70 and Sp-HSP70 mRNA expression level in blood and head kidney displayed a significant increase in vibrio-challenged group with the bacterium Aeromonas hydrophila at 24 h post-infection compared to a control group. Temporally, there was a clear time-dependent expression pattern of Sp-HSC70 or Sp-HSP70 gene after bacterial challenge, and the expression of Sp-HSC70 and Sp-HSP70 mRNAs reached a maximum level at 12 and 6 h post-challenge, respectively. Both returned to control level after 7 × 24 h. The results suggest that Sp-HSC70 and Sp-HSP70 genes may play important roles in mediating the immune responses of A. hydrophila-related diseases in the Prenant's schizothoracin.

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

Anabolic steroid and gender-dependent modulation of cytosolic HSP70s in fast- and slow-twitch skeletal muscle.

PubMed

González, B; Hernando, R; Manso, R

2000-09-01

Besides their clinical uses, anabolic steroids (AASs) are self-administered by athletes to improve muscle mass and sports performance. The biological basis for their presumed effectiveness at suprapharmacological doses, however, remains uncertain. Since the expression of high levels of some stress proteins (HSPs) has been associated with an increased tolerance to stress and chronic exercise up-regulates HSP72 in skeletal muscle, this investigation was aimed at testing whether the administration of suprapharmacological doses of AASs, either alone or in conjunction with chronic exercise, induced changes in HSP72. Nandrolone decanoate (ND), an estrene derivative, but not stanozolol (ST), a derivative of the androstane series, up-regulated the levels of HSP72 and changed the proportions of various charge variants of the cytosolic HSP70s in sedentary and exercise-trained rats, exclusively in fast-twitch fibres. Since the expression of HSP73-levels in skeletal muscle was dependent on gender but not on muscle type, and that of HSP72-levels was muscle type specific but gender-independent, ND effects on cytosolic HSP70s could not be explained solely by a functional relationship with sex steroids. The reported results indicate that, by up-regulating the expression levels of HSP72 in fast-twitch fibres, nandrolone decanoate could contribute to improving the tolerance of skeletal muscle to high-intensity training.

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

PubMed

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

2014-12-01

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

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

PubMed

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

2009-06-01

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

HSP70 reduces chronic hypoxia-induced neural suppression via regulating expression of syntaxin.

PubMed

Fei, Guanghe; Guo, Conghui; Sun, Hong-Shuo; Feng, Zhong-Ping

2008-01-01

Long-term exposure to modest hypoxia conditions may result in neural dysfunction; however, the involvement of presynaptic proteins has not been tested directly. Here, we reported that adult snails, Lymnaea stagnalis, developed a slow righting movement after placement in low O2 (approximately 5%) for 4 days. Semi-quantitative Western blot analysis showed that hypoxia induced heat shock protein 70 (HSP70) up-regulation and a reduction of syntaxin I. The inducible HSP70 occurs within 6 hours preceding the down-regulation of syntaxin I, suggesting that HSP70 may be involved in regulation of syntaxin expression. Injecting directly double-stranded RNAs (dsRNA) into the center ganglia region, we found that dsRNA HSP70, not the scrambled RNA, prevented the hypoxia-induced HSP70 expression, enhanced the hypoxia-dependent down-regulation of syntaxin I, and aggravated motor suppression. We thus provided the first evidence that early induction of HSP70 by chronic hypoxia is critical for maintaining expression levels of presynaptic proteins and neural function. These findings implicate a new molecular mechanism underlying chronic hypoxia-induced neurobehavioral adaptation and impairment.

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.

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

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.

[Anti-heat shock protein 70 (anti - Hsp 70) antibodies in alcohol use disorder patients].

PubMed

Michalak, Sławomir; Piorunek, Tomasz; Lenart-Jankowska, Danuta; Osztynowicz, Krystyna; Kozubski, Wojciech

2012-01-01

The expression of the most important chaperone protein - Hsp70 and autoimmunity directed against it is a risk factor of cardiovascular diseases, increased in subjects with alcohol use disorder (AUD). The aim of the study was to evaluate the level of anti-Hsp 70 protein antibodies (anti-Hsp 70) in sera of AUD patients during abstinence period. Material and methods. The study included 54 subjects with AUD diagnosed basing on DSM IV criteria. In the studied group clinimetric evaluation was performed, plasma lipids, basic transketolase activity in erythrocytes (TK), thiamine pyrophosphate (TPP) activation of transketolase and the level of anti-Hsp 70 antibodies were evaluated as well. Results. In AUD subjects anti-Hsp 70 level was decreased during abstinence period. During first month of abstinency it correlated negatively with total cholesterol concentration (rS=-0.8857, p=0.0188) and the percentage of TPP stimulation (rS=-0.5960, p<0.05), and during 6 months of abstinence with HDL cholesterol (rS=-0.6848, p=0.0289). After 1 year of abstinence anti-Hsp 70 correlated positively with basic TK activity (rS=0.9550, p=0.0008). Sex is an independent factor influencing anti-Hsp 70 level in AUD subjects (B=60.9469, p=0.0435). In multiple regression model including results of clinimetric evaluation and its effect on the level of anti-Hsp 70 antibodies in AUD patients during 1 month of abstinency anti-Hsp 70 correlated with TWEAK scale score (BETA=-1.4543, p=0.0144) and AUDIT score (BETA-=1.2255, p=0.0224). In 2-6 months of abstinency anti-Hsp 70 correlated with TWEAK score (BETA=1.1110, p=0.0418). After 1 year of abstinency anti-Hsp 70 correlated with AUDIT score (BETA=-1.2161, p=0.0210). Conclusion. The autoimmune reaction against Hsp 70 is decreased during abstinency in AUD patients. Its relation with plasma lipids and thiamine deficiency may lead to increased risk of cardiovascular disorders. TWEAK and AUDIT scoring seem to be most useful for clinimetric evaluation in the

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

PubMed

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

2013-01-01

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

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

PubMed

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

2006-01-01

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

Antarctic marine molluscs do have an HSP70 heat shock response

PubMed Central

Fraser, Keiron P. P.; Peck, Lloyd S.

2008-01-01

The success of any organism depends not only on niche adaptation but also the ability to survive environmental perturbation from homeostasis, a situation generically described as stress. Although species-specific mechanisms to combat “stress” have been described, the production of heat shock proteins (HSPs), such as HSP70, is universally described across all taxa. Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) members, plus GRP78 (glucose-regulated protein, 78 kDa), a related HSP70 family member, were cloned using degenerate polymerase chain reaction (PCR) from two evolutionary divergent Antarctic marine molluscs (Laternula elliptica and Nacella concinna), a bivalve and a gastropod, respectively. The expression of the HSP70 family members was surveyed via quantitative PCR after an acute 2-h heat shock experiment. Both species demonstrated significant up-regulation of HSP70 gene expression in response to increased temperatures. However, the temperature level at which these responses were induced varied with the species (+6–8°C for L. elliptica and +8–10°C for N. concinna) compared to their natural environmental temperature). L. elliptica also showed tissue-specific expression of the genes under study. Previous work on Antarctic fish has shown that they lack the classical heat shock response, with the inducible form of HSP70 being permanently expressed with an expression not further induced under higher temperature regimes. This study shows that this is not the case for other Antarctic animals, with the two molluscs showing an inducible heat shock response, at a level probably set during their temperate evolutionary past. PMID:18347940

Antarctic marine molluscs do have an HSP70 heat shock response.

PubMed

Clark, Melody S; Fraser, Keiron P P; Peck, Lloyd S

2008-01-01

The success of any organism depends not only on niche adaptation but also the ability to survive environmental perturbation from homeostasis, a situation generically described as stress. Although species-specific mechanisms to combat "stress" have been described, the production of heat shock proteins (HSPs), such as HSP70, is universally described across all taxa. Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) members, plus GRP78 (glucose-regulated protein, 78 kDa), a related HSP70 family member, were cloned using degenerate polymerase chain reaction (PCR) from two evolutionary divergent Antarctic marine molluscs (Laternula elliptica and Nacella concinna), a bivalve and a gastropod, respectively. The expression of the HSP70 family members was surveyed via quantitative PCR after an acute 2-h heat shock experiment. Both species demonstrated significant up-regulation of HSP70 gene expression in response to increased temperatures. However, the temperature level at which these responses were induced varied with the species (+6-8 degrees C for L. elliptica and +8-10 degrees C for N. concinna) compared to their natural environmental temperature). L. elliptica also showed tissue-specific expression of the genes under study. Previous work on Antarctic fish has shown that they lack the classical heat shock response, with the inducible form of HSP70 being permanently expressed with an expression not further induced under higher temperature regimes. This study shows that this is not the case for other Antarctic animals, with the two molluscs showing an inducible heat shock response, at a level probably set during their temperate evolutionary past.

Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

PubMed Central

Wiech, Milena; Olszewski, Maciej B.; Tracz-Gaszewska, Zuzanna; Wawrzynow, Bartosz; Zylicz, Maciej; Zylicz, Alicja

2012-01-01

Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes. PMID:23251530

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.

Luminescence diagnostics of conformational changes of the Hsp70 protein in the course of thermal denaturation

NASA Astrophysics Data System (ADS)

Bukina, M. N.; Bakulev, V. M.; Barmasov, A. V.; Zhakhov, A. V.; Ishchenko, A. M.

2015-06-01

The spectral luminescence properties of aqueous solutions of the Hsp70 protein are studied, the dependence of the luminescence spectrum on the excitation wavelength is revealed, and the temperature dependence of luminescence intensity of tyrosine and tryptophan residues in the temperature interval of 20-80° C is analyzed. The luminescence method is used to determine temperature interval (42-57° C) in which protein melting takes place. An increase in the fluorescence quantum yield of tryptophan and the bathochromic shift of the emission spectrum of denatured Hsp70 prove that transition takes place of tryptophanyls to the surface of the protein molecule.

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.

Conserved structure and expression of hsp70 paralogs in teleost fishes.

PubMed

Metzger, David C H; Hemmer-Hansen, Jakob; Schulte, Patricia M

2016-06-01

The cytosolic 70KDa heat shock proteins (Hsp70s) are widely used as biomarkers of environmental stress in ecological and toxicological studies in fish. Here we analyze teleost genome sequences to show that two genes encoding inducible hsp70s (hsp70-1 and hsp70-2) are likely present in all teleost fish. Phylogenetic and synteny analyses indicate that hsp70-1 and hsp70-2 are distinct paralogs that originated prior to the diversification of the teleosts. The promoters of both genes contain a TATA box and conserved heat shock elements (HSEs), but unlike mammalian HSP70s, both genes contain an intron in the 5' UTR. The hsp70-2 gene has undergone tandem duplication in several species. In addition, many other teleost genome assemblies have multiple copies of hsp70-2 present on separate, small, genomic scaffolds. To verify that these represent poorly assembled tandem duplicates, we cloned the genomic region surrounding hsp70-2 in Fundulus heteroclitus and showed that the hsp70-2 gene copies that are on separate scaffolds in the genome assembly are arranged as tandem duplicates. Real-time quantitative PCR of F. heteroclitus genomic DNA indicates that four copies of the hsp70-2 gene are likely present in the F. heteroclitus genome. Comparison of expression patterns in F. heteroclitus and Gasterosteus aculeatus demonstrates that hsp70-2 has a higher fold increase than hsp70-1 following heat shock in gill but not in muscle tissue, revealing a conserved difference in expression patterns between isoforms and tissues. These data indicate that ecological and toxicological studies using hsp70 as a biomarker in teleosts should take this complexity into account. Copyright © 2016 Elsevier Inc. All rights reserved.

OLA1 protects cells in heat shock by stabilizing HSP70

PubMed Central

Mao, R-F; Rubio, V; Chen, H; Bai, L; Mansour, O C; Shi, Z-Z

2013-01-01

The heat-shock response is an evolutionarily conserved cellular defense mechanism against environmental stresses, characterized by the rapid synthesis of heat-shock proteins (HSPs). HSP70, a highly inducible molecular chaperone, assists in refolding or clearance of damaged proteins, thereby having a central role in maintaining intracellular homeostasis and thermotolerance. To date, induction of HSP70 expression has been described extensively at the transcriptional level. However, post-translational regulation of HSP70, such as protein stability, is only partially understood. In this study, we investigated the role of OLA1 (Obg-like ATPase 1), a previously uncharacterized cytosolic ATPase, in regulating the turnover of HSP70. Downregulation of OLA1 in mammalian cells by either RNAi or targeted gene disruption results in reduced steady-state levels of HSP70, impaired HSP70 induction by heat, and functionally, increased cellular sensitivity to heat shock. Conversely, overexpression of OLA1 correlates with elevated HSP70 protein levels and improved thermal resistance. Protein–protein interaction assays demonstrated that binding of OLA1 to the HSP70 carboxyl terminus variable domain hinders the recruitment of CHIP (C-terminus of Hsp70-binding protein), an E3 ubiquitin ligase for HSP70, and thus prevents HSP70 from the CHIP-mediated ubiquitination. These findings suggest a novel molecular mechanism by which OLA1 stabilizes HSP70, leading to upregulation of HSP70 as well as increased survival during heat shock. PMID:23412384

Molecular cloning and characterization of a HSP70 gene from Schistosoma japonicum.

PubMed

Yang, Jie; Yang, Linlin; Lv, Zhiyue; Wang, Juan; Zhang, Qixian; Zheng, Huanqin; Wu, Zhongdao

2012-05-01

Schistosoma japonicum is the pathogen responsible for schistosomiasis japonica, one of the major infectious diseases targeted for prevention nationally in China. Expression of heat shock proteins (HSPs) following stress plays a very important biological role in many organisms including S. japonicum. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. Based on the published sequences in GenBank/EMBL (AF044412.1), open reading frame belonging to HSP70 protein corresponds to a full-length cDNA containing an open reading frame of 1,947 bp encoded of 648 amino acids was identified as HSP70 from schistosome. In this study, the coding region that we named rSj648/hsp70 was amplified from S. japonicum adult worm cDNA library, and the recombinant protein was expressed in vector pET32a(+) and purified using a Ni-NTA purification system. The target protein rSj648/hsp70 was determined by matrix-assisted laser desorption/ionization mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blotting analysis confirmed that Sj648/hsp70 could be expressed in the eggs, normal cercariae, ultraviolet-attenuated cercariae (UVAC), and adult worms of S. japonicum. Real-time quantitative PCR analysis indicated that Sj648/hsp70 was expressed significantly higher in eggs than that in cercariae and adult worms, and the expression in UVAC was higher than that in normal cercariae. A thermotolerance assay showed that rSj648/hsp70 could protect Escherichia coli cells from heat damage. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay demonstrated that mice immunized with rSj648/hsp70 induced higher level of specific anti-rSj648/hsp70 IgG1 compared with those vaccinated with adjuvant alone, indicating that rSj648/hsp70 was able to elicit Th2-type bias immune response. Our results suggest that Sj648/hsp70 might be an

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.

Expression analysis of HSP70 in the testis of Octopus tankahkeei under thermal stress.

PubMed

Long, Ling-Li; Han, Ying-Li; Sheng, Zhang; Du, Chen; Wang, You-Fa; Zhu, Jun-Quan

2015-09-01

The gene encoding heat shock protein 70 (HSP70) was identified in Octopus tankahkeei by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA (2471 bp) consists of a 5'-untranslated region (UTR) (89 bp), a 3'-UTR (426 bp), and an open reading frame (1956 bp) that encodes 651 amino acid residues with a predicted molecular mass of 71.8 kDa and an isoelectric point of 5.34. Based on the amino acid sequence analysis and multiple sequence alignment, this cDNA is a member of cytoplasmic hsp70 subfamily of the hsp70 family and was designated as ot-hsp70. Tissue expression analysis showed that HSP70 expression is highest in the testes when all examined organs were compared. Immunohistochemistry analysis, together with hematoxylin-eosin staining, revealed that the HSP70 protein was expressed in all spermatogenic cells, but not in fibroblasts. In addition, O. tankahkeei were heat challenged by exposure to 32 °C seawater for 2 h, then returned to 13 °C for various recovery time (0-24 h). Relative expression of ot-hsp70 mRNA in the testes was measured at different time points post-challenge by quantitative real-time PCR. A clear time-dependent mRNA expression of ot-hsp70 after thermal stress indicates that the HSP70 gene is inducible. Ultrastructural changes of the heat-stressed testis were observed by transmission electron microscopy. We suggest that HSP70 plays an important role in spermatogenesis and testis protection against thermal stress in O. tankahkeei. Copyright © 2015 Elsevier Inc. All rights reserved.

Serum Hsp70 Antigen: Early Diagnosis Marker in Perinatal Asphyxia.

PubMed

Boskabadi, Hassan; Omidian, Masoud; Tavallai, Shima; Mohammadi, Shabnam; Parizadeh, Mostafa; Ghayour Mobarhan, Majid; Ferns, Gordon Aa

2015-04-01

Perinatal asphyxia is an important cause of mortality and permanent neurological and developmental deficit. Early and accurate diagnosis would help to establish the likely prognosis and may also help in determining the most appropriate treatment. Studies in experimental animal models suggest that a protein called Hsp70 may be a good and potentially useful marker of cellular stress that may be clinically useful in determining the presence of neonatal asphyxia. Regarding the importance of early and accurate diagnosis of asphyxia, we conducted this study, which is the first investigation of the comparison of the serum Hsp70 antigen level between asphyxiated and healthy infants. In this observational study, the serum concentrations of Hsp70 antigen were compared between neonates suffering from perinatal asphyxia (n = 50) and normal neonates (n = 51). The inclusion criteria for the cases were neonates who had reached term and had at least two clinical criteria of asphyxia. Exclusion criteria were babies with gestational age < 37 weeks, infants with congenital abnormalities or positive blood culture. Exclusion criteria in this group were the requirement to hospital stay during first week of the life or babies whose mothers had difficulties during pregnancy or delivery. Term neonates without major anomalies who had asphyxia during delivery were enrolled in the first six hours after delivery, and control group consisted of healthy term neonates without problems and normal delivery process in the first week of life. The cord blood was taken during labor to measure Hsp70 antigen level by using an in-house ELISA (The enzyme-linked immunosorbent assay). The median values of serum anti Hsp70 titers were significantly higher in asphyxiated neonates compared with non-asphyxiated neonates (0.36 [0.04 - 1.14] vs 0.24 [0.01 - 0.63]). At cutoff point = 0.3125 ng/mL, sensitivity was 58% and specificity 76% based on ROC curve. A significant difference between the serum concentrations

Serum Hsp70 Antigen: Early Diagnosis Marker in Perinatal Asphyxia

PubMed Central

Boskabadi, Hassan; Omidian, Masoud; Tavallai, Shima; Mohammadi, Shabnam; Parizadeh, Mostafa; Ghayour Mobarhan, Majid; Ferns, Gordon AA

2015-01-01

Background: Perinatal asphyxia is an important cause of mortality and permanent neurological and developmental deficit. Early and accurate diagnosis would help to establish the likely prognosis and may also help in determining the most appropriate treatment. Studies in experimental animal models suggest that a protein called Hsp70 may be a good and potentially useful marker of cellular stress that may be clinically useful in determining the presence of neonatal asphyxia. Objectives: Regarding the importance of early and accurate diagnosis of asphyxia, we conducted this study, which is the first investigation of the comparison of the serum Hsp70 antigen level between asphyxiated and healthy infants. Patients and Methods: In this observational study, the serum concentrations of Hsp70 antigen were compared between neonates suffering from perinatal asphyxia (n = 50) and normal neonates (n = 51). The inclusion criteria for the cases were neonates who had reached term and had at least two clinical criteria of asphyxia. Exclusion criteria were babies with gestational age < 37 weeks, infants with congenital abnormalities or positive blood culture. Exclusion criteria in this group were the requirement to hospital stay during first week of the life or babies whose mothers had difficulties during pregnancy or delivery. Term neonates without major anomalies who had asphyxia during delivery were enrolled in the first six hours after delivery, and control group consisted of healthy term neonates without problems and normal delivery process in the first week of life. The cord blood was taken during labor to measure Hsp70 antigen level by using an in-house ELISA (The enzyme-linked immunosorbent assay). Results: The median values of serum anti Hsp70 titers were significantly higher in asphyxiated neonates compared with non-asphyxiated neonates (0.36 [0.04 - 1.14] vs 0.24 [0.01 - 0.63]). At cutoff point = 0.3125 ng/mL, sensitivity was 58% and specificity 76% based on ROC curve

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

NASA Technical Reports Server (NTRS)

Cubano, L. A.; Lewis, M. L.

