Heat shock cognate protein 70 is a cell fusion-enhancing factor but not an entry factor for human T-cell lymphotropic virus type I.

Heat shock cognate protein 70 (HSC70) has been shown to bind to the peptide corresponding to amino acids 197 to 216 of human T-cell lymphotropic virus type I (HTLV-I) envelope protein, gp46, and an anti-HSC70 monoclonal antibody (mAb) inhibits HTLV-I-induced syncytium formation. These findings suggest that HSC70 is necessary for the entry of HTLV-I into its target cells. Here we showed that HSC70 directly binds to gp46 by co-immunoprecipitation of HSC70 and gp46 from HTLV-I-producing human T-cell lysate. However, transduction of human HSC70 cDNA into BaF3 cells, which were found to be highly resistant to HTLV-I infection, did not support the HTLV-I entry, and HSC70 expressed in NIH3T3 cells, which were found to be almost resistant to syncytium formation upon cocultivation with HTLV-I-producing cells but sensitive to infection with cell-free HTLV-I, enhanced cell fusion induced by HTLV-I-producing cells, but did not enhance the entry of cell-free HTLV-I into these cells. The mAb against HSC70 inhibited syncytium formation in NIH3T3 cells expressing HSC70, but showed little effect on infection of these cells with cell-free HTLV-I. These findings indicate that HSC70 markedly enhances syncytium formation induced by HTLV-I but does not facilitate HTLV-I entry into target cells.     

N-myristoyltransferase inhibitor protein is homologous to heat shock cognate protein 70

Many of viral and eukaryotic proteins are required for signal transduction and regulatory functions which undergo a lipid modification by the enzyme N-myristoyltransferase (NMT). In this study, we demonstrated that heat shock cognate protein 70 (HSC70) is homologous to NMT inhibitor protein (NIP71), which was discovered in our laboratory, based on MALDI-TOF mass spectrometric analysis. The purified bovine cytosolic HSC70 and particulate NIP71 produced a dose-dependent inhibition of human NMT having half maximal inhibitions of 235 and 230 nM, respectively. Further, Western blot analysis revealed that the purified particulate NIP71 and cytosolic HSC70 cross-reacted with both anti-NIP71 and anti-HSC70 antibodies. The results we obtained imply that molecular chaperones could be involved in the regulation of NMT in normal and cancerous cells. Further studies directed to revealing the role of HSC70 in the regulation of NMT may lead to the development of gene based therapies of colon cancer.     

Heat shock cognate protein 70 controls Borna disease virus replication via interaction with the viral non-structural protein X

Borna disease virus (BDV) is a non-segmented, negative-sense RNA virus and has the property of persistently infecting the cell nucleus. BDV encodes a 10-kDa non-structural protein, X, which is a negative regulator of viral polymerase activity but is essential for virus propagation. Recently, we have demonstrated that interaction of X with the viral polymerase cofactor, phosphoprotein (P), facilitates translocation of P from the nucleus to the cytoplasm. However, the mechanism by which the intracellular localization of X is controlled remains unclear. In this report, we demonstrate that BDV X interacts with the 71 kDa molecular chaperon protein, Hsc70. Immunoprecipitation assays revealed that Hsc70 associates with the same region of X as P and, interestingly, that expression of P interferes competitively with the interaction between X and Hsc70. A heat shock experiment revealed that BDV X translocates into the nucleus, dependent upon the nuclear accumulation of Hsc70. Furthermore, we show that knockdown of Hsc70 by short interfering RNA decreases the nuclear localization of both X and P and markedly reduces the expression of viral genomic RNA in persistently infected cells. These data indicate that Hsc70 may be involved in viral replication by regulating the intracellular distribution of X.     

Heat shock protein 70 expression in myocardium is blunted with simulated weightlessness

Exposure of cardiac cells to a mild thermal or ischaemic stress, sufficient to induce HSP expression, protects them against a subsequent exposure to a more severe ischaemic stress. Over expression of HSPs by transfection of herpes simplex virus vectors in vitro or in transgenic animal in vivo can protect primary cardiac cells from subsequent exposure to severe thermal or hypoxic stress. The increases in myocardial and liver HSP70 accumulation in response to nonexertional heat stress are attenuated with senescence, and hearts from aged animals exhibit an impaired ability to produce the protective HSP. In our previous work, peculiar changes associated with aging, like lipofuscin accumulation and collagen deposition were shown in the myocardial tissue of long-term simulated weightless rats. We therefore designed the present study to examine whether the heat-stress induced HSP70 accumulation in myocardial tissue may decline with simulated weightlessness.     

