Protective Effect Of Hsp70 Research Articles

Conformational dynamics of free and membrane-bound human Hsp70 in model cytosolic and endo-lysosomal environments

The binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen–deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87–118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.

Роль білків теплового шоку в механізмах нейродегенерації та нейропротекції при первинній глаукомі: огляд літератури

Heat shock proteins (HSP) are important components of the defense mechanism that increases the survival of body cells in adverse conditions due to antiapoptotic and cytoprotective effects. Since their discovery, numerous studies and experimental models have proved the role of HSPs as a key link in the processes of both repair and coagulation of proteins, as well as in the protection of cells from oxidative stress. The potential for pharmacological induction of HSPs in the human body makes them an attractive therapeutic target for many neurodegenerative diseases. This review examines the role of HSPs, especially fraction 70, in the mechanisms of neuroprotection of retinal ganglion cells in primary open-angle glaucoma being one of the common neurodegenerative diseases that can lead to complete loss of visual functions. A number of studies have shown the protective effect of HSP70 on retinal ganglion cells in animals with artificially induced glaucoma. But in the course of experiments on animal models, it was also proved that direct immunization with HSP through intravitreal injections induced pressure-independent degeneration of retinal ganglion cells. This indicates the need for indirect stimulation of HSP70 in order to activate their neuroprotective properties. To date, there are insufficient data on the circulation of HSP70 in the body of a person with glaucoma. These data indicate the prospects for further study of the role of HSP70 in glaucoma degeneration and elucidation of the ways of their mediated induction. Keywords: heat shock protein, HSP70, glaucoma, ganglion cells, retina, neuroprotection.

Heat shock protein 70 expression protects against sepsis-associated cardiomyopathy by inhibiting autophagy.

Increasing evidence suggests that heat shock protein 70 (Hsp70) has a protective effect in sepsis-induced cardiomyopathy; however, the protective mechanism remains unclear. Previous studies have also implicated autophagy in sepsis-induced cardiomyopathy. The aim of the current study was to reveal the protective mechanisms of Hsp70 in sepsis-induced cardiomyopathy using a cecal ligation and puncture (CLP) rat sepsis model. The roles of Hsp70 and autophagy in sepsis-induced cardiomyopathy were investigated by pretreating rats with the Hsp70 inhibitor quercetin or the autophagy inhibitor 3-methyladenine (3-Ma) before CLP. We also investigated the protective mechanisms of Hsp70 and the relationship between Hsp70 and autophagy in vitro by stimulating H9c2 cells with lipopolysaccharide (LPS) to simulate sepsis. The result show that inhibition of Hsp70 promoted sepsis-induced death in rats, while inhibition of autophagy inhibited sepsis-induced death. These results suggested that both Hsp70 and autophagy were involved in sepsis-induced cardiomyopathy. Overexpression of Hsp70 in H9c2 myocardial cells in vitro suppressed LPS-induced apoptosis, while inhibition of autophagy with 3-Ma also decreased LPS-induced H9c2 cell apoptosis, suggesting that the protective effect of Hsp70 in sepsis-induced cardiomyopathy was related to autophagy regulation. Overall, these results suggested that Hsp70 protected against sepsis-induced cardiac impairment by attenuating sepsis-induced autophagy.

Leucinostatin acts as a co-inducer for heat shock protein 70 in cultured canine retinal pigment epithelial cells

Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 co-inducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.

Neuroprotective Effects of Heat Shock Protein70.

Heat Shock Proteins (HSPs) increase response to many stresses in cells. Stroke is a neural shock that leads to the destruction of a large number of brain cells, whereas induction and expression of HSPs can decrease the amount of damage, and in some conditions can cure damaged cells. HSP70 family is considered as the most important member of HSPs in normal and stress condition of cells. They are strongly up-regulated by stresses and have protective roles in under stressed cells. Therefore, in this review, we briefly consider the association between HSP70 and stroke. We searched in Pubmed and Scopus databases using the specified keywords and selected the articles based on the certain association between HSP70 and stroke. HSP70 protects cells from damage through a variety of cellular and biochemical processes such as chaperone function, anti-apoptotic, anti-necrotic and anti-inflammatory mechanisms. Protective effects of HSP70 in neurodegenerative shocks are illustrated in the review, and it can be concluded that the induction of HSP70 in stresses can be considered as a therapeutic factor, although it needs further studies.