2001-01-01

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat).

PubMed

Cubano, L A; Lewis, M L

2001-05-01

Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

The metazoan protein disaggregase and amyloid depolymerase system: Hsp110, Hsp70, Hsp40, and small heat shock proteins.

PubMed

Torrente, Mariana P; Shorter, James

2013-01-01

A baffling aspect of metazoan proteostasis is the lack of an Hsp104 ortholog that rapidly disaggregates and reactivates misfolded polypeptides trapped in stress induced disordered aggregates, preamyloid oligomers, or amyloid fibrils. By contrast, in bacteria, protozoa, chromista, fungi, and plants, Hsp104 orthologs are highly conserved and confer huge selective advantages in stress tolerance. Moreover, in fungi, the amyloid remodeling activity of Hsp104 has enabled deployment of prions for various beneficial modalities. Thus, a longstanding conundrum has remained unanswered: how do metazoan cells renature aggregated proteins or resolve amyloid fibrils without Hsp104? Here, we highlight recent advances that unveil the metazoan protein-disaggregase machinery, comprising Hsp110, Hsp70, and Hsp40, which synergize to dissolve disordered aggregates, but are unable to rapidly solubilize stable amyloid fibrils. However, Hsp110, Hsp70, and Hsp40 exploit the slow monomer exchange dynamics of amyloid, and can slowly depolymerize amyloid fibrils from their ends in a manner that is stimulated by small heat shock proteins. Upregulation of this system could have key therapeutic applications in various protein-misfolding disorders. Intriguingly, yeast Hsp104 can interface with metazoan Hsp110, Hsp70, and Hsp40 to rapidly eliminate disease associated amyloid. Thus, metazoan proteostasis is receptive to augmentation with exogenous disaggregases, which opens a number of therapeutic opportunities.

Noradrenaline and alpha-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells.

PubMed

Lacoste, A; De Cian, M C; Cueff, A; Poulet, S A

2001-10-01

Expression of heat shock proteins (hsp) is a homeostatic mechanism induced in both prokaryotic and eukaryotic cells in response to metabolic and environmental insults. A growing body of evidence suggests that in mammals, the hsp response is integrated with physiological responses through neuroendocrine signaling. In the present study, we have examined the effect of noradrenaline (NA) on the hsp70 response in mollusc immune cells. Oyster and abalone hemocytes transfected with a gene construct containing a gastropod hsp70 gene promoter linked to the luciferase reporter-gene were exposed to physiological concentrations of NA, or to various alpha- and beta-adrenoceptor agonists and antagonists. Results show that NA and alpha-adrenergic stimulations induced the expression of luciferase in transfected mollusc immunocytes. Furthermore, exposure of hemocytes to NA or to the alpha-adrenoceptor agonist phenylephrine (PE) resulted in the expression of the inducible isoform of the hsp70 protein. Pertussis toxin (PTX), the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor calphostin C, the Ca(2+)-dependent PKC inhibitor Gö 6976 and the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 blocked the PE-mediated induction of the hsp70 gene promoter. These results suggest that alpha-adrenergic signaling induces the transcriptionnal upregulation of hsp70 in mollusc hemocytes through a PTX-sensitive G-protein, PLC, Ca(2+)-dependent PKC and PI 3-kinase. Thus, a functional link exists between neuroendocrine signaling and the hsp70 response in mollusc immune cells.

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

Reactive oxygen species generated by a heat shock protein (Hsp) inducing product contributes to Hsp70 production and Hsp70-mediated protective immunity in Artemia franciscana against pathogenic vibrios.

PubMed

Baruah, Kartik; Norouzitallab, Parisa; Linayati, Linayati; Sorgeloos, Patrick; Bossier, Peter

2014-10-01

The cytoprotective role of heat shock protein (Hsp70) described in a variety of animal disease models, including vibriosis in farmed aquatic animals, suggests that new protective strategies relying upon the use of compounds that selectively turn on Hsp genes could be developed. The product Tex-OE® (hereafter referred to as Hspi), an extract from the skin of the prickly pear fruit, Opuntia ficus indica, was previously shown to trigger Hsp70 synthesis in a non-stressful situation in a variety of animals, including in a gnotobiotically (germ-free) cultured brine shrimp Artemia franciscana model system. This model system offers great potential for carrying out high-throughput, live-animal screens of compounds that have health benefit effects. By using this model system, we aimed to disclose the underlying cause behind the induction of Hsp70 by Hspi in the shrimp host, and to determine whether the product affects the shrimp in inducing resistance towards pathogenic vibrios. We provide unequivocal evidences indicating that during the pretreatment period with Hspi, there is an initial release of reactive oxygen species (hydrogen peroxide and/or superoxide anion), generated by the added product, in the rearing water and associated with the host. The reactive molecules generated are the triggering factors responsible for causing Hsp70 induction within Artemia. We have also shown that Hspi acts prophylactically at an optimum dose regimen to confer protection against pathogenic vibrios. This salutary effect was associated with upregulation of two important immune genes, prophenoloxidase and transglutaminase of the innate immune system. These findings suggest that inducers of stress protein (e.g. Hsp70) are potentially important modulator of immune responses and might be exploited to confer protection to cultured shrimp against Vibrio infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Structural Basis of J Cochaperone Binding and Regulation of Hsp70

PubMed Central

Jiang, Jianwen; Maes, E. Guy; Taylor, Alex B; Wang, Liping; Hinck, Andrew P; Lafer, Eileen M; Sousa, Rui

2007-01-01

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) towards a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, while both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles. PMID:17996706

A hitchhiker's guide to the human Hsp70 family

PubMed Central

Tavaria, Michael; Gabriele, Tim; Kola, Ismail; Anderson, Robin L.

1996-01-01

The human Hsp70 family encompasses at least 11 genes which encode a group of highly related proteins. These proteins include both cognate and highly inducible members, at least some of which act as molecular chaperones. The location of cognate Hsp70s within all the major subcellular compartments is an indication of the importance of these proteins. The expression of several inducible Hsp70 genes is also an indication of the importance of these proteins in the stres response. The existence of multiple genes and protein isoforms has created confusion in the identification and naming of particular family members. We have compiled, from the literature, a list of genes and genetic loci and produced a two-dimensional protein map of the known human Hsp70 family members. This will enable researchers in the field to quickly and reliably identify human Hsp70s. We have also devised a more rational nomenclature for these genes and gene products which, subject to general acceptance, could be extended to Hsp70 families from other species. PMID:9222585

Autophosphorylation of the 16 kDa and 70 kDa antigens (Hsp 16.3 and Hsp 70) of Mycobacterium tuberculosis.

PubMed

Preneta, Rachel; Papavinasasundaram, K G; Cozzone, Alain J; Duclos, Bertrand

2004-07-01

Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been previously cloned and are being exploited in the development of improved vaccines and diagnostic reagents. In this study, the molecular characteristics of two of these antigens, the immunodominant proteins Hsp 16.3 and Hsp 70, were analysed in further detail by assessing their capacity to undergo protein phosphorylation, a chemical modification frequently used by organisms to adjust to environmental variations. Hsp 16.3 was overproduced in an Escherichia coli expression system and purified to homogeneity. Upon incubation in the presence of radioactive ATP, it was shown to possess autophosphorylation activity. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at serine residues. In addition, cross-linking experiments demonstrated that it could tightly bind to ATP. Purified Hsp 70 was also shown to autophosphorylate but phosphorylation occurred exclusively at threonine residues. This reaction was found to be strongly stimulated by calcium ions. These data indicate that both structural and functional similarities exist between Hsp 16.3 (Acr) and alpha-crystallin, a eukaryotic protein which plays an important role in maintaining the transparency of the vertebrate eye, and that the functional properties of Hsp 70 from M. tuberculosis are similar to those of other bacterial members of the Hsp 70 family, particularly the E. coli homologue DnaK.

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.

Tumor-derived inducible heat-shock protein 70 (HSP70) is an essential component of anti-tumor immunity.

PubMed

Dodd, K; Nance, S; Quezada, M; Janke, L; Morrison, J B; Williams, R T; Beere, H M

2015-03-05

The anti-apoptotic function and tumor-associated expression of heat-shock protein 70 (HSP70) is consistent with HSP70 functioning as a survival factor to promote tumorigenesis. However, its immunomodulatory activities to induce anti-tumor immunity predict the suppression of tumor growth. Using the Hsp70.1/3(-/-)(Hsp70(-/-)) mouse model, we observed that tumor-derived HSP70 was neither required for cellular transformation nor for in vivo tumor growth. Hsp70(-/-) murine embryonic fibroblasts (MEFs) were transformed by E1A/Ras and generated tumors in immunodeficient hosts as efficiently as wild-type (WT) transformants. Comparison of Bcr-Abl-mediated transformation of WT and Hsp70(-/-) bone marrow and progression of B-cell leukemogenesis in vivo revealed no differences in disease onset or survival rates, and Eμ-Myc-driven lymphoma in Hsp70(-/-) mice was phenotypically indistinguishable from that in WT Eμ-Myc mice. However, Hsp70(-/-) E1A/Ras MEFs generated significantly larger tumors than their WT counterparts in C57BL/6 J immune-competent hosts. Concurrent with this was a reduction in intra-tumoral infiltration of innate and adaptive immune cells, including macrophages and CD8(+) T cells. Evaluation of several potential mechanisms revealed an HSP70-chemokine-like activity to promote cellular migration. These observations support a role for tumor-derived HSP70 in facilitating anti-tumor immunity to limit tumor growth and highlight the potential consequences of anti-HSP70 therapy as an efficacious anti-cancer strategy.

Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

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

Koizumi, Shinya; Ohama, Naohiko; Mizoi, Junya

2014-07-18

Highlights: • HKL, a Hikeshi homologous gene is identified in Arabidopsis. • HKL interacts with two HSP70 isoforms and regulates the subcellular localization of HSC70-1. • The two HSP70 translocate into nucleus in response to heat stress. • Overexpression of HKL confers thermotolerance in transgenic plants. - Abstract: Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier proteinmore » Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.« less

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

Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome.

PubMed

Weiss, Yoram G; Maloyan, Alina; Tazelaar, John; Raj, Nichelle; Deutschman, Clifford S

2002-09-01

The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

Ste20-like kinase, SLK, activates the heat shock factor 1 - Hsp70 pathway.

PubMed

Cybulsky, Andrey V; Guillemette, Julie; Papillon, Joan

2016-09-01

Expression and activation of SLK increases during renal ischemia-reperfusion injury. When highly expressed, SLK signals via c-Jun N-terminal kinase and p38 to induce apoptosis, and it exacerbates apoptosis induced by ischemia-reperfusion injury. Overexpression of SLK in glomerular epithelial cells (GECs)/podocytes in vivo induces injury and proteinuria. In response to various stresses, cells enhance expression of chaperones or heat shock proteins (e.g. Hsp70), which are involved in the folding and maturation of newly synthesized proteins, and can refold denatured or misfolded proteins. We address the interaction of SLK with the heat shock factor 1 (HSF1)-Hsp70 pathway. Increased expression of SLK in GECs (following transfection) induced HSF1 transcriptional activity. Moreover, HSF1 transcriptional activity was increased by in vitro ischemia-reperfusion injury (chemical anoxia/recovery) and heat shock, and in both instances was amplified further by SLK overexpression. HSF1 binds to promoters of target genes, such as Hsp70 and induces their transcription. By analogy to HSF1, SLK stimulated Hsp70 expression. Hsp70 was also enhanced by anoxia/recovery and was further amplified by SLK overexpression. Induction of HSF1 and Hsp70 was dependent on the kinase activity of SLK, and was mediated via polo-like kinase-1. Transfection of constitutively active HSF1 enhanced Hsp70 expression and inhibited SLK-induced apoptosis. Conversely, the proapoptotic action of SLK was augmented by HSF1 shRNA, or the Hsp70 inhibitor, pifithrin-μ. In conclusion, increased expression/activity of SLK activates the HSF1-Hsp70 pathway. Hsp70 attenuates the primary proapoptotic effect of SLK. Modulation of chaperone expression may potentially be harnessed as cytoprotective therapy in renal cell injury. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2018-03-01

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

Hsp70-Bag3 interactions regulate cancer-related signaling networks.

PubMed

Colvin, Teresa A; Gabai, Vladimir L; Gong, Jianlin; Calderwood, Stuart K; Li, Hu; Gummuluru, Suryaram; Matchuk, Olga N; Smirnova, Svetlana G; Orlova, Nina V; Zamulaeva, Irina A; Garcia-Marcos, Mikel; Li, Xiaokai; Young, Z T; Rauch, Jennifer N; Gestwicki, Jason E; Takayama, Shinichi; Sherman, Michael Y

2014-09-01

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target. ©2014 American Association for Cancer Research.

Hsp70-Bag3 interactions regulate cancer-related signaling networks

PubMed Central

Colvin, T.A.; Gabai, V.L.; Gong, J.; Calderwood, S.K.; Li, H.; Gummuluru, S.; Matchuk, O.N; Smirnova, S.G; Orlova, N.V; Zamulaeva, I.A; Garcia-Marcos, M.; Li, X.; Young, Z.T.; Rauch, J.N.; Gestwicki, J.E.; Takayama, S.; Sherman, M.Y.

2014-01-01

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling, including by modulating the activity of the transcription factors NF-kB, FoxM1 and Hif1α, the translation regulator HuR and the cell cycle regulators p21 and survivin. We also identified a small molecule inhibitor, YM-1, that disrupts Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anti-cancer target. PMID:24994713

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

PubMed

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

2017-02-01

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

Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection

PubMed Central

Taguwa, Shuhei; Maringer, Kevin; Li, Xiaokai; Bernal-Rubio, Dabeiba; Rauch, Jennifer N.; Gestwicki, Jason E.; Andino, Raul; Fernandez-Sesma, Ana; Frydman, Judith

2015-01-01

Summary Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals. PMID:26582131

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.

Variation in Hsp70-1A expression contributes to skin color diversity

PubMed Central

Murase, Daiki; Hachiya, Akira; Fullenkamp, Rachel; Beck, Anita; Moriwaki, Shigeru; Hase, Tadashi; Takema, Yoshinori; Manga, Prashiela

2017-01-01

The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either White or African American (AA) skin. Two dimensional gel electrophoresis (2-DGE) and mass spectrometry demonstrated that Heat Shock Protein 70-1A (Hsp70-1A) protein levels were significantly higher in AA melanocytes compared to White melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70-family members in tyrosinase post-translational modification. Additionally, pharmacologic inhibition and siRNA-mediated down-regulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes. PMID:27094592

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

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

The interaction between a HSP-70 gene variant with dietary calories in determining serum markers of inflammation and cardiovascular risk.

PubMed

Mehramiz, Mehrane; Hassanian, Seyed Mahdi; Mardan-Nik, Maryam; Pasdar, Alireza; Jamialahmadi, Khadijeh; Fiuji, Hamid; Moetamani-Ahmadi, Mehrdad; Parizadeh, Seyed Mohammad Reza; Moohebati, Mohsen; Heidari-Bakavoli, Alireza; Ebrahimi, Mahmoud; Ferns, Gordon A; Ghayour-Mobarhan, Majid; Avan, Amir

2017-10-24

The high prevalence of cardiovascular disease (CVD) globally is attributable to an interaction between environmental and genetic factors. Gene × diet interaction studies aim to explore how a modifiable factor interacts with genetic predispositions. Here we have explored the interaction of a heat shock protein (HSP70) gene polymorphism (+1267A > G) with dietary intake and their possible association with serum C-reactive protein (CRP), an inflammatory marker, that is a major component of CVD risk. HSP70 genotype was determined using a TaqMan real time PCR based method.Dietary intake was assessed using a dietary questionnaire. Serum high sensitivity (Hs) CRP and other cardiovascular risk factors were assessed by routine methods. This included coronary angioplasty to determine the presence of coronary artery stenosis. There were significant differences between serum lipid profile and Hs-CRP across the genotypes for Hsp70. The carriers of G allele had higher serum hs-CRP concentrations, compared with the AA homozygotes, with the wild genotype. Interaction analysis showed the association was modulated by total energy intake; the interaction of high energy intake with GG genotype: RERI = 0.77, AP = 0.26, S = 1.6. We have found a significant association between the +1267A > G variant of the HSP70 gene with cardiovascular risk factors and serum hs-CRP concentrations. It is possible that a low energy diet could ameliorate the unfavorable effects of G allele of HSP70. Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Ultrastructure of rabbit embryos exposed to hyperthermia and anti-Hsp 70.

PubMed

Olexikova, L; Makarevich, A V; Pivko, J; Chrenek, P

2013-08-01

The aim of the study was to determine the effect of short-term hyperthermia and Hsp70 blockage on ultrastructural changes in cell organelles and nucleoli of rabbit preimplantation embryos. The embryos were cultured either at 37.5°C (control, C) or 41.5°C (hyperthermia, HT) during 6 h. The antibody against Hsp70 was added into the culture medium (4 μg/ml) of morula stage embryos from C and HT groups. After termination of the culture, the embryos were processed for transmission electron microscopy. The embryos exposed to hyperthermia showed increased volume of lipid droplets, considerable occurrence of cellular debris in the perivitelline space and slight changes in the occurrence of microvilli on the surface of trophoblastic cells. In the embryos exposed to anti-Hsp 70 at 37.5°C, there were considerable changes in mitochondria morphology, decreased volume of dense bodies in the cytoplasm and considerable changes in the occurrence of microvilli on the surface of trophoblastic cells. In the group of embryos exposed simultaneously to hyperthermia and anti-Hsp 70, mitochondria were also expanded and swollen; the volume of flocculent vesicles and lipid droplets was increased and the volume of dense bodies in the cytoplasm was diminished. General organization of the cytoplasm in groups with anti-Hsp70 was characterized by cell organelle segregation. Averaged size of the nucleolar area was significantly increased in the embryos exposed to hyperthermia, whereas in the group exposed to the anti-Hsp70 without hyperthermia it was significantly diminished. Hyperthermia also caused disintegration of compact status of the nucleoli. In presence of anti-Hsp 70, the structural changes, described within the nucleoli during hyperthermia, were not observed. In conclusion, these results document ultrastructural changes in cell organelles of rabbit preimplantation embryo caused by hyperthermia, and also changes in the nucleolar structures, at which presence of Hsp-70 inhibit these

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

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.

Significance of serum antibodies against HSP 60 and HSP 70 for the diagnostic of infectious diseases.

PubMed

Bleotu, Coralia; Chifiriuc, Mariana Carmen; Pircalabioru, Gratiela; Berteşteanu, Şerban Vifor Gabriel; Grigore, Raluca; Ruta, Simona Maria; Lazar, Veronica

2014-01-01

Heat shock proteins (HSP) represent important antigenic targets for the immune response, playing an important role in the pathology and infectious diseases control. The purpose of this work was to investigate the levels of HSP60 and HSP70 specific antibodies in the bloodstream of patients with different bacterial infections and cancer, in order to evaluate their potential role as diagnosis markers of different infectious diseases. Detection of specific anti-HSP 60 and HSP 70 serum levels was performed by ELISA. Statistical analysis of data by multivariate logistic regression was performed using GraphPadPrism software and statistical tests based on chi-square and Student t-test. High levels of anti-HSP60 were found in patients with localized infections, while the levels of anti- HSP70 were higher in the group with generalized infections. The serum levels of both anti-HSP 60 and anti-HSP70 were significantly increased in patients with Gram-negative bacterial infections, as compared with patients harbouring infections produced by Gram-positive and fungal strains, demonstrating their potential use as additional diagnosis and prognosis markers in infections with this etiology.

Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle.

PubMed

Bharati, Jaya; Dangi, S S; Chouhan, V S; Mishra, S R; Bharti, M K; Verma, V; Shankar, O; Yadav, V P; Das, K; Paul, A; Bag, S; Maurya, V P; Singh, G; Kumar, P; Sarkar, M

2017-06-01

Six male Tharparkar cattle aged 2-3 years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15 days and then exposed to 42 °C temperature up to 23 days followed by 12 days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6 h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P < 0.05) and showed two peaks on day 17 and day 32 (2nd and 17th days of thermal challenge, respectively). The HSP70 mRNA expression was increased (P < 0.05) in a temperature- and time-dependent manner in heat stress challenge treatment as compared to control in cultured PBMCs. HSP70 expression was found to be higher (P < 0.05) after 10 days of heat exposure (corresponds to chronic heat stress) as compared to the first 5 days of heat stress (corresponds to short-term heat stress) and control period at TNZ. The present findings indicate that HSP70 is possibly involved in heat stress adaptive response in Tharparkar cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

Variation in Hsp70-1A Expression Contributes to Skin Color Diversity.

PubMed

Murase, Daiki; Hachiya, Akira; Fullenkamp, Rachel; Beck, Anita; Moriwaki, Shigeru; Hase, Tadashi; Takema, Yoshinori; Manga, Prashiela

2016-08-01

The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes, and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either white or African American skin. Two-dimensional gel electrophoresis and mass spectrometry demonstrated that heat shock protein 70-1A (Hsp70-1A) protein levels were significantly higher in African American melanocytes compared with white melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70 family members in tyrosinase post-translational modification. In addition, pharmacologic inhibition and small interfering RNA-mediated downregulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes, and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

BAG3 Expression in Glioblastoma Cells Promotes Accumulation of Ubiquitinated Clients in an Hsp70-dependent Manner*

PubMed Central

Gentilella, Antonio; Khalili, Kamel

2011-01-01

Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners. PMID:21233200

Induction of hsp70 by the herbicide oxyfluorfen (Goal) in the Egyptian Nile fish, Oreochromis niloticus.

PubMed

Hassanein, H M; Banhawy, M A; Soliman, F M; Abdel-Rehim, S A; Müller, W E; Schröder, H C

1999-07-01

This paper deals with the expression of the biomarker hsp70 in the liver and kidney of the freshwater fish Oreochromis niloticus following exposure to the herbicide oxyfluorfen (Goal). Fishes were exposed to three concentrations, the 96-h LC50 (3 mg/L), the 96-h (1/2)LC50 (1.5 mg/L), and the 96-h (1/4)LC50 (0.75 mg/L) of oxyfluorfen for 6, 15, and 24 days, respectively, and samples were taken at three different time periods for each concentration. The livers responded to the herbicide by an induction of the expression of both the constitutive (hsp75; Mr 75 kDa) and the inducible (hsp73; Mr 73 kDa) hsp70 proteins. In kidney, the herbicide induced a time-dependent increase in the expression of the constitutive hsp70 (hsp75) as well, but the inducible hsp70 (hsp73) required much longer incubation periods to reach maximal levels (15 and 24 days). Our results suggest that expression of hsp70 in fish is a sensitive indicator of cellular responses to herbicide exposure in the aquatic environment.

The growth transformation of human B cells involves superinduction of hsp70 and hsp90.

PubMed

Cheung, R K; Dosch, H M

1993-04-01

Epstein-Barr virus (EBV) is a latent human herpes virus associated with a range of malignant and non-malignant disorders. EBV binds to CD21 virus receptors on B lymphocytes and growth transforms these cells; in susceptible (e.g., immunodeficient) hosts such cells rapidly expand into fatal lymphomas. Virus binding and infection trigger a cascade of cellular events which are transformation prerequisite and analogous to non-oncogenic cell activation events but which differ in several quantitative or qualitative respects. Unique trans-membrane Ca2+ currents, Na+/H+ exchange, as well as tyrosine phosphorylation and p56lck-gene induction suggest that even early on the transformation process has oncogenic specificity. In this report we describe that two additional cellular gene families, the stress proteins hsp70 and hsp90, are coordinately induced at mRNA and protein levels and, quite different from hsp induction by thermal stress, this induction is dependent on EBV-induced trans-membrane Ca2+ currents. Blockade of hsp induction prevents transformation. The kinetics and induction prerequisites set this response well apart from reported responses to thermal or viral stress protein induction. Like p56lck-, hsp induction is purely a post-receptor binding event and not dependent on expression of any viral gene. The induction kinetics, with a peak at approximately 12-16 hr and subsequent decline to control levels, considerably extend the chronological map of elements in the CD21-dependent branch of the transformation pathway and suggest a specific role of induced hsp different from the cell cycle-related functions observed in other cell systems.

Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways

PubMed Central

Kondrikov, Dmitry; Fulton, David; Dong, Zheng; Su, Yunchao

2015-01-01

Exposure of pulmonary artery endothelial cells (PAECs) to hyperoxia results in a compromise in endothelial monolayer integrity, an increase in caspase-3 activity, and nuclear translocation of apoptosis-inducing factor (AIF), a marker of caspase-independent apoptosis. In an endeavor to identify proteins involved in hyperoxic endothelial injury, we found that the protein expression of heat-shock protein 70 (Hsp70) was increased in hyperoxic PAECs. The hyperoxia-induced Hsp70 protein expression is from hspA1B gene. Neither inhibition nor overexpression of Hsp70 affected the first phase barrier disruption of endothelial monolayer. Nevertheless, inhibition of Hsp70 by using the Hsp70 inhibitor KNK437 or knock down Hsp70 using siRNA exaggerated and overexpression of Hsp70 prevented the second phase disruption of lung endothelial integrity. Moreover, inhibition of Hsp70 exacerbated and overexpression of Hsp70 prevented hyperoxia-induced apoptosis, caspase-3 activation, and increase in nuclear AIF protein level in PAECs. Furthermore, we found that Hsp70 interacted with AIF in the cytosol in hyperoxic PAECs. Inhibition of Hsp70/AIF association by KNK437 correlated with increased nuclear AIF level and apoptosis in KNK437-treated PAECs. Finally, the ROS scavenger NAC prevented the hyperoxia-induced increase in Hsp70 expression and reduced the interaction of Hsp70 with AIF in hyperoxic PAECs. Together, these data indicate that increased expression of Hsp70 plays a protective role against hyperoxia-induced lung endothelial barrier disruption through caspase-dependent and AIF-dependent apoptotic pathways. Association of Hsp70 with AIF prevents AIF nuclear translocation, contributing to the protective effect of Hsp70 on hyperoxia-induced endothelial apoptosis. The hyperoxia-induced increase in Hsp70 expression and Hsp70/AIF interaction is contributed to ROS formation. PMID:26066050

Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection.

PubMed

Taguwa, Shuhei; Maringer, Kevin; Li, Xiaokai; Bernal-Rubio, Dabeiba; Rauch, Jennifer N; Gestwicki, Jason E; Andino, Raul; Fernandez-Sesma, Ana; Frydman, Judith

2015-11-19

Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals. Copyright © 2015 Elsevier Inc. All rights reserved.

Increased Anti-HSP60 and Anti-HSP70 Antibodies in Women with Unexplained Recurrent Pregnancy Loss.

PubMed

Matsuda, Miwa; Sasaki, Aiko; Shimizu, Keiko; Kamada, Yasuhiko; Noguchi, Soichi; Hiramatsu, Yuji; Nakatsuka, Mikiya

2017-06-01

　Vascular dysfunction has been reported in women with recurrent pregnancy loss (RPL). We investigated the severity of vascular dysfunction in non-pregnant women with RPL and its correlation with anti-heat shock protein (HSP) antibodies that are known to induce arteriosclerosis. We measured the serum anti-HSP60 antibodies, anti-HSP70 antibodies, and anti-phospholipid antibodies (APA) in 68 women with RPL and 29 healthy controls. Among the women with RPL, 14 had a diagnosis of antiphospholipid syndrome (APS), and in the remaining 54, the causes for RPL were unexplained. Compared to the controls, the brachial-ankle pulse wave velocity (baPWV), carotid augmentation index (cAI), and uterine artery pulsatility index (PI) were all significantly higher in the women with both APS and unexplained RPL. Compared to the controls, the anti-HSP60 antibody levels were significantly higher in the APA-positive group of women with unexplained RPL, and the anti-HSP70 antibody levels were significantly higher in APS and APA-positive group of women with unexplained RPL. However, the anti-HSP60 and anti-HSP70 antibody levels did not correlate with the values of baPWV or cAI. Our results demonstrated anti-HSP60 and anti-HSP70 antibodies are increased in women with unexplained RPL. Further studies are needed to elucidate the roles of anti-HSP antibodies and their pathophysiology in unexplained RPL.

HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress.

PubMed

González-Aravena, Marcelo; Calfio, Camila; Mercado, Luis; Morales-Lange, Byron; Bethke, Jorn; De Lorgeril, Julien; Cárdenas, César A

2018-03-27

Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5  and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract. Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is non-induced by heat stress in S. neumayeri.

Association of plasma IL-6 and Hsp70 with HRV at different levels of PAHs metabolites.

PubMed

Ye, Jian; Zhu, Rui; He, Xiaosheng; Feng, Yingying; Yang, Liangle; Zhu, Xiaoyan; Deng, Qifei; Wu, Tangchun; Zhang, Xiaomin

2014-01-01

Exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with reduced heart rate variability (HRV), a strong predictor of cardiovascular diseases, but the mechanism is not well understood. We hypothesized that PAHs might induce systemic inflammation and stress response, contributing to altered cardiac autonomic function. HRV indices were measured using a 3-channel digital Holter monitor in 800 coke oven workers. Plasma levels of interleukin-6 (IL-6) and heat shock protein 70 (Hsp70) were determined using ELISA. Twelve urinary PAHs metabolites (OH-PAHs) were measured by gas chromatography-mass spectrometry. We found that significant dose-dependent relationships between four urinary OH-PAHs and IL-6 (all Ptrend<0.05); and an increase in quartiles of IL-6 was significantly associated with a decrease in total power (TP) and low frequency (LF) (Ptrend = 0.014 and 0.006, respectively). In particular, elevated IL-6 was associated in a dose-dependent manner with decreased TP and LF in the high-PAHs metabolites groups (all Ptrend<0.05), but not in the low-PAHs metabolites groups. No significant association between Hsp70 and HRV in total population was found after multivariate adjustment. However, increased Hsp70 was significantly associated with elevated standard deviation of NN intervals (SDNN), TP and LF in the low-PAHs metabolites groups (all Ptrend<0.05). We also observed that both IL-6 and Hsp70 significantly interacted with multiple PAHs metabolites in relation to HRV. In coke oven workers, increased IL-6 was associated with a dose-response decreased HRV in the high-PAHs metabolites groups, whereas increase of Hsp70 can result in significant dose-related increase in HRV in the low-PAHs metabolites groups.

Association of Plasma IL-6 and Hsp70 with HRV at Different Levels of PAHs Metabolites

PubMed Central

He, Xiaosheng; Feng, Yingying; Yang, Liangle; Zhu, Xiaoyan; Deng, Qifei; Wu, Tangchun; Zhang, Xiaomin

2014-01-01

Background Exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with reduced heart rate variability (HRV), a strong predictor of cardiovascular diseases, but the mechanism is not well understood. Objectives We hypothesized that PAHs might induce systemic inflammation and stress response, contributing to altered cardiac autonomic function. Methods HRV indices were measured using a 3-channel digital Holter monitor in 800 coke oven workers. Plasma levels of interleukin-6 (IL-6) and heat shock protein 70 (Hsp70) were determined using ELISA. Twelve urinary PAHs metabolites (OH-PAHs) were measured by gas chromatography-mass spectrometry. Results We found that significant dose-dependent relationships between four urinary OH-PAHs and IL-6 (all P trend<0.05); and an increase in quartiles of IL-6 was significantly associated with a decrease in total power (TP) and low frequency (LF) (P trend = 0.014 and 0.006, respectively). In particular, elevated IL-6 was associated in a dose-dependent manner with decreased TP and LF in the high-PAHs metabolites groups (all P trend<0.05), but not in the low-PAHs metabolites groups. No significant association between Hsp70 and HRV in total population was found after multivariate adjustment. However, increased Hsp70 was significantly associated with elevated standard deviation of NN intervals (SDNN), TP and LF in the low-PAHs metabolites groups (all P trend<0.05). We also observed that both IL-6 and Hsp70 significantly interacted with multiple PAHs metabolites in relation to HRV. Conclusions In coke oven workers, increased IL-6 was associated with a dose-response decreased HRV in the high-PAHs metabolites groups, whereas increase of Hsp70 can result in significant dose-related increase in HRV in the low-PAHs metabolites groups. PMID:24722336

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

PubMed

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

2015-12-08

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

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

PubMed

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

2013-09-01

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

Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma.

PubMed

Antonoff, Mara B; Chugh, Rohit; Skube, Steven J; Dudeja, Vikas; Borja-Cacho, Daniel; Clawson, Kimberly A; Vickers, Selwyn M; Saluja, Ashok K

2010-09-01

Our recent work demonstrated that treatment of neuroblastoma with triptolide causes apoptotic cell death in vitro and decreases tumor size in vivo. Triptolide therapy has been associated with reduced expression of Hsp-70, suggesting a mechanism of cell killing involving Hsp-70 inhibition. The principal objective of this study was to investigate the role of Hsp-70 in triptolide-mediated cell death in neuroblastoma. Neuroblastoma cells were transfected with Hsp-70-specific siRNA. Viability, caspase activity, and phosphatidylserine externalization were subsequently measured. An orthotopic, syngeneic murine tumor model was developed, and randomized mice received daily injections of triptolide or vehicle. At 21 d, mice were sacrificed. Immunohistochemisty was used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis. Targeted silencing of Hsp-70 with siRNA significantly decreased cellular viability, augmented caspase-3 activity, and resulted in increased annexin-V staining. These effects parallel those findings obtained following treatment with triptolide. Residual tumors from triptolide-treated mice showed minimal staining with Hsp-70 immunohistochemistry, while control tumors stained prominently. Tumors from treated mice demonstrated marked staining with the TUNEL assay, while control tumors showed no evidence of apoptosis. Use of siRNA to suppress Hsp-70 expression in neuroblastoma resulted in apoptotic cell death, similar to the effects of triptolide. Residual tumors from triptolide-treated mice expressed decreased levels of Hsp-70 and demonstrated significant apoptosis. These findings support the hypothesis that Hsp-70 inhibition plays a significant role in triptolide-mediated neuroblastoma cell death. Copyright 2010 Elsevier Inc. All rights reserved.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

PubMed Central

Dix, D J; Allen, J W; Collins, B W; Mori, C; Nakamura, N; Poorman-Allen, P; Goulding, E H; Eddy, E M

1996-01-01

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8622925

Mechanism underlying berberine's effects on HSP70/TNFα under heat stress: Correlation with the TATA boxes.

PubMed

Jiang, Jing-Fei; Lei, Fan; Yuan, Zhi-Yi; Wang, Yu-Gang; Wang, Xin-Pei; Yan, Xiao-Jin; Yu, Xuan; Xing, Dong-Ming; DU, Li-Jun

2017-03-01

Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Stress-induced localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells.

PubMed

Khalouei, Sam; Chow, Ari M; Brown, Ian R

2014-05-01

The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.

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

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

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

Sudden infant death syndrome: no significant expression of heat-shock proteins (HSP27, HSP70).

PubMed

Doberentz, Elke; Führing, Sarah; Madea, Burkhard

2016-03-01

In industrialized countries, sudden infant death is the most common cause of death in young children. Although prone sleeping position is a well-known risk factor, hyperthermia might also be important. Pathognomonic findings of premortem hyperthermia do not exist. During stress, including thermal effects, heat-shock protein (HSP) expression increases. This study investigated hyperthermia as a contributing or pathogenic factor for sudden infant death syndrome (SIDS). Immunohistochemical staining for HSP27 and HSP70 in the kidney, heart, and lung from 120 SIDS cases was examined. HSP70 immunostaining was negative in kidney, heart, and lung tissues in all cases and in tissues from the control group. HSP27 staining was positive in the kidney from one case, and was positive in the lungs (respiratory epithelia in 27% of cases; vascular endothelia in 19% of cases) and was negative in the heart. In the control group HSP27 was positive in 8% of renal tubular tissues and in 29% of renal vascular endothelia. Staining for HSP27 in lung tissues was positive in respiratory epithelia in 8% of cases and for vascular endothelia in 29%, whereas tissues from the heart were positive in only 4%. The hypothesis of hyperthermia being a pathogenic factor for SIDS was not supported by immunohistochemical visualization of HSP70 or HSP27.