The increased expression of the inducible Hsp70 (HSP70A1A) in serum of patients with heart failure and its protective effect against the cardiotoxic agent doxorubicin.

The aim of this study was to examine the potential association between the expression of Hsp70 protein and heart failure and to investigate the possible protective effect of Hsp70 against the doxorubicin-induced toxicity. Initially, at clinical level, the expression levels of the inducible Hsp70 were quantified in serum from patients with heart failure. Our results showed that in heart failure, Hsp70 concentration appeared to be increased in blood sera of patients compared to that of healthy individuals. The enhanced expression of Hsp70 in serum of patients with heart failure seemed to be associated with various features, such as gender, age and the type of heart failure, but not with its etiology. Next, in our study at cellular level, we used primary cell cultures isolated from embryos of Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70 and wild-type mice (F1/F1). After exposure to a wide range of doxorubicin concentrations and incubation times, the dose- and time-dependent toxicity of the drug, which appeared to be reduced in Tg/Tg cells, was demonstrated. In addition, doxorubicin administration appeared to result in a dose- and time-dependent decrease in the activity of two of the major endogenous antioxidant enzymes (SOD and GPx). The increased activity of these enzymes in Tg/Tg cells compared to the control F1/F1 cells was obvious, suggesting that the presence of Hsp70 confers enhanced tolerance against DOX-induced oxidative stress. Overall, it has been indicated that Hsp70 protein exerts a very important protective action and renders cells more resistant to the harmful effects of doxorubicin.

Protective effects of HSP70, HO-1 and HSP27 on spermatogenic function in rats with varicocele

Objective To investigate the protective effects of HSP70、HO-1 and HSP27 on spermatogenic function in rats with varicocele. Methods Forty adult male SD rats were randomly divided into 4 groups with 10 rats in each group: sham group (group A), sham + quercetin group (group B), VC group (group C) and VC+ quercetin group (group D). An experimental varicocele model was successfully created in group C and D and quercetin(100 mg/kg) was injected intraperitoneally. HE staining was used to observe the pathological changes of testis. Malondialdehyde (MDA) and superoxide dismutase (SOD) content were determined. Cell apoptosis index was detected by dUTP nick end labeling. The expression of HSP70 in testis tissue was detected by immunohistochemistry. The mRNA expression of HSP27 and HO-1 in testis tissue was measured by RT-PCR. Results Testicular structure and spermatogenic function were seriously affected, and histological damage was significantly increased caused by VC in group D. AI was (5.26±0.45)% , (5.83±0.52)% , (14.64±0.98)% , (25.37±1.46)%, respectively.The SOD activities were(0.96±0.06), (0.88±0.05), (0.45±0.04), (0.23±0.03) U/mg protein, MDA content was(2.24±0.35), (2.69±0.32), (4.78±0.41), (7.37±0.68) nmol/g protein.The expression of HSP70 in group C was significantly higher than that in group A and B, and the expression of HSP70 in group D was significantly lower than that of group C. Compared with group A and B, the mRNA expression level of HSP27 and HO-1 was significantly increased in group C. Compared with group C, the expression level of HSP27 and HO-1 in group D was significantly decreased.The comparison between B、C and D groups was statistically significant (P<0.05). Conclusions HSP70、HO-1 and HSP27 reduces the level of oxidative stress and inhibits the apoptosis.Thus, it plays a protective role on the spermatogenic function in rats with varicocele. Key words: Varicocele; Disease Models, Animal; HSC70 Heat-Shock Proteins; Heat-Shock Proteins; Rats

Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress

Heat shock protein 70 (Hsp70) can physically interact with and prevent thermal inactivation of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) 1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. This study examined whether Hsp70 could physically interact with and prevent thermal inactivation of SERCA2a, the SERCA isoform expressed in heart. HEK-293 cells were cotransfected with cDNAs encoding human Hsp70 and rabbit SERCA2a (S2a/Hsp70). Cells cotransfected with SERCA2a cDNA and pMT2 (S2a/pMT2) were used as control. One-half of the cells was heat shocked at 40 degrees C for 1 h (HS), and one-half was maintained at 37 degrees C before harvesting the cells and isolating microsomes. Western blot analysis showed that Hsp70 and SERCA2a were colocalized in the microsomal fraction. The levels of Hsp70 were approximately fivefold higher (P < 0.05) in S2a/Hsp70 compared with S2a/pMT2 and approximately twofold higher (P < 0.05) following HS in all cells. Coimmunoprecipitation demonstrated that Hsp70 directly binds to SERCA2a. Following HS, maximal SERCA2a activity was reduced ( approximately 52%, P < 0.05) in S2a/pMT2 but was increased ( approximately 33%, P < 0.05) in S2a/Hsp70. Thermal inactivation of SERCA2a in S2a/pMT2 was associated with decreased ( approximately 49%, P < 0.05) binding capacity for fluorescein isothiocyanate (FITC) and increased carbonyl ( approximately 42%, P < 0.05) and nitrotyrosine ( approximately 40%, P < 0.05) levels in SERCA2a. By contrast, the HS-induced increase in maximal SERCA2a activity observed in S2a/Hsp70 corresponded with no change (P > 0.05) in FITC-binding capacity and reductions in carbonyl ( approximately 40%, P < 0.05) and nitrotyrosine ( approximately 23%, P < 0.05) levels in SERCA2a compared with S2a/pMT2. These results show that Hsp70 forms a protective interaction with SERCA2a during HS actually reducing oxidation and nitrosylation of SERCA2a thus increasing its maximal activity.

Protective effect of HSP70 on gastric mucosal cells against apoptosis

Protective effect of HSP70 on gastric mucosal cells against apoptosis

Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na(+),K(+),2Cl(-)-cotransporter (NKCC1) to RVI. Hypertonic stress induced caspase-3 activity in WEHI cells and iMEFs, an effect potentiated by Hsp70 in WEHI cells but inhibited by Hsp70 in iMEFs. Osmotic shrinkage-induced PCD was associated with Hsp70-inhibitable cysteine cathepsin release in iMEFs and attenuated by caspase and cathepsin inhibitors in WEHI cells. Treatment with TNF-alpha or the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced the viability of WEHI cells further under isotonic and mildly, but not severely, hypertonic conditions. Thus, it is concluded that shrinkage-induced PCD involves both caspase- and cathepsin-dependent death mechanisms and is potently counteracted by Hsp70.

Downstream caspases are novel targets for the antiapoptotic activity of the molecular chaperone Hsp70

The response of cancer cells to apoptosis-inducing agents can be characterized by 2 opposing factors, the proapoptotic caspase cascade and the antiapoptotic stress protein Hsp70. We show here that these factors interact in U-937 leukemia cells induced to apoptosis with anticancer drugs, etoposide and adriamycin (ADR). The protective effect of Hsp70 was verified using 2 approaches: mild heat stress and transfection-mediated overexpression of the Hsp70 gene. The increase in Hsp70 levels attained by these 2 methods was found to postpone caspase activation for 12-18 hours. An in vitro assay was developed using mouse myeloma NS0/1 cells, which lack the expression of Hsp70. Measurement of DEVD-ase activity in extracts of apoptotic NS0/1 cells incubated with purified Hsp70 showed that Hsp70 reduced caspase activity by up to 50% of its control value in a dose-dependent manner. The hypothesis that the inhibitory effect of Hsp70 on caspase-3/7 activity related to a direct interaction between Hsp70 and the caspases was tested by reciprocal immunoprecipitations and Far-western analyses. These tests were performed with extracts of Hsp70-overexpressing, control, and ADR-treated U-937 cells and using anti-caspase-3, caspase-7, and anti-Hsp70 antibodies, and the data clearly showed that Hsp70 was able to interact with the proforms of these caspases in cell lysates and with reconstituted purified proteins but did not bind the activated forms of either caspase-3 or -7. This association was also corroborated by a novel, enzyme-linked immunosorbent assay-like assay, protein interaction assay, that combined the advantages of immunoprecipitation and immunoblotting in a 96-well microplate-based assay. Thus, Hsp70 may act to suppress caspase-dependent apoptotic signaling through binding the precursor forms of both caspase-3 and caspase-7 and preventing their maturation.