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis

NASA Technical Reports Server (NTRS)

Gruppi, C. M.; Wolgemuth, D. J.

1993-01-01

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells.

Lysosomal Rerouting of Hsp70 Trafficking as a Potential Immune Activating Tool for Targeting Melanoma

PubMed Central

Juhász, Kata; Thuenauer, Roland; Spachinger, Andrea; Duda, Ernő; Horváth, Ibolya; Vígh, László; Sonnleitner, Alois; Balogi, Zsolt

2013-01-01

Tumor specific cell surface localization and release of the stress inducible heat shock protein 70 (Hsp70) stimulate the immune system against cancer cells. A key immune stimulatory function of tumor-derived Hsp70 has been exemplified with the murine melanoma cell model, B16 overexpressing exogenous Hsp70. Despite the therapeutic potential mechanism of Hsp70 transport to the surface and release remained poorly understood. We investigated principles of Hsp70 trafficking in B16 melanoma cells with low and high level of Hsp70. In cells with low level of Hsp70 apparent trafficking of Hsp70 was mediated by endosomes. Excess Hsp70 triggered a series of changes such as a switch of Hsp70 trafficking from endosomes to lysosomes and a concomitant accumulation of Hsp70 in lysosomes. Moreover, lysosomal rerouting resulted in an elevated concentration of surface Hsp70 and enabled active release of Hsp70. In fact, hyperthermia, a clinically applicable approach triggered immediate active lysosomal release of soluble Hsp70 from cells with excess Hsp70. Furthermore, excess Hsp70 enabled targeting of internalized surface Hsp70 to lysosomes, allowing in turn heat-induced secretion of surface Hsp70. Altogether, we show that excess Hsp70 expressed in B16 melanoma cells diverts Hsp70 trafficking from endosomes to lysosomes, thereby supporting its surface localization and lysosomal release. Controlled excess-induced lysosomal rerouting and secretion of Hsp70 is proposed as a promising tool to stimulate anti-tumor immunity targeting melanoma. PMID:22920897

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

Co-expression of heat shock protein (HSP) 40 and HSP70 in Pinctada martensii response to thermal, low salinity and bacterial challenges.

PubMed

Li, Jun; Zhang, Yuehuan; Liu, Ying; Zhang, Yang; Xiao, Shu; Yu, Ziniu

2016-01-01

Heat shock protein (HSP) 40 proteins are a family of molecular chaperones that bind to HSP70 through their J-domain and regulate the function of HSP70 by stimulating its adenosine triphosphatase activity. In the present study, a HSP40 homolog named PmHSP40 was cloned from the hemocytes of pearl oyster Pinctada martensii using EST and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PmHSP40 was 1251 bp in length, which included a 5' untranslated region (UTR) of 75 bp, an open reading frame (ORF) of a 663 bp, and a 3' UTR of 513 bp. The deduced amino acid sequence of PmHSP40 contains a J domain in the N-terminus. In response to thermal and low salinity stress challenges, the expression of PmHSP40 in hemocytes and the gill were inducible in a time-dependent manner. After bacterial challenge, PmHSP40 transcripts in hemocytes increased and peaked at 6 h post injection. In the gill, PmHSP40 expression increased, similar to expression in hemocytes; however, transcript expression of PmHSP40 was significantly up-regulated at 12 h post injection. Furthermore, the transcripts of PmHSP70 showed similar kinetics as that of PmHSP40, with highest induction during thermal, low salinity stress and bacterial challenges. Altogether these results demonstrate that PmHSP40 is an inducible protein under thermal, low salinity and bacterial challenges, suggesting its involvement in both environmental and biological stresses, and in the innate immunity of the pearl oyster. Copyright © 2015 Elsevier Ltd. All rights reserved.

Physical activity, muscle, and the HSP70 response.

PubMed

Kilgore, J L; Musch, T I; Ross, C R

1998-06-01

Selye (1936) described how organisms react to various external stimuli (i.e., stressors). These reactions generally follow a programmed series of events and help the organism adapt to the imposed stress. The heat shock response is a common cellular reaction to external stressors, including physical activity. A characteristic set of proteins is synthesised shortly after the organism is exposed to stress. Researchers have not determined how heat shock proteins affect the exercise response. However, their role in adaptation to exercise and training might be inferred, since the synthetic patterns correlate well with the stress adaptation syndrome that Selye described. This review addresses the 70 kilodalton heat shock protein family (HSP70), the most strongly induced heat shock proteins. This paper provides an overview of the general heat shock response and a brief review of literature on HSP70 function, structure, regulation, and potential applications. Potential applications in health, exercise, and medicine are provided.

Hierarchical functional specificity of cytosolic heat shock protein 70 (Hsp70) nucleotide exchange factors in yeast.

PubMed

Abrams, Jennifer L; Verghese, Jacob; Gibney, Patrick A; Morano, Kevin A

2014-05-09

Heat shock protein 70 (Hsp70) molecular chaperones play critical roles in protein homeostasis. In the budding yeast Saccharomyces cerevisiae, cytosolic Hsp70 interacts with up to three types of nucleotide exchange factors (NEFs) homologous to human counterparts: Sse1/Sse2 (Heat shock protein 110 (Hsp110)), Fes1 (HspBP1), and Snl1 (Bag-1). All three NEFs stimulate ADP release; however, it is unclear why multiple distinct families have been maintained throughout eukaryotic evolution. In this study we investigate NEF roles in Hsp70 cell biology using an isogenic combinatorial collection of NEF deletion mutants. Utilizing well characterized model substrates, we find that Sse1 participates in most Hsp70-mediated processes and is of particular importance in protein biogenesis and degradation, whereas Fes1 contributes to a minimal extent. Surprisingly, disaggregation and resolubilization of thermally denatured firefly luciferase occurred independently of NEF activity. Simultaneous deletion of SSE1 and FES1 resulted in constitutive activation of heat shock protein expression mediated by the transcription factor Hsf1, suggesting that these two factors are important for modulating stress response. Fes1 was found to interact in vivo preferentially with the Ssa family of cytosolic Hsp70 and not the co-translational Ssb homolog, consistent with the lack of cold sensitivity and protein biogenesis phenotypes for fes1Δ cells. No significant consequence could be attributed to deletion of the minor Hsp110 SSE2 or the Bag homolog SNL1. Together, these lines of investigation provide a comparative analysis of NEF function in yeast that implies Hsp110 is the principal NEF for cytosolic Hsp70, making it an ideal candidate for therapeutic intervention in human protein folding disorders.

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.

The role of heat shock protein 70 (Hsp 70) in male infertility: is it a line of defense against sperm DNA fragmentation?

PubMed

Erata, Gül Ozdemirler; Koçak Toker, Necla; Durlanik, Ozgür; Kadioğlu, Ateş; Aktan, Gülşen; Aykaç Toker, Gülçin

2008-08-01

To clarify the role of heat shock protein 70 (Hsp 70) and its relation with DNA damage in male infertility. Prospective study. Andrology laboratory of Istanbul Medical Faculty. Semen samples from 37 infertile men and 13 fertile men (as controls). The percentage of DNA fragmentation was assayed with the use of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Sperm Hsp 70 expression was determined by using Western blot analysis. Both the percentages of sperm DNA fragmentation and Hsp 70 expression were correlated with semen analysis parameters. TUNEL-positive spermatozoa in the infertile group (18.7% for asthenospermics and 13.0% for oligoasthenospermics) were higher than the fertile group (4.9%). Significant inverse correlations were detected between percentage of TUNEL-positive cells and both concentration (r = -0.487) and motility (r = -0.377) of spermatozoa. No expression of Hsp 70 was observed in azospermic group, whereas Hsp 70 levels were found increased significantly in infertile group (U = 62 for asthenospermics and U = 38 for oligoasthenospermics) compared to fertile group as analyzed by using Mann-Whitney U Wilcoxon rank sum test. Furthermore, significant positive correlation was found between percentage of TUNEL-positive cells and Hsp 70 expression (r = 0.357). Hsp 70 expression may have been increased as a protective mechanism against apoptosis in spermatozoa of infertile men.

HSP70: therapeutic potential in acute and chronic cardiac disease settings.

PubMed

Bernardo, Bianca C; Weeks, Kate L; Patterson, Natalie L; McMullen, Julie R

2016-12-01

Heat shock proteins are a family of proteins that are produced by cells in response to exposure to stressful conditions. The best studied heat shock protein is HSP70, which is known to act as a molecular chaperone to maintain cellular homeostasis and inhibit protein aggregation in response to stress. While early animal studies suggested that increasing HSP70 in the heart (using a transgenic, gene transfer or pharmacological approach) provided cardiac protection against acute cardiac stress, recent studies have found no benefit of increasing HSP70 in mouse models of chronic cardiac stress. As HSP70 has been considered a potential therapeutic target, it is important to comprehensively assess HSP70 therapies in preclinical models of acute and chronic cardiac disease.

Effects of cadmium on MAPK signalling pathways and HSP70 expression in a human trophoblast cell line.

PubMed

Valbonesi, P; Ricci, L; Franzellitti, S; Biondi, C; Fabbri, E

2008-08-01

The aim of this work was to provide a greater insight into the possible effects of Cd on signal transduction and stress-related pathways in reproductive tissues. Cd is a known placental toxin in both animals and humans. Our experiments were designed to study the influence of Cd on MAPK (ERK1/2, JNK1/2 and p38MAPK) activation in the extravillous trophoblast cell line, HTR-8/SVneo, used as an experimental model. We also studied the HSP70 response in cells exposed to Cd, since these proteins may have an important role in conferring protection and tolerance against teratogenic concentrations of the metal. The effects of Cd were compared with those of a well-known toxic agent, H2O2. The metal triggered MAPK activation in a dose- and time-dependent manner. At 30 microM Cd, stimulations of about 300%, 550% and 250% were observed for ERK1/2, JNK1/2, and p38MAPK, respectively. Phosphorylation of ERK1/2 and JNK1/2 was significantly induced after a 1-h exposure to 30 microM Cd, while that of p38MAPK occurred only after 8h. Similarly, H2O2 caused dose- and time-dependent activation of MAPK pathways. Cd potently stimulated HSP70 expression and that of related genes HSP70 A, B and C. H2O2 did not increase HSP70 and HSP70 A and B expression, while temporarily increasing HSP70C transcript levels. In conclusion, Cd triggers different stress responses in trophoblast cells involving HSP70 and SAPK, and also enhances ERK1/2 phosphorylation. Since MAPK dependent pathways play a crucial role during pregnancy, non-physiological activation by Cd exposure may disrupt normal functions in trophoblast cells.

Oxalate exposure provokes HSP 70 response in LLC-PK1 cells, a line of renal epithelial cells: protective role of HSP 70 against oxalate toxicity.

PubMed

Koul, Sweaty; Huang, Meiyi; Bhat, Sidarth; Maroni, Paul; Meacham, Randall B; Koul, Hari K

2008-02-01

We investigated the effects of oxalate on immediate early genes (IEGs) and stress protein HSP 70, commonly induced genes in response to a variety of stresses. LLC-PK1 cells were exposed to oxalate. Gene transcription and translation were monitored by Northern and Western blot analysis. RNA and DNA synthesis were assessed by [(3)H]-uridine and [(3)H]-thymidine incorporation, respectively. Oxalate exposure selectively increased the levels of mRNA encoding IEGs c-myc and c-jun as well as stress protein HSP 70. While expression of c-myc and c-jun was rapid (within 15 min to 2 h) and transient, HSP 70 expression was delayed (approximately 8 h) and stable. Furthermore, oxalate exposure resulted in delayed induction of generalized transcription by 18 h and reinitiation of the DNA synthesis by 24 h of oxalate exposure. Moreover, we show that prior induction of HSP 70 by mild hypertonic exposure protected the cells from oxalate toxicity. To the best of our knowledge this is the first study to demonstrate rapid IEG response and delayed heat-shock response to oxalate toxicity and protective role of HSP 70 against oxalate toxicity to renal epithelial cells. Oxalate, a metabolic end product, induces IEGs c-myc and c-jun and a delayed HSP 70 expression; While IEG expression may regulate additional genetic responses to oxalate, increased HSP 70 expression would serve an early protective role during oxalate stress.

Assessment of heat shock protein (HSP60, HSP72, HSP90, and HSC70) expression in cultured limbal stem cells following air lifting

PubMed Central

Mohammadi, Parvaneh; Daryadel, Arezoo; Baharvand, Hossein

2010-01-01

Objectives The aim of this study is to create an ex vivo model to examine the expression of major heat-shock protein (HSP) families; HSP60, HSP72, and HSP90, and heat-shock cognate 70 (HCS70) at the mRNA and protein level in differentiating corneal cells from limbal stem cells (LSC) following air exposure. Methods Limbal biopsies taken from cadaveric normal human limbus were cultivated as explants on human amniotic membrane (HAM) and plastic dish (PD). Corneal differentiation was induced by air lifting for 16 days. The expression of putative LSC markers (P63 and ATP-binding cassette G2 [ABCG2]), corneal markers (keratin 3 [K3/12] and connexin 43 [CX43]), and HSP60, HSP72, HSP90, and HSC70 were tested by RT–PCR, immunofluorescence, and flow cytometry pre- and post-air exposure. Fresh limbal and corneal tissues were used as control groups. Results Air lifting induced corneal differentiation with a decrease in the number of P63+ cells and an increase in the number of K3+/CX43+ cells, which characterized transient amplifying cells (TACs). Moreover, denuded HAM provided a superior niche for LSC proliferation and phenotype maintenance in vitro. Additionally, we have evidence that expressions of HSC70 as well as HSP72 were enhanced through corneal differentiation and HSP90 post-air lifting in vitro and in vivo. HSP60, however, was not detected in either LSC or corneal cells, in vivo and in vitro. Conclusions These results suggest that corneal differentiation following air exposure may regulate HSP72 and HSC70 expression. In addition, HSP72 and HSP90 may protect LSC and corneal cells against oxidative stress. PMID:20806039

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.

A furoviral replicase recruits host HSP70 to membranes for viral RNA replication

PubMed Central

Yang, Jian; Zhang, Fen; Cai, Nian-Jun; Wu, Ne; Chen, Xuan; Li, Jing; Meng, Xiang-Feng; Zhu, Tong-Quan; Chen, Jian-Ping; Zhang, Heng-Mu

2017-01-01

Many host factors have been identified to be involved in viral infection. However, although furoviruses cause important diseases of cereals worldwide, no host factors have yet been identified that interact with furoviral genes or participate in the viral infection cycle. In this study, both TaHSP70 and NbHSP70 were up-regulated in Chinese wheat mosaic furovirus (CWMV)-infected plants. Their overexpression and inhibition were correlated with the accumulation of viral genomic RNAs, suggesting that the HSP70 genes could be necessary for CWMV infection. The subcellular distributions of TaHSP70 and NbHSP70 were significantly affected by CWMV infection or by infiltration of RNA1 alone. Further assays showed that the viral replicase encoded by CWMV RNA1 interacts with both TaHSP70 and NbHSP70 in vivo and vitro and that its region aa167–333 was responsible for the interaction. Subcellular assays showed that the viral replicase could recruit both TaHSP70 and NbHSP70 from the cytoplasm or nucleus to the granular aggregations or inclusion-like structures on the intracellular membrane system, suggesting that both HSP70s may be recruited into the viral replication complex (VRC) to promote furoviral replication. This is the first host factor identified to be involved in furoviral infection, which extends the list and functional scope of HSP70 chaperones. PMID:28367995

Plasmodium falciparum-Infected Erythrocytes Induce Granzyme B by NK Cells through Expression of Host-Hsp70

PubMed Central

Böttger, Evelyn; Multhoff, Gabriele; Kun, Jürgen F. J.; Esen, Meral

2012-01-01

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo. PMID:22438997

Plasmodium falciparum-infected erythrocytes induce granzyme B by NK cells through expression of host-Hsp70.

PubMed

Böttger, Evelyn; Multhoff, Gabriele; Kun, Jürgen F J; Esen, Meral

2012-01-01

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo.

Yeast Two-Hybrid and One-Hybrid Screenings Identify Regulators of hsp70 Gene Expression.

PubMed

Saito, Youhei; Nakagawa, Takanobu; Kakihana, Ayana; Nakamura, Yoshia; Nabika, Tomomi; Kasai, Michihiro; Takamori, Mai; Yamagishi, Nobuyuki; Kuga, Takahisa; Hatayama, Takumi; Nakayama, Yuji

2016-09-01

The mammalian stress protein Hsp105β, which is specifically expressed during mild heat shock and localizes to the nucleus, induces the major stress protein Hsp70. In the present study, we performed yeast two-hybrid and one-hybrid screenings to identify the regulators of Hsp105β-mediated hsp70 gene expression. Six and two proteins were detected as Hsp105β- and hsp70 promoter-binding proteins, respectively. A luciferase reporter gene assay revealed that hsp70 promoter activation is enhanced by the transcriptional co-activator AF9 and splicing mediator SNRPE, but suppressed by the coiled-coil domain-containing protein CCDC127. Of these proteins, the knockdown of SNRPE suppressed the expression of Hsp70 irrespective of the presence of Hsp105β, indicating that SNRPE essentially functions as a transcriptional activator of hsp70 gene expression. The overexpression of HSP70 in tumor cells has been associated with cell survival and drug resistance. We here identified novel regulators of Hsp70 expression in stress signaling and also provided important insights into Hsp70-targeted anti-cancer therapy. J. Cell. Biochem. 117: 2109-2117, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family

PubMed Central

2013-01-01

Background Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses

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.

Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population.

PubMed

Mardan-Nik, Maryam; Pasdar, Alireza; Jamialahmadi, Khadijeh; Biabangard-Zak, Atefeh; Mirhafez, Seyed Reza; Ghalandari, Marzieh; Tajfard, Mohammad; Mohebati, Mohsen; Esmaily, Habibollah; Ferns, Gordon A; Ghayour-Mobarhan, Majid

2014-10-25

Coronary artery disease (CAD) is an inflammatory process and a major cause of mortality and morbidity. The (heat shock protein70-2) HSP70-2 gene is reported to be associated with coronary artery disease possibly by affecting the regulation of pro-inflammatory cytokines such as TNF-α. The association between CAD and the HSP70-2 gene +1267A>G polymorphism has been studied in some populations but there are no data about this association in the Iranian population. We have investigated the association between the HSP70-2 gene +1267A>G polymorphism and angiographically defined CAD within an Iranian population. We determined the presence of the HSP70-2 gene +1267A>G polymorphism in 628 patients with CAD and 307 healthy individuals using PCR-RFLP. Of the patients, 433 (68%) had >50% stenosis (CAD+) and the remaining 195 patients had <50% stenosis (CAD-), based on coronary angiography. Angiogram positive patients were subdivided into three groups: those with single (n=113), double (n=134), and triple vessels (n=186) disease. A significant higher frequency of AG+GG genotypes (G allele carriers) was observed in angiogram positive and angiogram negative groups compared to controls in a dominant analysis model of the HSP70-2 gene +1267A>G position (51.2 vs. 43.2, P=0.002, OR=1.37) (51.0 vs. 43.2, P=0.01, OR=1.37). The allele frequency of the HSP70-2 G was also significantly higher in angiogram positive and angiogram negative groups compared to the control group (51.2 vs. 43.2, P=0.002, OR=1.37) (51.0 vs. 43.2, P=0.01, OR=1.37). These results suggest that HSP70-2 +1267 polymorphism may influence the risk of CAD in Iranian population, however further studies are needed to clarify the role of other HSP70-2 gene polymorphisms in the pathogenesis of the CAD. Copyright © 2014 Elsevier B.V. All rights reserved.

Different Relationship between hsp70 mRNA and hsp70 Levels in the Heat Shock Response of Two Salmonids with Dissimilar Temperature Preference

PubMed Central

Lewis, Mario; Götting, Miriam; Anttila, Katja; Kanerva, Mirella; Prokkola, Jenni M.; Seppänen, Eila; Kolari, Irma; Nikinmaa, Mikko

2016-01-01

The heat shock response (HSR) refers to the rapid production of heat shock proteins (hsps) in response to a sudden increase in temperature. Its regulation by heat shock factors is a good example of how gene expression is transcriptionally regulated by environmental stresses. In contrast, little is known about post-transcriptional regulation of the response. The heat shock response is often used to characterize the temperature tolerance of species with the rationale that whenever the response sets on, a species is approaching its lethal temperature. It has commonly been considered that an increase in hsp mRNA gives an accurate indication that the same happens to the protein level, but this need not be the case. With climate change, understanding the effects of temperature on gene expression of especially polar organisms has become imperative to evaluate how both biodiversity and commercially important species respond, since temperature increases are expected to be largest in polar areas. Here we studied the HSR of two phylogenetically related Arctic species, which differ in their temperature tolerance with Arctic charr having lower maximally tolerated temperature than Atlantic salmon. Arctic charr acclimated to 15°C and exposed to 7°C temperature increase for 30 min showed both an increase in hsp70 mRNA and hsp70 whereas in salmon only hsp70 mRNA increased. Our results indicate that the temperature for transcriptional induction of hsp can be different from the one required for a measurable change in inducible hsp level. The species with lower temperature tolerance, Arctic charr, are experiencing temperature stress already at the higher acclimation temperature, 15°C, as their hsp70 mRNA and hsp70 levels were higher, and they grow less than fish at 8°C (whereas for salmon the opposite is true). Consequently, charr experience more drastic heat shock than salmon. Although further studies are needed to establish the temperature range and length of exposure where hsp

Ethanol increases HSP70 concentrations in honeybee (Apis mellifera L.) brain tissue.

PubMed

Hranitz, John M; Abramson, Charles I; Carter, Richard P

2010-05-01

Previous research on the honeybee ethanol model established how acute ethanol exposure altered function at different levels of organization: behavior and learning, ecology, and physiology. The purpose of this study was to evaluate whether ethanol doses that affect honeybee behavior also induce a significant stress response, measured by heat shock protein 70 (HSP70) concentrations, in honeybee brain tissues. Experiment 1 examined how pretreatment handling influenced brain HSP70 concentrations in three pretreatment groups of bees; immediately after being collected, after being harnessed and fed, and after 22-24h in a harness. HSP70 concentrations did not differ among pretreatment groups within replicates, although we observed significantly different HSP70 concentrations between the two replicates. Experiment 2 investigated the relationship between ethanol dose and brain HSP70 concentrations. Bees were placed in seven experimental groups, the three pretreatment groups as in Experiment 1 and four ethanol-fed groups. Bees in ethanol treatments were fed 1.5M sucrose (control) and 1.5M sucrose-ethanol solutions containing 2.5, 5, and 10% ethanol, allowed to sit for 4h, and dissected brains were assayed for HSP70. We observed ethanol-induced increases in honeybee brain HSP70 concentrations from the control group through the 5% ethanol group. Only bees in the 5% ethanol group had HSP70 concentrations significantly higher than the control group. The inverted U-shaped ethanol dose-HSP70 concentration response curve indicated that ingestion of 2.5% ethanol and 5% ethanol stimulated the stress response, whereas ingestion of 10% ethanol inhibited the stress response. Doses that show maximum HSP70 concentration (5% ethanol) or HSP70 inhibition (10% ethanol) correspond to those (> or =5% ethanol) that also impaired honeybees in previous studies. We conclude that acute ethanol intoxication by solutions containing > or =5% ethanol causes significant ethanol-induced stress in brain

Unraveling the CHIP:Hsp70 complex as an information processor for protein quality control.

PubMed

VanPelt, Jamie; Page, Richard C

2017-02-01

The CHIP:Hsp70 complex stands at the crossroads of the cellular protein quality control system. Hsp70 facilitates active refolding of misfolded client proteins, while CHIP directs ubiquitination of misfolded client proteins bound to Hsp70. The direct competition between CHIP and Hsp70 for the fate of misfolded proteins leads to the question: how does the CHIP:Hsp70 complex execute triage decisions that direct misfolded proteins for either refolding or degradation? The current body of literature points toward action of the CHIP:Hsp70 complex as an information processor that takes inputs in the form of client folding state, dynamics, and posttranslational modifications, then outputs either refolded or ubiquitinated client proteins. Herein we examine the CHIP:Hsp70 complex beginning with the structure and function of CHIP and Hsp70, followed by an examination of recent studies of the interactions and dynamics of the CHIP:Hsp70 complex. Copyright © 2016 Elsevier B.V. All rights reserved.

Bos indicus cattle possess greater basal concentrations of HSP27, alpha B-crystallin, and HSP70 in skeletal muscle in vivo compared with cattle.

PubMed

Mullins, C R; Zerby, H N; Fitzpatrick, L A; Parker, A J

2016-01-01

The objectives of the present study were to evaluate the basal concentrations of heat shock proteins (HSP) between and cattle and to determine if HSP basal concentrations change as an animal matures. A total of 40 cattle were used in a 2 × 2 factorial design to evaluate the effects of genotype and age (heifers and mature cows) on basal concentrations of heat shock protein 27 (HSP27), α B-crystallin (Cryab), and heat shock protein 70 (HSP70). Each experimental group of 10 animals was sampled on a separate day over a period of 4 wk during July 2014. A muscle sample was collected from the longissimus thoracis (LT) and concentrations of HSP were quantified using ELISA. There were no significant differences in HSP concentration for the interaction between age and genotype or for age alone. cattle had greater ( < 0.05) basal concentrations of HSP27, Cryab, and HSP70 in the LT than cattle. The results of this study show that basal in vivo HSP concentrations differ between and cattle. However, further studies are needed to investigate the relationship between HSP concentrations and meat tenderness with respect to genotypes to see if HSP concentrations account for at least some variability in tenderness differences.

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

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

Yamagishi, Nobuyuki; Goto, Kazumasa; Nakagawa, Satomi

2010-09-10

Hsp105{alpha} and Hsp105{beta} are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105{alpha} is expressed constitutively in the cytoplasm of cells, while Hsp105{beta}, an alternatively spliced form of Hsp105{alpha}, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105{beta} but also Hsp105{alpha} accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105{alpha} and Hsp105{beta} with changes in the nuclearmore » localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105{alpha} and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105{alpha} and Hsp105{beta} on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105{alpha} and Hsp105{beta} suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.« less

Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation*

PubMed Central

Tranter, Michael; Helsley, Robert N.; Paulding, Waltke R.; McGuinness, Michael; Brokamp, Cole; Haar, Lauren; Liu, Yong; Ren, Xiaoping; Jones, W. Keith

2011-01-01

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3′-UTR. This shortening of the 3′-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3′-UTR relieves translational suppression observed in the long 3′-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression. PMID:21757701

In Vivo Suppression of Heat Shock Protein (HSP)27 and HSP70 Accelerates DMBA-Induced Skin Carcinogenesis by Inducing Antigenic Unresponsiveness to the Initiating Carcinogenic Chemical.

PubMed

Yusuf, Nabiha; Nasti, Tahseen H; Ahmad, Israr; Chowdhury, Sanim; Mohiuddin, Hasan; Xu, Hui; Athar, Mohammad; Timares, Laura; Elmets, Craig A

2015-05-15

Heat shock proteins (HSPs) are constitutively expressed in murine skin. HSP27 is present in the epidermis, and HSP70 can be found in both the epidermis and dermis. The purpose of this study was to investigate the role of these proteins in cutaneous chemical carcinogenesis and to determine whether their effects on cell-mediated immune function were a contributing factor. In vivo inhibition of HSP27 and HSP70 produced a reduction in the T cell-mediated immune response to 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene in C3H/HeN mice and resulted in a state of Ag-specific tolerance. When mice were pretreated with anti-HSP27 and anti-HSP70 Abs in vivo prior to subjecting them to a standard two-stage DMBA/12-O-tetradecanoylphorbol-13-acetate cutaneous carcinogenesis protocol, the percentage of mice with tumors was much greater (p < 0.05) in anti-HSP27- and HSP70-pretreated animals compared with mice pretreated with control Ab. Similar results were obtained when the data were evaluated as the cumulative number of tumors per group. Mice pretreated with HSP27 and HSP70 Abs developed more H-ras mutations and fewer DMBA-specific cytotoxic T lymphocytes. These findings indicate that in mice HSP27 and HSP70 play a key role in the induction of cell-mediated immunity to carcinogenic polyaromatic hydrocarbons. Bolstering the immune response to carcinogenic polyaromatic hydrocarbons may be an effective method for prevention of the tumors that they produce. Copyright © 2015 by The American Association of Immunologists, Inc.

Targeting HSP70 and GRP78 in canine osteosarcoma cells in combination with doxorubicin chemotherapy.

PubMed

Asling, Jonathan; Morrison, Jodi; Mutsaers, Anthony J

2016-11-01

Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

Role of Hsp-70 responses in cold acclimation of HUVEC-12 cells.

PubMed

Guan, Hao; Hu, Dahai; Zhao, Zhijing; Cai, Weixia; Zhou, Qin; Yang, Ximing; Yan, Ying; Zhu, Xiongxiang

2015-01-01

Endothelial recovery is a central feature of tissues after frostbite injuries. Thermo tolerance plays an important role in protecting cells against injury after frozen and thawing. The present study aimed to quantitatively assess the injury of human umbilical vein endothelial cells HUVEC-12 after repeated low temperature. Pretreatments (HUVEC-12) cells were repeatedly exposed to cold (1°C/min decrement to -20°C). Their proliferation, death, apoptosis, and protein and mRNA expressions of HSP70 were determined. Endothelial cells after repeated cold exposures were more resistant to apoptosis and necrosis than normal cells. The expressions of HSP70 in cells after repeated cold exposures were significantly higher than in normal HUVEC-12 cells (P < 0.05). Cold acclimation may induce the expression of HSP-70 which plays a protective role in the temperature tolerance.

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.

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.

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.

Slower skeletal muscle phenotypes are critical for constitutive expression of Hsp70 in overloaded rat plantaris muscle.

PubMed

O'Neill, David E T; Aubrey, F Kris; Zeldin, David A; Michel, Robin N; Noble, Earl G

2006-03-01

Heat shock protein 72 (Hsp70) is constitutively expressed in rat hindlimb muscles, reportedly in proportion to their content of type I myosin heavy chain. This distribution pattern has been suggested to result from the higher recruitment and activity of such muscles and/or a specific relationship between myosin phenotype and Hsp70 content. To differentiate between these possibilities, the fiber-specific distribution of Hsp70 was examined in male Sprague-Dawley rat plantaris under control conditions, following a fast-to-slow phenotypic shift in response to surgically induced overload (O) and in response to O when the phenotypic shift was prevented by 3,5,3'-triiodo-dl-thyronine administration. Constitutive expression of Hsp70 was restricted to type I and IIa fibers in plantaris from control rats, and this fiber-specific pattern of expression was maintained following O of up to 28 days, although Hsp70 content in the O muscle doubled. When O (for 40 days) of the plantaris was combined with 3,5,3'-triiodo-dl-thyronine administration, despite typical hypertrophy in the overloaded plantaris, prevention of the normal phenotypic transformation also blocked the increased expression of Hsp70 observed in euthyroid controls. Collectively, these data suggest that chronic changes in constitutive expression of Hsp70 with altered contractile activity appear critically dependent on fast-to-slow phenotypic remodeling.

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

PubMed

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

2016-03-01

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

Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4.

PubMed

Asea, Alexzander; Rehli, Michael; Kabingu, Edith; Boch, Jason A; Bare, Olivia; Auron, Philip E; Stevenson, Mary Ann; Calderwood, Stuart K

2002-04-26

Recent studies have initiated a paradigm shift in the understanding of the function of heat shock proteins (HSP). It is now clear that HSP can and do exit mammalian cells, interact with cells of the immune system, and exert immunoregulatory effects. We recently demonstrated that exogenously added HSP70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca(2+) flux, activate NF-kappaB, and up-regulate the expression of pro-inflammatory cytokines in human monocytes. Here for the first time, we report that HSP70-induced proinflammatory cytokine production is mediated via the MyD88/IRAK/NF-kappaB signal transduction pathway and that HSP70 utilizes both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion. These studies now pave the way for the development of highly effective pharmacological or molecular tools that will either up-regulate or suppress HSP70-induced functions in conditions where HSP70 effects are desirable (cancer) or disorders where HSP70 effects are undesirable (arthritis and arteriosclerosis).

High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis

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

Miao, Zongyu; Xu, Delin; Cui, Miao

2016-06-10

HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed bymore » EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl{sub 2} stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression. -- Highlights: •HMG protein ChDSP1 shows affinity to ChHSP70 promoter in Crassostrea hongkongensis. •ChDSP1 negatively regulates ChHSP70 transcription. •ChHSP70 and ChDSP1 transcriptions were coordinately induced by thermal/Cd stress. •ChDSP1 may contribute to the recovery of the induced ChHSP70 to its original state. •This is the first report regarding negative regulator of HSP70 transcription.« less

Drug- and/or trauma-induced hyperthermia? Characterization of HSP70 and myoglobin expression

PubMed Central

Rosinsky, Franziska; Trauer, Heiner; Schneider, Eckhardt; Dreßler, Jan; Franke, Heike

2018-01-01

Introduction Heat shock protein 70 (HSP70) expression could be discussed as an adaption that promotes repair and counteracts cell damage. Myoglobin is released upon muscle damage of several pathways. The purpose of the present study was to determine whether the expression of HSP70 in kidney, heart and brain and of myoglobin in the kidney were associated with the cause of death and the survival times after lethal intoxications with three of the drugs most widely used in our local area (Saxony, Germany) as well as after fatal traumatic brain injury (TBI). Methods We retrospectively collected kidney, heart and brain samples of 50 autopsy cases with toxicological proved lethal intoxication (main drugs methamphetamine, morphine, alcohol), 14 TBI cases and 15 fatalities with acute myocardial injury in age- and gender-matched compilations. Results Our main findings suggest that HSP70 is associated with hyperthermal and other stress factors of most cell populations. HSP70 expressions in kidney and heart muscle are useful for a differentiation between fatal intoxications and cases without toxicological influence (p < 0.05). There were significant differences in the cerebral expression patterns between methamphetamine- and morphine-associated deaths compared to alcohol fatalities (p < 0.05). An intensive staining of HSP70 in the pericontusional zone and the hippocampus after TBI (especially neuronal and vascular) was shown even after short survival times and may be useful as an additional marker in questions of vitality or wound age. A relevant myoglobin decoration of renal tubules was only shown for methamphetamine abuse in the study presented. Conclusion In sum, the immunohistochemical characteristics presented can be supportive for determining final death circumstances and minimal trauma survival times but are not isolated usefully for the detection of drug- or trauma-induced hyperthermia. PMID:29566034

HSP70 production patterns in coastal and estuarine organisms facing increasing temperatures

NASA Astrophysics Data System (ADS)

Madeira, D.; Narciso, L.; Cabral, H. N.; Vinagre, C.; Diniz, M. S.

2012-10-01

Heat shock proteins are important components in the cellular defense against proteotoxic stress. This work aimed to reveal HSP70 (hsc70 plus hsp70) expression patterns in several marine species (fish, crabs and shrimps) within a community along a temperature gradient and at the upper thermal limit. The organisms were collected in the Tagus estuary and adjacent shore (in Cabo Raso), Portugal. Exposure trials were performed using the critical thermal maximum (CTMax) method in order to recreate a stress gradient of ecological relevance. Protein analysis was performed using an enzyme linked immunosorbent assay (ELISA). Organisms within each community (estuary, coast; subtidal, intertidal, supratidal) responded in several different ways: no change in HSP70 levels, an increase in HSP70 levels, or increases and decreases in HSP70 levels. These patterns of response occurred independently of taxa, CTMax and habitat type. Magnitude of expression relates to the habitat's thermal conditions. Species from highly variable and hot habitats i.e. intertidal/supratidal zone, and living in greater shore heights produce higher amounts of HSP70. Demersal and subtidal species inhabit colder and more stable waters thus they seem to have a slower heat shock response. No clear pattern was observed for species of the same group (fish, crabs and shrimps) or congeneric species. HSP70 expression showed high intraspecific variability potentially due to genetic traits, environmental traits and condition status.

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

Organization and evolution of hsp70 clusters strikingly differ in two species of Stratiomyidae (Diptera) inhabiting thermally contrasting environments.

PubMed

Garbuz, David G; Yushenova, Irina A; Zatsepina, Olga G; Przhiboro, Andrey A; Bettencourt, Brian R; Evgen'ev, Michael B

2011-03-22

Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp) expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters. Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form tight clusters in this species. In the case of the other

Organization and evolution of hsp70 clusters strikingly differ in two species of Stratiomyidae (Diptera) inhabiting thermally contrasting environments

PubMed Central

2011-01-01

Background Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp) expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters. Results Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. Conclusions The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form tight clusters in this

Encapsulated Hsp70 decreases endotoxin-induced production of ROS and TNFα in human phagocytes.