Heat Shock Protein 70 Protects from Caspase-Independent Programmed Cell Death via Suppression of Stress Kinase JNK.

METHODS. To increase levels of Hsp70 we used adenovirus expressing human Hsp72 under control of tet-promoter, and adenovirus expressing dominant-negative mutant of SEK1 was used to inhibit JNK activation. Cell death was assessed by acridine orange/ethidium bromide staining under fluorescent microscope, or by poly-ADP ribose (PARP) cleavage and caspase-3 activation by immunoblotting. Activities of MAP kinases (ERK1/2, p38, and JNK) were measured by immunoblotting with antibodies to phosphorylated (activated) form of the kinases. RESULTS. Although the protective effect of Hsp70 against caspase-dependent apoptosis is well established, little is known about its role in caspase-independent cell death. We studied the effect of Hsp70 in two models of caspase-independent cell death: heat-induced killing of normal human fibroblasts and death of myogenic cells after transient ATP depletion. Morphology of heat-treated IMR90 diploid fibroblasts resembled that of apoptotic cells (e.g., after TNF or staurosporine treatment), and the plasma membrane remained intact. Morphology of H9c2 myogenic cells after transient ATP depletion was also similar to that of apoptotic cells, but some cells lost plasma membrane integrity. Both forms of cell death were not accompanied either by caspase-3 activation or PARP cleavage. Moreover, pancaspase inhibitor zVAD-fmk did not protect either from heat-induced killing of fibroblasts or ischemic death of H9c2 cells. Both heating and transient energy deprivation activated several MAP kinases, Erk1/2, p38, and JNK in these cells. As with classical apoptosis, inhibition of kinase, Erk1/2, decreased cell survival in both models. In contrast, inhibition of JNK (but not p38) almost completely protected from heat-induced killing of fibroblasts and markedly reduced death of H9c2 after energy deprivation. These types of caspase-independent cell death were effectively prevented by increased levels of Hsp70. Hsp70 overexpression greatly diminished activation of JNK after heat shock or transient energy deprivation; on the other hand, it did not increase activity of Erk1/2. Therefore, the protection of cells from the caspase

Protective effects of hsp70 in inflammation.

Inflammation results from the recruitment to a given tissue or organ and the activation of leucocytes, among which the monocytes-macrophages play a major role. These phagocytic cells produce high levels of reactive oxygen species (ROS) as well as cytokines. Whereas both ROS and cytokines have the potential to regulate the expression of heat shock (HS)/stress proteins (HSP), it appears that these proteins in turn have the ability to protect cells and tissues from the deleterious effects of inflammation. The mechanisms by which such protection occurs include prevention of ROS-induced DNA strand breaks and lipid peroxidation as well as protection from mitochondrial structure and function. In vivo, HS protects organs against a number of lesions associated with the increased production of ROS and/or cytokines. In an animal model for adult respiratory distress syndrome, an acute pulmonary inflammatory condition, HS completely prevented mortality. HSP (hsp70 in particular) may also exert protective effects in the immune system by contributing to the processing and presentation of bacterial and tumoral antigens. The analysis of the expression of hsp70 may prove of diagnostic and prognostic value in inflammatory conditions and therapeutical applications are being considered.