PubMed

Yurinskaya, M M; Kochetkova, O Yu; Shabarchina, L I; Antonova, O Yu; Suslikov, A V; Evgen'ev, M B; Vinokurov, M G

2017-01-01

Human heat shock protein Hsp70 was experimentally inserted into polyelectrolyte microcapsules. Encapsulated recombinant Hsp70 was studied in terms of its effects on neutrophil apoptosis, the production of reactive oxygen species, and the secretion of tumor necrosis factor alpha by promonocytic THP-1 cells. It was found that encapsulated Hsp70 effectively inhibits neutrophil apoptosis, unlike free exogenous protein used in solution. In THP-1 cells, encapsulated and free Hsp70 reduced LPS-induced tumor necrosis factor alpha production with a similar efficiency. Encapsulated Hsp70 reduces LPS-induced reactive oxygen species production by neutrophils in the course of its release from the microcapsules but not as much as free Hsp70. Thus, the polyelectrolyte microcapsules can be used as containers for the effective delivery of Hsp70 to neutrophils and monocytes to significantly improve the functioning of the innate immune system.

Differential GFP expression patterns induced by different heavy metals in Tg(hsp70:gfp) transgenic medaka (Oryzias latipes).

PubMed

Ng, Grace Hwee Boon; Xu, Hongyan; Pi, Na; Kelly, Barry C; Gong, Zhiyuan

2015-06-01

Heat shock protein 70 (Hsp70) is one of the most widely used biomarker for monitoring environment perturbations in biological systems. To facilitate the analysis of hsp70 expression as a biomarker, we generated a Tg(hsp70:gfp) transgenic medaka line in which green fluorescence protein (GFP) reporter gene was driven by the medaka hsp70 promoter. Here, we characterized Tg(hsp70:gfp) medaka for inducible GFP expression by seven environment-relevant heavy metals, including mercury, arsenic, lead, cadmium, copper, chromium, and zinc. We found that four of them (mercury, arsenic, lead, and cadmium) induced GFP expression in multiple and different organs. In general, the liver, kidney, gut, and skin are among the most frequent organs to show induced GFP expression. In contrast, no detectable GFP induction was observed to copper, chromium, or zinc, indicating that the transgenic line was not responsive to all heavy metals. RT-qPCR determination of hsp70 mRNA showed similar induction and non-induction by these metals, which also correlated with the levels of metal uptake in medaka exposed to these metals. Our observations suggested that these heavy metals have different mechanisms of toxicity and/or differential bioaccumulation in various organs; different patterns of GFP expression induced by different metals may be used to determine or exclude metals in water samples tested. Furthermore, we also tested several non-metal toxicants such as bisphenol A, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 4-introphenol, and lindane; none of them induced significant GFP expression in Tg(hsp70:gfp) medaka, further suggesting that the inducibility of Tg(hsp70:gfp) for GFP expression is specific to a subset of heavy metals.

Induction of Hsp 70 in Vero cells in response to mycotoxins cytoprotection by sub-lethal heat shock and by Vitamin E.

PubMed

El Golli, Emna; Hassen, Wafa; Bouslimi, Amel; Bouaziz, Chayma; Ladjimi, M Moncef; Bacha, Hassen

2006-10-10

This paper analysed the toxicity mechanisms of several mycotoxins using Hsp 70 expression, cytoprotection of Vero cells by sub-lethal heat shock (sub-LHS) and Vitamin E. Our aim was (i) to determine whether Citrinin (CTN), Zearalenone (ZEN) and T2 toxin (T2) could induce the expression of Hsp 70, (ii) to check whether or not elevated levels of Hsp and Vitamin E pre-treatment could provide cytoprotection from these mycotoxins, and finally (iii) to emphasize the eventual involvement of oxidative stress on mycotoxin's toxicity. Our study demonstrated that the three examined mycotoxins induced Hsp 70 expression in a dose-dependent manner. A cytoprotective effect of Hsp 70 was obtained when Vero cells were exposed to sub-lethal heat shock followed by a 12h recovery prior to mycotoxins treatment and evidenced by a reduction of their cytolethality. This cytoprotection suggested that Hsp 70 might constitute an important cellular defence mechanism. A cytoprotective action was also obtained although at lesser extent, when cells were pre-treated with an antioxidant agent, the Vitamin E before mycotoxins treatment. This Vitamin E cytoprotection evoked the involvement of oxidative stress in mycotoxins induced toxicity, which was further, confirmed by the reduction of Hsp 70 expression when cells were pre-treated with Vitamin E prior to mycotoxins. Our data clearly shows that oxidative stress is certainly involved in the toxicity of the three studied mycotoxins, Citrinin, Zearalenone and T2 toxin and may therefore constitutes a relevant part in their toxicities; however, at variable extent from one mycotoxin to another.

Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica.

PubMed

Martynova, M G; Bystrova, O A; Shabelnikov, S V; Margulis, B A; Prokofjeva, D S

2007-04-01

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.

Hsp70 May Be a Molecular Regulator of Schistosome Host Invasion.

PubMed

Ishida, Kenji; Jolly, Emmitt R

2016-09-01

Schistosomiasis is a debilitating disease that affects over 240 million people worldwide and is considered the most important neglected tropical disease following malaria. Free-swimming freshwater cercariae, one of the six morphologically distinct schistosome life stages, infect humans by directly penetrating through the skin. Cercariae identify and seek the host by sensing chemicals released from human skin. When they reach the host, they burrow into the skin with the help of proteases and other contents released from their acetabular glands and transform into schistosomula, the subsequent larval worm stage upon skin infection. Relative to host invasion, studies have primarily focused on the nature of the acetabular gland secretions, immune response of the host upon exposure to cercariae, and cercaria-schistosomulum transformation methods. However, the molecular signaling pathways involved from host-seeking through the decision to penetrate skin are not well understood. We recently observed that heat shock factor 1 (Hsf1) is localized to the acetabular glands of infectious schistosome cercariae, prompting us to investigate a potential role for heat shock proteins (HSPs) in cercarial invasion. In this study, we report that cercarial invasion behavior, similar to the behavior of cercariae exposed to human skin lipid, is regulated through an Hsp70-dependent process, which we show by using chemical agents that target Hsp70. The observation that biologically active protein activity modulators can elicit a direct and clear behavioral change in parasitic schistosome larvae is itself interesting and has not been previously observed. This finding suggests a novel role for Hsp70 to act as a switch in the cercaria-schistosomulum transformation, and it allows us to begin elucidating the pathways associated with cercarial host invasion. In addition, because the Hsp70 protein and its structure/function is highly conserved, the model that Hsp70 acts as a behavior transitional

Hsp70 May Be a Molecular Regulator of Schistosome Host Invasion

PubMed Central

Ishida, Kenji; Jolly, Emmitt R.

2016-01-01

Schistosomiasis is a debilitating disease that affects over 240 million people worldwide and is considered the most important neglected tropical disease following malaria. Free-swimming freshwater cercariae, one of the six morphologically distinct schistosome life stages, infect humans by directly penetrating through the skin. Cercariae identify and seek the host by sensing chemicals released from human skin. When they reach the host, they burrow into the skin with the help of proteases and other contents released from their acetabular glands and transform into schistosomula, the subsequent larval worm stage upon skin infection. Relative to host invasion, studies have primarily focused on the nature of the acetabular gland secretions, immune response of the host upon exposure to cercariae, and cercaria-schistosomulum transformation methods. However, the molecular signaling pathways involved from host-seeking through the decision to penetrate skin are not well understood. We recently observed that heat shock factor 1 (Hsf1) is localized to the acetabular glands of infectious schistosome cercariae, prompting us to investigate a potential role for heat shock proteins (HSPs) in cercarial invasion. In this study, we report that cercarial invasion behavior, similar to the behavior of cercariae exposed to human skin lipid, is regulated through an Hsp70-dependent process, which we show by using chemical agents that target Hsp70. The observation that biologically active protein activity modulators can elicit a direct and clear behavioral change in parasitic schistosome larvae is itself interesting and has not been previously observed. This finding suggests a novel role for Hsp70 to act as a switch in the cercaria-schistosomulum transformation, and it allows us to begin elucidating the pathways associated with cercarial host invasion. In addition, because the Hsp70 protein and its structure/function is highly conserved, the model that Hsp70 acts as a behavior transitional

Hsp70/J-protein machinery from Glossina morsitans morsitans, vector of African trypanosomiasis

PubMed Central

Bentley, Stephen J.

2017-01-01

Tsetse flies (Glossina spp.) are the sole vectors of the protozoan parasites of the genus Trypanosoma, the causative agents of African Trypanosomiasis. Species of Glossina differ in vector competence and Glossina morsitans morsitans is associated with transmission of Trypanosoma brucei rhodesiense, which causes an acute and often fatal form of African Trypanosomiasis. Heat shock proteins are evolutionarily conserved proteins that play critical roles in proteostasis. The activity of heat shock protein 70 (Hsp70) is regulated by interactions with its J-protein (Hsp40) co-chaperones. Inhibition of these interactions are emerging as potential therapeutic targets. The assembly and annotation of the G. m. morsitans genome provided a platform to identify and characterize the Hsp70s and J-proteins, and carry out an evolutionary comparison to its well-studied eukaryotic counterparts, Drosophila melanogaster and Homo sapiens, as well as Stomoxys calcitrans, a comparator species. In our study, we identified 9 putative Hsp70 proteins and 37 putative J-proteins in G. m. morsitans. Phylogenetic analyses revealed three evolutionarily distinct groups of Hsp70s, with a closer relationship to orthologues from its blood-feeding dipteran relative Stomoxys calcitrans. G. m. morsitans also lacked the high number of heat inducible Hsp70s found in D. melanogaster. The potential localisations, functions, domain organisations and Hsp70/J-protein partnerships were also identified. A greater understanding of the heat shock 70 (Hsp70) and J-protein (Hsp40) families in G. m. morsitans could enhance our understanding of the cell biology of the tsetse fly. PMID:28902917

Neurotherapeutic activity of the recombinant heat shock protein Hsp70 in a model of focal cerebral ischemia in rats.

PubMed

Shevtsov, Maxim A; Nikolaev, Boris P; Yakovleva, Ludmila Y; Dobrodumov, Anatolii V; Dayneko, Anastasiy S; Shmonin, Alexey A; Vlasov, Timur D; Melnikova, Elena V; Vilisov, Alexander D; Guzhova, Irina V; Ischenko, Alexander M; Mikhrina, Anastasiya L; Galibin, Oleg V; Yakovenko, Igor V; Margulis, Boris A

2014-01-01

Recombinant 70 kDa heat shock protein (Hsp70) is an antiapoptotic protein that has a cell protective activity in stress stimuli and thus could be a useful therapeutic agent in the management of patients with acute ischemic stroke. The neuroprotective and neurotherapeutic activity of recombinant Hsp70 was explored in a model of experimental stroke in rats. Ischemia was produced by the occlusion of the middle cerebral artery for 45 minutes. To assess its neuroprotective capacity, Hsp70, at various concentrations, was intravenously injected 20 minutes prior to ischemia. Forty-eight hours after ischemia, rats were sacrificed and brain tissue sections were stained with 2% triphenyl tetrazolium chloride. Preliminary treatment with Hsp70 significantly reduced the ischemic zone (optimal response at 2.5 mg/kg). To assess Hsp70's neurotherapeutic activity, we intravenously administered Hsp70 via the tail vein 2 hours after reperfusion (2 hours and 45 minutes after ischemia). Rats were then kept alive for 72 hours. The ischemic region was analyzed using a high-field 11 T MRI scanner. Administration of the Hsp70 decreased the infarction zone in a dose-dependent manner with an optimal (threefold) therapeutic response at 5 mg/kg. Long-term treatment of the ischemic rats with Hsp70 formulated in alginate granules with retarded release of protein further reduced the infarct volume in the brain as well as apoptotic area (annexin V staining). Due to its high neurotherapeutic potential, prolonged delivery of Hsp70 could be useful in the management of acute ischemic stroke.

The effects of icariin on the expression of HIF-1α, HSP-60 and HSP-70 in PC12 cells suffered from oxygen-glucose deprivation-induced injury.

PubMed

Mo, Zhen-Tao; Li, Wen-Na; Zhai, Yu-Rong; Gao, Shu-Ying

2017-12-01

The effects of icariin, a chief constituent of flavonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown. To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability. PC12 cells were treated with icariin (10 -7 , 10 -6 or 10 -5  mol/L) for 3 h (1 h before oxygen-glucose deprivation (OGD) plus 2 h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 2 h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3 ± 1.9 ng/L, HSP-60: 199 ± 16 ng/L, HSP-70: 195 ± 17 ng/L, NSE: 1487 ± 125 ng/L), and cell viability was significantly decreased (0.26 ± 0.03), while icariin (10 -7 , 10 -6 , or 10 -5  mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1 ± 1.4, 22.6 ± 1.8, 15.7 ± 2.1, HSP-60: 100 ± 12, 89 ± 6, 113 ± 11, HSP-70: 139 ± 9, 118 ± 7, 95 ± 9 and NSE: 1121 ± 80, 1019 ± 52, 731 ± 88), and improved cell viability (0.36 ± 0.03, 0.38 ± 0.04, 0.37 ± 0.03) in OGD-treated PC12 cells. These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.

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

PubMed

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

2017-02-25

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

Potential contributions of heat shock proteins to temperature-dependent sex determination in the American alligator.

PubMed

Kohno, S; Katsu, Y; Urushitani, H; Ohta, Y; Iguchi, T; Guillette, L J

2010-01-01

Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be 'reversed' by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90alpha, HSP90beta, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90 alpha (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed. (c) 2009 S. Karger AG, Basel.

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

PubMed

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

2017-07-11

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

Functional Organization of hsp70 Cluster in Camel (Camelus dromedarius) and Other Mammals

PubMed Central

Garbuz, David G.; Astakhova, Lubov N.; Zatsepina, Olga G.; Arkhipova, Irina R.; Nudler, Eugene; Evgen'ev, Michael B.

2011-01-01

Heat shock protein 70 (Hsp70) is a molecular chaperone providing tolerance to heat and other challenges at the cellular and organismal levels. We sequenced a genomic cluster containing three hsp70 family genes linked with major histocompatibility complex (MHC) class III region from an extremely heat tolerant animal, camel (Camelus dromedarius). Two hsp70 family genes comprising the cluster contain heat shock elements (HSEs), while the third gene lacks HSEs and should not be induced by heat shock. Comparison of the camel hsp70 cluster with the corresponding regions from several mammalian species revealed similar organization of genes forming the cluster. Specifically, the two heat inducible hsp70 genes are arranged in tandem, while the third constitutively expressed hsp70 family member is present in inverted orientation. Comparison of regulatory regions of hsp70 genes from camel and other mammals demonstrates that transcription factor matches with highest significance are located in the highly conserved 250-bp upstream region and correspond to HSEs followed by NF-Y and Sp1 binding sites. The high degree of sequence conservation leaves little room for putative camel-specific regulatory elements. Surprisingly, RT-PCR and 5′/3′-RACE analysis demonstrated that all three hsp70 genes are expressed in camel's muscle and blood cells not only after heat shock, but under normal physiological conditions as well, and may account for tolerance of camel cells to extreme environmental conditions. A high degree of evolutionary conservation observed for the hsp70 cluster always linked with MHC locus in mammals suggests an important role of such organization for coordinated functioning of these vital genes. PMID:22096537

The expression and correlation of Hsp 70 and Hsp 27 in serous middle ear effusion fluids of pediatric patients-a preliminary study.

PubMed

Min, Hyun Jin; Choe, Ji Won; Chang, Moon Young; Kim, Kyung Soo; Lee, Sei Young; Mun, Seog-Kyun

2017-10-01

Several cytokines and innate immune-associated molecules are present in middle ear effusions, but damage-associated molecular patterns (DAMPs) in middle ear effusion have not been studied. Therefore, we evaluated the role of heat shock proteins (Hsps) in the development of otitis media with effusion (OME). Serous middle ear effusions from 22 pediatric patients who were diagnosed with OME and underwent ventilation tube insertion from June 2015 to March 2017 were evaluated in our study. The levels of Hsp 90, 70, 27, IL-8, and TNF-α in effusion fluids were evaluated by enzyme-linked immunosorbent assays. The associations between the levels of these molecules and the degree of tympanic membrane inflammation were statistically evaluated. Finally, the relationships among these molecules were also evaluated. Hsp 70 and Hsp 27 were detected in all middle ear effusions, but Hsp 90 was detected in only five effusion fluid samples. IL-8 was also detected in all middle ear effusions, but TNF-α was detected in only four effusion fluid samples. When we compared the degree of tympanic membrane inflammation with the levels of Hsp 70, Hsp 27, and IL-8, which were detected in all effusion fluids, we could not find statistical significance. However, Hsp 70, Hsp 27, and IL-8 were significantly associated with each other (p < 0.05). Hsp 70 and Hsp 27 were expressed in middle ear effusions. Furthermore, the levels of Hsp 70 and Hsp 27 were positively correlated with each other, and were also positively associated with the neutrophil chemoattractant, IL-8. Our findings suggested that Hsp 70 and Hsp 27 might be involved in the pathophysiology of pediatric OME. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Heat shock protein 70 (Hsp70) interacts with the Notch1 intracellular domain and contributes to the activity of Notch signaling in myelin-reactive CD4 T cells.

PubMed

Juryńczyk, Maciej; Lewkowicz, Przemysław; Domowicz, Małgorzata; Mycko, Marcin P; Selmaj, Krzysztof W

2015-10-15

Notch receptors (Notch1-4) are involved in the differentiation of CD4 T cells and the development of autoimmunity. Mechanisms regulating Notch signaling in CD4 T cells are not fully elucidated. In this study we investigated potential crosstalk between Notch pathway molecules and heat shock protein 70 (Hsp70), the major intracellular chaperone involved in the protein transport during immune responses and other stress conditions. Using Hsp70(-/-) mice we found that Hsp70 is critical for up-regulation of NICD1 and induction of Notch target genes in Jagged1- and Delta-like1-stimulated CD4 T cells. Co-immunoprecipitation analysis of wild-type CD4 T cells stimulated with either Jagged1 or Delta-like1 showed a direct interaction between NICD1 and Hsp70. Both molecules co-localized within the nucleus of CD4 T cells stimulated with Notch ligands. Molecular interaction and nuclear colocalization of NICD1 and Hsp70 were also detected in CD4 T cells reactive against myelin oligodendrocyte glycoprotein (MOG)35-55, which showed Hsp70-dependent up-regulation of both NICD1 and Notch target genes. In conclusion, we demonstrate for the first time that Hsp70 interacts with NICD1 and contributes to the activity of Notch signaling in CD4 T cells. Interaction between Hsp70 and NICD1 may represent a novel mechanism regulating Notch signaling in activated CD4 T cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Heat shock proteins HSP70 and MRJ cooperatively regulate cell adhesion and migration through urokinase receptor.

PubMed

Lin, Yuli; Peng, Nana; Zhuang, Hongqin; Zhang, Di; Wang, Yao; Hua, Zi-Chun

2014-08-30

The urokinase-type plasminogen activator receptor (uPAR) is an important regulator of ECM proteolysis, cell-ECM interactions and cell signaling. uPAR and heat shock proteins HSP70 and MRJ (DNAJB6) have been implicated in tumor growth and metastasis. We have reported recently that MRJ (DNAJB6, a heat shock protein) can interact with uPAR and enhance cell adhesion. Here, we identified another heat shock protein HSP70 as a novel uPAR-interacting protein. We performed co-immunoprecipitation in human embryonic kidney (HEK) 293 and colon cancer HCT116 cells as well as immunofluorence assays in HEK293 cells stably transfected with uPAR to investigate the association of suPAR with HSP70/MRJ. To understand the biological functions of the triple complex of suPAR/HSP70/MRJ, we determined whether HSP70 and/or MRJ regulated uPAR-mediated cell invasion, migration, adhesion to vitronectin and MAPK pathway in two pair of human tumor cells (uPAR negative HEK293 cells vs HEK293 cells stably transfected with uPAR and HCT116 cells stably transfected with antisense-uPAR vs HCT116 mock cells transfected with vector only) using transwell assay, wound healing assay, quantitative RT-PCR analyzing mmp2 and mmp9 transcription levels, cell adhesion assay and Western blotting assay. HSP70 and MRJ formed a triple complex with uPAR and over-expression of MRJ enhanced the interaction between HSP70 and uPAR, while knockdown of MRJ decreased soluble uPAR in HCT116 cells (P < 0.05) and reduced the formation of the triple complex, suggesting that MRJ may act as an uPAR-specific adaptor protein to link uPAR to HSP70. Further experiments showed that knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR-mediated cell adhesion to vitronectin as well as suppressed cell invasion and migration. Knockdown of HSP70 and/or MRJ inhibited expression of invasion related genes mmp2 and mmp9. Finally, HSP70 and/or MRJ up-regulated phosphorylation levels of ERK1/2 and FAK suggesting MAPK pathway was involved

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion.

PubMed

Garg, Manoj; Kanojia, Deepika; Seth, Amlesh; Kumar, Rajive; Gupta, Anju; Surolia, Avadhesha; Suri, Anil

2010-01-01

Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma.

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta).

PubMed

Tedeschi, J N; Kennington, W J; Berry, O; Whiting, S; Meekan, M; Mitchell, N J

2015-01-01

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34°C and 36°C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36°C, 3h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression of stress response HSP70 gene in Asian paddle crabs, Charybdis japonica, exposure to endocrine disrupting chemicals, bisphenol A (BPA) and 4-nonylphenol (NP)

NASA Astrophysics Data System (ADS)

Park, Kiyun; Kwak, Ihn-Sil

2013-06-01

The Asian paddle crab, Charybdis japonica, is a potential bio-indicator reflecting marine sediment toxicity as well as a commercially important species living along coastal areas in Korea. This study investigated its stress response by looking at the heat shock protein (HSP70) gene of C. japonica when the organism is exposed to bisphenol A (BPA) and 4-nonylphenol (NP). We characterized partial sequence of HSP70 as the stressresponse gene of C. japonica. The nucleotide sequence of C. japonica HSP70 is over 90% homologous with the corresponding gene of other crabs. Phylogenetic tree analysis revealed a close relationship between C. japonica HSP70 and HSP70 in other species of lobster and shrimps. HSP70 mRNA transcripts were detected in all the examined tissues of C. japonica, with the highest level in gills, the organ that most frequently came into contact with the external BPA or NP-laden water. As no reference data were available for C. japonica crab exposure, the BPA and NP 24-h LC50 values have not been previously determined. The expression of the C. japonica HSP70 gene to various BPA or NP concentrations during short and longer times was assessed. Gene expression was significantly induced in concentration- and time-dependent manners after BPA or NP exposures. These results support the postulation that crab C. japonica HSP70 could be a potential stress response molecular marker to monitor marine ecosystems.

Non-Lethal Heat Shock of the Asian Green Mussel, Perna viridis, Promotes Hsp70 Synthesis, Induces Thermotolerance and Protects Against Vibrio Infection.

PubMed

Aleng, Nor Afiqah; Sung, Yeong Yik; MacRae, Thomas H; Abd Wahid, Mohd Effendy

2015-01-01

Mild heat stress promotes thermotolerance and protection against several different stresses in aquatic animals, consequences correlated with the accumulation of heat shock protein 70 (Hsp70). The purpose of this study was to determine if non-lethal heat shock (NLHS) of the Asian green mussel, Perna viridis, an aquatic species of commercial value, promoted the production of Hsp70 and enhanced its resistance to stresses. Initially, the LT50 and LHT for P. viridis were determined to be 42°C and 44°C, respectively, with no heat shock induced death of mussels at 40°C or less. Immunoprobing of western blots revealed augmentation of constitutive (PvHsp70-1) and inducible (PvHsp70-2) Hsp70 in tissue from adductor muscle, foot, gill and mantel of P. viridis exposed to 38°C for 30 min followed by 6 h recovery, NLHS conditions for this organism. Characterization by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that PvHsp70-1 and PvHsp70-2 respectively corresponded most closely to Hsp70 from P. viridis and Mytilus galloprovincialis. Priming of adult mussels with NLHS promoted thermotolerance and increased resistance to V. alginolyticus. The induction of Hsp70 in parallel with enhanced thermotolerance and improved protection against V. alginolyticus, suggests Hsp70 functions in P. viridis as a molecular chaperone and as a stimulator of the immune system.

Functional evaluation of Heat Shock Proteins 70 (HSP70/HSC70) on Rhodnius prolixus (Hemiptera, Reduviidae) physiological responses associated with feeding and starvation.

PubMed

Paim, Rafaela M M; Araujo, Ricardo N; Leis, Miguel; Sant'anna, Mauricio R V; Gontijo, Nelder F; Lazzari, Claudio R; Pereira, Marcos H

2016-10-01

Blood-sucking vectors must overcome thermal stress caused by intake of proportionally large amounts of warm blood from their hosts. In response to this, Heat Shock Proteins (HSPs) such as the widely studied HSP70 family (the inducible HSP70 and the cognate form HSC70, known for their role in preserving essential cellular functions) are rapidly up-regulated in their tissues. The triatomine Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative pathogen of Chagas' disease, and is also a model organism for studying insect biology and physiology. In this work, we observed that the expression of Rhodnius prolixus HSP70 was rapidly up-regulated in response to thermal shocks (0 °C and 40 °C) and also during the first hours after feeding on blood. HSP70/HSC70 RNAi knockdown elicited important alterations in R. prolixus physiological responses triggered by blood meal and starvation. HSP70/HSC70 knockdown insects showed lower resistance to prolonged starvation in comparison to appropriate controls, dying between 32 and 40 days after dsRNA injection. After blood feeding, the physiological effects of HSP70/HSC70 knockdown were more prominent and the insects died even earlier, within 14-20 days after feeding (21-27 days after dsRNA injection). These bugs showed impaired blood processing and digestion, reduced energetic metabolism and the midgut immune responses were compromised. Our findings suggest that HSP70/HSC70 depletion affected R. prolixus in starvation or fed conditions. After feeding, the arrival of blood in the digestive tract of knockdown insects fails to activate essential signaling pathways involved in blood processing, producing several alterations in their physiological processes enough to generate a premature death. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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

PubMed

Gunter, Helen M; Degnan, Bernard M

2007-08-01

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

Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation*

PubMed Central

Chotewutmontri, Prakitchai; Bruce, Barry D.

2015-01-01

Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences. PMID:25645915

Cloning of heat shock protein genes (hsp70, hsc70 and hsp90) and their expression in response to larval diapause and thermal stress in the wheat blossom midge, Sitodiplosis mosellana.

PubMed

Cheng, Weining; Li, Dan; Wang, Yue; Liu, Yang; Zhu-Salzman, Keyan

2016-12-01

Sitodiplosis mosellana Géhin, one of the most important pests of wheat, undergoes obligatory diapause as a larva to survive unfavorable temperature extremes during hot summers and cold winters. To explore the potential roles of heat shock proteins (hsp) in this process, we cloned full-length cDNAs of hsp70, hsc70 and hsp90 from S. mosellana larvae, and examined their expression in response to diapause and short-term temperature stresses. Three hsps included all signature sequences of corresponding protein family and EEVD motifs. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known classification of insects. Expression of hsp70 and hsp90 were highly induced by diapause, particularly pronounced during summer and winter. Interestingly, hsp70 was more strongly expressed in summer than in winter whereas hsp90 displayed the opposite pattern. Abundance of hsc70 mRNA was comparable prior to and during diapauses and was highly up-regulated when insects began to enter the stage of post-diapause quiescence. Heat-stressed over-summering larvae (⩾30°C) or cold-stressed over-wintering larvae (⩽0°C) could further elevate expression of these three genes, but temperature extremes i.e. as high as 45°C or as low as -15°C failed to trigger such expression patterns. Notably, hsp70 was most sensitive to heat stress and hsp90 was most sensitive to cold stress. These results suggested that hsp70 and hsp90 play key roles in diapause maintenance and thermal stress; the former may be more prominent contributor to heat tolerance and the latter for cold tolerance. In contrast, hsc70 most likely is involved in developmental transition from diapause to post-diapause quiescence, and thus may serve as a molecular marker to predict diapause termination. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heat shock protein 70-2 (HSP70-2) is a novel therapeutic target for colorectal cancer and is associated with tumor growth.

PubMed

Jagadish, Nirmala; Parashar, Deepak; Gupta, Namita; Agarwal, Sumit; Suri, Vaishali; Kumar, Rajive; Suri, Vitusha; Sadasukhi, Trilok Chand; Gupta, Anju; Ansari, Abdul S; Lohiya, Nirmal Kumar; Suri, Anil

2016-07-29

Colorectal cancer (CRC) is the third leading cause of cancer related deaths worldwide both in men and women. Our recent studies have indicated an association of heat shock protein 70-2 (HSP70-2) with bladder urothelial carcinoma. In the present study, we investigated the association of HSP70-2 with various malignant properties of colorectal cancer cells and clinic-pathological features of CRC in clinical specimens. HSP70-2 mRNA and protein was investigated expression by RT-PCR, immunohistochemistry, immunofluorescence, flow cytometry and Western blotting in CRC clinical specimens and COLO205 and HCT116 cell lines. Plasmid-based gene silencing approach was employed to study the association of HSP70-2 with various malignant properties of COLO205 and HCT116 cells in in vitro and with tumor progression in in vivo COLO205 human xenograft mice model. HSP70-2 expression was detected in 78 % of CRC patients irrespective of various stages and grades by RT-PCR and IHC. Our analysis further revealed that HSP70-2 expression was detected in both COLO205 and HCT116 cell lines. Ablation of HSP70-2 expression resulted in reduced cellular growth, colony forming ability, migratory and invasive ability of CRC cells. In addition, ablation of HSP70-2 expression showed significant reduction in tumor growth in COLO205 human xenograft in in vivo mouse model. Collectively, our results indicate that HSP70-2 is associated with CRC clinical specimens. In addition, down regulation of HSP70-2 expression reduces cellular proliferation and tumor growth indicating that HSP70-2 may be a potential therapeutic target for CRC treatment.

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

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.

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

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.

Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction.

PubMed

Dhamad, Ahmed E; Zhou, Zhenqi; Zhou, Jianhong; Du, Yuchun

2016-01-01

Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90β) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2-3 times more abundant than Hsp90β in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17β-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.

Targeting the testis-specific heat-shock protein 70-2 (HSP70-2) reduces cellular growth, migration, and invasion in renal cell carcinoma cells.

PubMed

Singh, Swarnendra; Suri, Anil

2014-12-01

Renal cell carcinoma (RCC) represents one of the most resistant tumors to radiotherapy and chemotherapy. Current therapies for the RCC patients are limited owing to lack of diagnosis and therapeutic treatments. Testis-specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, has been shown to be associated with various cancers. In the present study, we investigated the putative role of HSP70-2 in various malignant properties of the RCC cells. HSP70-2 messenger RNA (mRNA) and protein expression was investigated in A704, ACHN, and Caki-1 cells derived from the RCC patients. We assessed the expression of HSP70-2 gene and protein by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The expression of HSP70-2 protein was further validated by performing indirect immunofluorescence (IIF) and flow cytometry. The malignant properties of high-grade invasive A704 and Caki-1 cells, such as cellular proliferation, colony formation, migration, invasion, and wound healing, were evaluated by silencing the expression of HSP70-2 gene in these cells. Statistical significance was defined using Student's t test. Our RT-PCR and Western blotting data showed the expression of HSP70-2 in all RCC cells. Our results showed that HSP70-2 was predominantly expressed in cytoplasm and found to be colocalized with endoplasmic reticulum, mitochondria, Golgi body, and plasma membrane but not the nuclear envelope. Knockdown of HSP70-2 expression with specific short hairpin RNA (shRNA) demonstrated significant reduction in cell growth and colony formation. Further, a marked reduction in cell migration and invasion was also observed, indicating the potential role of HSP70-2 in metastasis. Collectively, our data suggest that HSP70-2 plays a key role in cancerous growth and invasive potential of RCC cells. Thus, HSP70-2 could serve as a novel potential therapeutic target for the RCC.

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

Critical analysis of eukaryotic phylogeny: a case study based on the HSP70 family.

PubMed

Germot, A; Philippe, H

1999-01-01

Trichomonads, together with diplomonads and microsporidia, emerge at the base of the eukaryotic tree, on the basis of the small subunit rRNA phylogeny. However, phylogenies based on protein sequences such as tubulin are markedly different with these protists emerging much later. We have investigated 70 kDa heat-shock protein (HSP70), which could be a reliable phylogenetic marker. In eukaryotes, HSP70s are found in cytosol, endoplasmic reticulum, and organelles (mitochondria and chloroplasts). In Trichomonas vaginalis we identified nine different HSP70-encoding genes and sequenced three nearly complete cDNAs corresponding to cytosolic, endoplasmic reticulum, and mitochondrial-type HSP70. Phylogenies of eukaryotes were reconstructed using the classical methods while varying the number of species and characters considered. Almost all the undoubtedly monophyletic groups, defined by ultrastructural characters, were recovered. However, due to the long branch attraction phenomenon, the evolutionary rates were the main factor determining the position of species, even with the use of a close outgroup, which is an important advantage of HSP70 with respect to many other markers. Numerous variable sites are peculiar to Trichomonas and probably generated the artefactual placement of this species at the base of the eukaryotes or as the sister group of fast-evolving species. The inter-phyla relationships were not well supported and were sensitive to the reconstruction method, the number of species; and the quantity of information used. This lack of resolution could be explained by the very rapid diversification of eukaryotes, likely after the mitochondrial endosymbiosis.

Loss of the Inducible Hsp70 Delays the Inflammatory Response to Skeletal Muscle Injury and Severely Impairs Muscle Regeneration

PubMed Central

Howard, Travis M.; Ahn, Bumsoo; Ferreira, Leonardo F.

2013-01-01

depending on the nature and severity of muscle injury, therapeutics which differentially target both intracellular and extracellular localized Hsp70 may optimally preserve muscle tissue and promote muscle functional recovery. PMID:23626847

Diversity of cytosolic HSP70 Heat Shock Protein from decapods and their phylogenetic placement within Arthropoda.

PubMed

Baringou, Stephane; Rouault, Jacques-Deric; Koken, Marcel; Hardivillier, Yann; Hurtado, Luis; Leignel, Vincent

2016-10-10

The 70kDa heat shock proteins (HSP70) are considered the most conserved members of the HSP family. These proteins are primordial to the cell, because of their implications in many cellular pathways (e. g., development, immunity) and also because they minimize the effects of multiple stresses (e. g., temperature, pollutants, salinity, radiations). In the cytosol, two ubiquitous HSP70s with either a constitutive (HSC70) or an inducible (HSP70) expression pattern are found in all metazoan species, encoded by 5 or 6 genes (Drosophila melanogaster or yeast and human respectively). The cytosolic HSP70 protein family is considered a major actor in environmental adaptation, and widely used in ecology as an important biomarker of environmental stress. Nevertheless, the diversity of cytosolic HSP70 remains unclear amongst the Athropoda phylum, especially within decapods. Using 122 new and 311 available sequences, we carried out analyses of the overall cytosolic HSP70 diversity in arthropods (with a focus on decapods) and inferred molecular phylogenies. Overall structural and phylogenetic analyses showed a surprisingly high diversity in cytosolic HSP70 and revealed the existence of several unrecognised groups. All crustacean HSP70 sequences present signature motifs and molecular weights characteristic of non-organellar HSP70, with multiple specific substitutions in the protein sequence. The cytosolic HSP70 family in arthropods appears to be constituted of at least three distinct groups (annotated as A, B and C), which comprise several subdivisions, including both constitutive and inducible forms. Group A is constituted by several classes of Arthropods, while group B and C seem to be specific to Malacostraca and Hexapoda/Chelicerata, respectively. The HSP70 organization appeared much more complex than previously suggested, and far beyond a simple differentiation according to their expression pattern (HSC70 versus HSP70). This study proposes a new classification of cytosolic

Hsp70 and gama-Semino protein as possible prognostic marker of prostate cancer.

PubMed

Kumar, Sanjay; Gurshaney, Sanjeev; Adagunodo, Yori; Gage, Erica; Qadri, Shezreen; Sharma, Mahak; Malik, Shalie; Manne, Upender; Singh, Udai P; Singh, Rajesh; Mishra, Manoj K

2018-06-01

In the United States, Prostate Cancer (PCa) is the leading cause of cancer-related mortality in men. PCa resulted in abnormal growth and function of prostate gland such as secretion of high level of gamma-seminoprotein (gama-SM)/Prostate-Specific Antigen (PSA) which could be detected in the blood. Beside gama-SM protein, the levels of heat shock proteins (Hsp70) were also observed significantly high. Therefore, gama-SM and Hsp70 are unique proteins with high potential for PCa therapeutics and diagnostics. High level of Hsp70 suppresses apoptosis, thus allowing PCa cells to exist; however, depletion of Hsp70 induces apoptosis in PCa cells. Gama-SM is the most prominent biomarker for PCa screening; however, its accuracy is still questionable. Thus, a more suitable streamline biomarker for PCa screening is urgently needed. Hsp70 and gama-SM proteins could be used as a revolutionary biomarker for PCa, and could help to identify possible therapeutic target(s). In this review article we will discuss the relationship between the Hsp70 and gama-SM proteins with PCa, their potential as a dual biomarker, and the possibility for both proteins being used as therapeutic targets.

Cytotoxicity and the induction of the stress protein Hsp 70 in Chang liver cells in response to zearalenone-induced oxidative stress.

PubMed

Lee, Hyungkyoung; Kang, Changgeun; Yoo, Yong-San; Hah, Do-Yun; Kim, Chung Hui; Kim, Euikyung; Kim, Jong Shu

2013-09-01

Zearalenone (ZEN) has been implicated in several cases of mycotoxicosis in farm animals and humans. The toxic effects of ZEN have been well characterized, but little is known regarding the mechanisms of ZEN toxicity, including the involvement of the oxidative stress pathway. Using Chang liver cells as a model, the aim of this study was to determine if ZEN could elevate the expression of the heat shock protein Hsp 70, induce cytotoxicity and modulate the levels of glutathione (GSH) and thiobarbituric acid reactive substance (TBARS). In addition, the cytoprotective effects of N-acetylcysteine amide (NACA) pre-treatment were assessed. Finally, the involvement of oxidative stress in ZEN-induced toxicity was confirmed. The results of this study demonstrated that ZEN-induced Hsp 70 expression in a dose- and time-dependent manners. This effect occurred at low-ZEN concentrations, and could therefore be considered a biomarker of ZEN-induced toxicity. The cytotoxicity was reduced when Chang liver cells were exposed to sub-lethal heat shock prior to ZEN treatment, demonstrating a cytoprotective effect of Hsp 70. This cytoprotective effect suggested that Hsp 70 might play a key role in the cellular defense mechanism. When cells were pre-treated with NACA prior to ZEN treatment, the cells were also protected from toxicity. This NACA cytoprotective effect suggested the involvement of oxidative stress in ZEN-induced toxicity, and this mechanism was supported by reduced Hsp 70 expression, inhibited cytolethality, increased GSH levels and decreased TBARS formation when cells were pre-treated with NACA prior to ZEN exposure. Our data clearly demonstrated that ZEN induced cytotoxicity in Chang liver cells by inhibiting cell proliferation, decreasing GSH levels and increasing TBARS formation in a dose-dependent manner. ZEN also, induced Hsp 70 expression, and the side effects of ZEN were significantly alleviated by pre-treatment with NACA. Oxidative stress is likely to be one of the

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

PubMed

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

2013-11-01

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

TAAR 6 and HSP-70 variations associated with bipolar disorder.

PubMed

Pae, Chi-Un; Drago, Antonio; Mandelli, Laura; De Ronchi, Diana; Serretti, Alessandro

2009-11-20

We report on the impact of a set of variations located in the HSP-70 (heat shock protein 70) and TAAR6 (trace amine associated receptors 6 gene) in a sample of bipolar patients. Holding a diagnosis of BPD was the first outcome measure. Response to pharmacotreatment in bipolar patients was the secondary outcome measure. One hundred seventy-one bipolar patients and 288 controls were enrolled for the study. Patients were administered HAM-D, YMRS and CGI at baseline and discharge by independent psychiatrists blind to genotypes. As a result, homozygosis at rs2075799 (HSP-70) was found to be more represented in controls than in cases (p=0.000009). The investigated variations did not show impact on treatment outcome. This study provides preliminary evidence that HSP-70 may play a role in the disrupted mechanisms that lead to BPD. Further confirmatory analyses in this direction are mandatory.

Expression of HSP 70 and its mRNAS during ischemia-reperfusion in the rat bladder.

PubMed

Saito, Motoaki; Tominaga, Lika; Nanba, Eiji; Kinoshita, Yukako; Housi, Daisuke; Miyagawa, Ikuo; Satoh, Keisuke

2004-08-27

HSP 70 is an important protein that repairs damaged tissue after injury. In the present study, we investigated the expression of HSP 70 and its mRNAs during ischemia-reperfusion in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder dome. Male Wistar rats, 8 weeks old, were divided into six groups: controls, 30-min ischemia, 30-min ischemia and 30-, 60-minute, 1- and 7-day reperfusion, groups A, B, C, D, E, and F, respectively. In functional studies, contractile responses to carbachol were measured in these groups. The expression of HSP 70-1/2 mRNAs was quantified using a real-time PCR method, and that of HSP 70 proteins was measured using ELISA in the bladders. In the functional study, Emax values of carbachol to bladders in the A, B, C, D, E and F groups were 9.3 +/- 1.3, 7.9 +/- 1.7, 4.3 +/- 0.8, 4.2 +/- 0.7, 4.5 +/- 0.6, and 8.1 +/- 1.2 g/mm2, respectively. In the control group, the expression of HSP 70-1/2 mRNA was detected, and the expression of HSP 70-1 mRNAs was significantly higher than that of HSP 70-2 mRNAs in each group. The expression of HSP 70-1 mRNA increased in groups B and C, but decreased in groups D, E, and F. The expression of HSP 70-2 mRNA in group C was significantly higher than that of groups A, D, E, and F. The expression of HSP 70-1/2 mRNAs after 1 day or 1 week of reperfusion was similar to control levels. The expression of HSP 70 proteins was increased shortly after the expression of their mRNAs. The expression of HSP 70 after 1 day or 1 week of reperfusion was almost identical to control levels. Our data indicate that contractile responses of the bladder were decreased by ischemia reperfusion, and that expression of HSP 70 and its mRNAs appeared to increase after a short period of the insult.

[A study on the expression of HSP70 in endothelial cells pretreated with ethanol and fluid shear stress].

PubMed

Yan, Hong-tao; Zhang, Yi; Liao, Ga; Zhang, Kui; Li, Bin; Wang, Ye; Liao, Zhi-gang

2006-07-01

To detect whether ethanol can affect the expression of HSP70 in endothelial cells under fluid shear stress. Ethanol at different concentrations was added to the culture medium of endothelial cells, EA. Hy926, which was treated statically or under 1Pa fluid shear stress. After the incubation of 1 h, 2 h, 4 h and 6 h, the expression of HSP70 was detected by immunohistochemical method(SP). In the control group, the expression of HSP70 was negative under static state, while it was positive under 1Pa fluid shear stress lasting 4 h even without ethanol. No statistic difference was found between the 50 mg/dL ethanol group and the control group with the same treatment time of fluid shear stress. HSP70 expression was found under static state with 150 mg/dL ethanol after 4 h or 300 mg/dL ethanol after 2 h respectively. The expression increased greatly under 1Pa fluid shear stress in the same ethanol concentrations. Medium to high ethanol concentration in coordination with fluid shear stress can strongly stimulates the expression of HSP70 by a kinetic mechanism of time-dependent.

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.

secHsp70 as a tool to approach amyloid-β42 and other extracellular amyloids.

PubMed

De Mena, Lorena; Chhangani, Deepak; Fernandez-Funez, Pedro; Rincon-Limas, Diego E

2017-07-03

Self-association of amyloidogenic proteins is the main pathological trigger in a wide variety of neurodegenerative disorders. These aggregates are deposited inside or outside the cell due to hereditary mutations, environmental exposures or even normal aging. Cumulative evidence indicates that the heat shock chaperone Hsp70 possesses robust neuroprotection against various intracellular amyloids in Drosophila and mouse models. However, its protective role against extracellular amyloids was largely unknown as its presence outside the cells is very limited. Our recent manuscript in PNAS revealed that an engineered form of secreted Hsp70 (secHsp70) is highly protective against toxicity induced by extracellular deposition of the amyloid-β42 (Aβ42) peptide. In this Extra View article, we extend our analysis to other members of the heat shock protein family. We created PhiC31-based transgenic lines for human Hsp27, Hsp40, Hsp60 and Hsp70 and compared their activities in parallel against extracellular Aβ42. Strikingly, only secreted Hsp70 exhibits robust protection against Aβ42-triggered toxicity in the extracellular milieu. These observations indicate that the ability of secHsp70 to suppress Aβ42 insults is quite unique and suggest that targeted secretion of Hsp70 may represent a new therapeutic approach against Aβ42 and other extracellular amyloids. The potential applications of this engineered chaperone are discussed.

Hsp70-1: upregulation via selective phosphorylation of heat shock factor 1 during coxsackieviral infection and promotion of viral replication via the AU-rich element.

PubMed

Qiu, Ye; Ye, Xin; Hanson, Paul J; Zhang, Huifang Mary; Zong, Jeff; Cho, Brian; Yang, Decheng

2016-03-01

Coxsackievirus B3 (CVB3) is the primary pathogen of viral myocarditis. Upon infection, CVB3 exploits the host cellular machineries, such as chaperone proteins, to benefit its own infection cycles. Inducible heat shock 70-kDa proteins (Hsp70s) are chaperone proteins induced by various cellular stress conditions. The internal ribosomal entry site (IRES) within Hsp70 mRNA allows Hsp70 to be translated cap-independently during CVB3 infection when global cap-dependent translation is compromised. The Hsp70 protein family contains two major members, Hsp70-1 and Hsp70-2. This study showed that Hsp70-1, but not Hsp70-2, was upregulated during CVB3 infection both in vitro and in vivo. Then a novel mechanism of Hsp70-1 induction was revealed in which CaMKIIγ is activated by CVB3 replication and leads to phosphorylation of heat shock factor 1 (HSF1) specifically at Serine 230, which enhances Hsp70-1 transcription. Meanwhile, phosphorylation of Ser230 induces translocation of HSF1 from the cytoplasm to nucleus, thus blocking the ERK1/2-mediated phosphorylation of HSF1 at Ser307, a negative regulatory process of Hsp70 transcription, further contributing to Hsp70-1 upregulation. Finally, we demonstrated that Hsp70-1 upregulation, in turn, stabilizes CVB3 genome via the AU-rich element (ARE) harbored in the 3' untranslated region of CVB3 genomic RNA.

Albumin stimulates renal tubular inflammation through an HSP70-TLR4 axis in mice with early diabetic nephropathy

PubMed Central

Jheng, Huei-Fen; Tsai, Pei-Jane; Chuang, Yi-Lun; Shen, Yi-Ting; Tai, Ting-An; Chen, Wen-Chung; Chou, Chuan-Kai; Ho, Li-Chun; Tang, Ming-Jer; Lai, Kuei-Tai A.; Sung, Junne-Ming; Tsai, Yau-Sheng

2015-01-01

ABSTRACT Increased urinary albumin excretion is not simply an aftermath of glomerular injury, but is also involved in the progression of diabetic nephropathy (DN). Whereas Toll-like receptors (TLRs) are incriminated in the renal inflammation of DN, whether and how albumin is involved in the TLR-related renal inflammatory response remains to be clarified. Here, we showed that both TLR2 and TLR4, one of their putative endogenous ligands [heat shock protein 70 (HSP70)] and nuclear factor-κB promoter activity were markedly elevated in the kidneys of diabetic mice. A deficiency of TLR4 but not of TLR2 alleviated albuminuria, tubulointerstitial fibrosis and inflammation induced by diabetes. The protection against renal injury in diabetic Tlr4−/− mice was associated with reduced tubular injuries and preserved cubilin levels, rather than amelioration of glomerular lesions. In vitro studies revealed that albumin, a stronger inducer than high glucose (HG), induced the release of HSP70 from proximal tubular cells. HSP70 blockade ameliorated albumin-induced inflammatory mediators. HSP70 triggered the production of inflammatory mediators in a TLR4-dependent manner. Moreover, HSP70 inhibition in vivo ameliorated diabetes-induced albuminuria, inflammatory response and tubular injury. Finally, we found that individuals with DN had higher levels of TLR4 and HSP70 in the dilated tubules than non-diabetic controls. Thus, activation of the HSP70-TLR4 axis, stimulated at least in part by albumin, in the tubular cell is a newly identified mechanism associated with induction of tubulointerstitial inflammation and aggravation of pre-existing microalbuminuria in the progression of DN. PMID:26398934

The Molecular Chaperone HSP70 Binds to and Stabilizes NOD2, an Important Protein Involved in Crohn Disease*

PubMed Central

Mohanan, Vishnu; Grimes, Catherine Leimkuhler

2014-01-01

Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. PMID:24790089

[Hsp70 Genes of the Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) Parasitic Wasp].

PubMed

Chuvakova, L N; Sharko, F S; Nedoluzhko, A V; Polilov, A A; Prokhorchuk, E B; Skryabin, K G; Evgen'ev, M B

2017-01-01

Miniaturization is an evolutionary process that is widely represented in both invertebrates and vertebrates. Miniaturization frequently affects not only the size of the organism and its constituent cells, but also changes the genome structure and functioning. The structure of the main heat shock genes (hsp70 and hsp83) was studied in one of the smallest insects, the Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) parasitic wasp, which is comparable in size with unicellular organisms. An analysis of the sequenced genome has detected six genes that relate to the hsp70 family, some of which are apparently induced upon heat shock. Both induced and constitutively expressed hsp70 genes contain a large number of introns, which is not typical for the genes of this family. Moreover, none of the found genes form clusters, and they are all very heterogeneous (individual copies are only 75-85% identical), which indicates the absence of gene conversion, which provides the identity of genes of this family in Drosophila and other organisms. Two hsp83 genes, one of which contains an intron, have also been found in the M. amalphitanum genome.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

PubMed

Mandal, Soumit Sankar; Merz, Dale R; Buchsteiner, Maximilian; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

2017-06-06

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

PubMed Central

Mandal, Soumit Sankar; Buchsteiner, Maximilian; Dima, Ruxandra I.; Rief, Matthias; Žoldák, Gabriel

2017-01-01

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD’s ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements. PMID:28533394

Andrographolide induces degradation of mutant p53 via activation of Hsp70.

PubMed

Sato, Hirofumi; Hiraki, Masatsugu; Namba, Takushi; Egawa, Noriyuki; Baba, Koichi; Tanaka, Tomokazu; Noshiro, Hirokazu

2018-05-22

The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein (Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

4-Phenylbutyrate stimulates Hsp70 expression through the Elp2 component of elongator and STAT-3 in cystic fibrosis epithelial cells.

PubMed

Suaud, Laurence; Miller, Katelyn; Panichelli, Ashley E; Randell, Rachel L; Marando, Catherine M; Rubenstein, Ronald C

2011-12-30

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0-24 h with 1 mM 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA.

4-Phenylbutyrate Stimulates Hsp70 Expression through the Elp2 Component of Elongator and STAT-3 in Cystic Fibrosis Epithelial Cells*

PubMed Central

Suaud, Laurence; Miller, Katelyn; Panichelli, Ashley E.; Randell, Rachel L.; Marando, Catherine M.; Rubenstein, Ronald C.

2011-01-01

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0–24 h with 1 mm 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA. PMID:22069317

Hsp70 Expression Profile in Preeclampsia Model of Pregnant Rat (Rattus norvegicus) after Giving the EVOO

NASA Astrophysics Data System (ADS)

Irianti, E.; ilyas, S.; Rosidah; Hutahaean, S.

2017-03-01

Heat shock protein (Hsp) has long been known to protect cells from oxidative stress. In this case an increased expression is found on several cases of preeclampsia. One of the efforts to prevent preeclampsia is by giving antioxidants such as Extra Virgin Olive Oil (EVOO) or it’s better known as olive oil (Oleoa europaea), in the form of extra virgin known for its rich antioxidant content of tocopherols (vitamin E). The purpose of this study is to determine the expression levels of Hsp70 serum on pregnant white rat model of preeclampsia after being given EVOO. This type of research is true experiment; the subjects were female white rats and male virgin with Sprague Dawley, ± 8-11 weeks old, 180g BB s / d 200g, healthy and didn’t show any physical defects. Samples were 25 animals, divided into 5 groups, which consisted of different control and treatment given to T2 (rat model of preeclampsia), T3 (rat model of preeclampsia + EVOO 0.45g/bw/day), T4 (rat model of preeclampsia + EVOO 0.9g/bw/day) and T5 (rat model of preeclampsia + EVOO 1.8g/bw/day). The determination of each group was done by simple random sampling. Result on serum levels of Hsp70 that were tested by Elisa test in rats showed the average control was 14.64 mg / ml, group T2: 22:51 mg/ml, T3: 13.62 mg/ml, T4: 15.92 mg/ml, T5: 16:09 mg/ml. ANOVA test showed the P value was 0.001 <0.005, which meant there were significant differences on serum Hsp70 levels in the control and treatment pregnant rats group. It was known that there was a significant difference level of Hsp70 serum in group of control rats with T2 (P value <0.001) after LSD test was conducted, but not so with the group T3, T4, and T5, where the difference was not significant. There was a significant difference in the levels of Hsp70 serum on group T2 and T3 (P value 0.000), T4 (0004), T5 (0000). The gift of EVOO in the treatment group which was given EVOO with even low doses was able to control the induction of Hsp70 serum levels, which

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.

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

PubMed

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

2007-04-01

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

Heat shock proteins (Hsp70) and water content in the estivating Mediterranean Grunt Snail (Cantareus apertus).

PubMed

Reuner, Andy; Brümmer, Franz; Schill, Ralph O

2008-09-01

Pulmonate land snails often are able to estivate to survive dry hot seasons were water and food are scarce. The aperture of the shell is closed with an epiphragm, and metabolism is depressed to approximately one fourth of basal metabolism. We investigated a molecular aspect of estivation focussing on the heat shock protein 70 (Hsp70) stress response during estivation in the Mediterranean Grunt Snail Cantareus apertus. Sequences of a new inducible hsp70 and of actin are presented and expression of the hsp70 gene as well as Hsp70 protein content was measured in estivating animals. Both Hsp70 protein and mRNA do not show a significant change from the control, although there is a trend that hsp70 mRNA is less abundant in estivating specimens. After heat shock, the expression of hsp70 increased and a higher Hsp70 protein content was detected. Water relations were also investigated. After a period of 6 months in the dormant state, the snails contained 14% less water than active ones, implying a constricted protection against desiccation, compared to the desert snail Sphincterochila zonata, and a Mediterranean-type water economy.

HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1).

PubMed

Ding, Xuan Z; Feng, Xiao R; Borschel, Richard H; Nikolich, Mikeljon P; Feng, Jie; Li, Yan S; Hoover, David L

2010-06-01

Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 microg/ml) and sphingosine kinase inhibitor (SKI, 5 microM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death. Published by Elsevier Inc.

Influence of encapsulated heat shock protein HSP70 on the basic functional properties of blood phagocytes.

PubMed

Kochetkova, O Yu; Yurinskaya, M M; Evgen'ev, M B; Zatsepina, O G; Shabarchina, L I; Suslikov, A V; Tikhonenko, S A; Vinokurov, M G

2015-11-01

Microencapsulated heat shock proteins HSP 70 were studied in terms of their effects on neutrophil apoptosis, production of reactive oxygen species, and secretion of TNF-α by human neurtrophils and monocytes. Encapsulated HSP70 inhibited neutrophil apoptosis by 65% as compared to the effect of nonencapsulated HSP70; TNF-α production by the promonocytic THP-1 cells was similarly inhibited by the non-encapsulated and encapsulated HSP70. Thus, the polyelectrolyte micromolecules can be used as containers for effective delivery of HSP70 up to neutrophils and monocytes to correct the innate immunity functions.

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

PubMed

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

2013-11-01

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