Heat shock protein 70 and anti-heat shock protein 70 antibodies in patients with chronic glomerulonephritis

We evaluated the heat shock system 70 (HSP70) in patients with chronic glomerulonephritis (CGN). Seventy-six patients with CGN patients were included in our study. Ten patients with mild proteinuria (median 0.48 [0.16–0.78] g/24 h) and ten healthy subjects served as positive and negative controls, respectively. Urinary levels of HSP70, interleukin-10, and serum levels of anti-HSP70 were measured by ELISA. The immunohistochemical peroxidase method was used to study the expression of HSP70 and Foxp3+ in kidney biopsies. Treg^FoxP3+ cells in the interstitium were determined morphometrically. Median urinary HSP70 levels in patients with nephrotic syndrome (NS) [6.57 (4.49–8.33) pg/mg] and subnephrotic range proteinuria [5.7 (4.12–6.9) pg/mg] were higher (p?<?0.05) than in positive [3.7 (2.5–4.82) pg/mg] and negative [3.78 (2.89–4.84) pg/mg] controls. HSP70 expression index in tubular cells positively correlated with urinary HSP70 (Rs?=?0.948, р?<?0.05) and proteinuria (Rs?=?0.362, p?<?0.05). The number of Treg^Foxp3+ cells in the kidney interstitium and interleukin-10 excretion were lower in patients with NS. Anti-HSP70 antibody serum levels in patients with NS [21.1 (17.47–29.72) pg/ml] and subnephrotic range proteinuria [24.9 (18.86–30.92) pg/ml] were significantly higher than in positive [17.8 (12.95–23.03) pg/ml] and negative [18.9 (13.5–23.9) pg/ml] controls. In patients with CGN, increasing proteinuria was associated with higher HSP70 renal tissue and urinary levels. However, activation of HSP70 in patients with nephrotic syndrome did not lead to an increase in tissue levels of Treg^Foxp3+ cells or to the release of IL-10.     

Heat shock protein 70 is necessary for Rice stripe virus infection in plants

Heat shock proteins 70 (HSP70s) are a highly conserved family of genes in eukaryotes, and are involved in a remarkable variety of cellular processes. In many plant positive‐stranded RNA viruses, HSP70 participates in the construction of a viral replication complex and plays various roles during viral infection. Here, we found increased expression of HSP70 following infection by Rice stripe virus (RSV), a negative‐stranded RNA virus, in both rice (the natural host) and Nicotiana benthamiana (an experimental host). Heat treatment of N. benthamiana (Nb) plants enhanced viral infection, whereas RSV infection was retarded and viral RNAs accumulated at a low level when HSP70 was silenced. In both bimolecular fluorescence complement and in vitro pull‐down assays, the N‐terminus of RSV RNA‐dependent RNA polymerase (RdRp) interacted and co‐localized with the HSP70s of both plants (OsHSP70 and NbHSP70). The localization of the N‐terminus of RdRp when expressed alone was not obviously different from when it was co‐expressed with OsHSP or NbHSP, and vice versa. RSV infection also had no effect on the localization of host HSP70. These results demonstrate that host HSP70 is necessary for RSV infection and probably plays a role in viral replication by interacting with viral RdRp, which provides the first evidence of an interacting host protein related to RSV replication, which has been little studied to date.     

Heat shock Hsp70 protein is chloroplast-encoded in the chromophytic alga Pavlova lutherii

Heat shock proteins are ubiquitous and highly conserved. Recently they have become implicated in the import of proteins into organelles. All the heat shock genes characterized to date, however, are known or assumed to be encoded in the nuclear genome even if the corresponding protein can be localised in the mitochondrion or chloroplast. In contrast, we identify here an hsp70 gene in the unicellular chromophytic alga Pavlova lutherii which is located on the chloroplast genome. Localisation of this gene to the chloroplast chromosome is confirmed by Southern blot analysis and pulse-field gel electrophoresis which also reveals that the length of the P. lutherii chloroplast chromosome is 115 kb. We compare the predicted protein of this hsp70 gene with that of maize and of the analogous proteins in the prokaryotic organisms Escherichia coli and Synechocystis PCC6803. The greatest identity is found with the cyanobacterium Synechocystis PCC6803.     

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite leishmania

Antimony-containing drugs are still the drugs of choice in the treatment of infections caused by the parasite Leishmania. Resistance to antimony is now common in some parts of the world, and several mechanisms of resistance have been described. By transfecting cosmid banks and selecting with potassium antimonyl tartrate (SbIII), we have isolated a cosmid associated with resistance. This cosmid contains 2 copies of the heat shock protein 70 (HSP70) and 1 copy of the heat shock cognate protein 70 (HSC70). Several data linked HSP70 to antimony response and resistance. First, several Leishmania species, both as promastigotes and amastigotes, increased the expression of their HSP70 proteins when grown in the presence of 1 or 2 times the Effect Concentration 50% of SbIII. In several mutants selected for resistance to either SbIII or to the related metal arsenite, the HSP70 proteins were found to be overexpressed. This increase was also observed in revertant cells grown for several passages in the absence of SbIII, suggesting that this increased production of HSP70 is stable. Transfection of HSP70 or HSC70 in Leishmania cells does not confer resistance directly, though these transfectants were better able to tolerate a shock with SbIII. Our results are consistent with HSP70 and HSC70 being a first line of defense against SbIII until more specific and efficient resistance mechanisms take over.     

Regulation of the Dbl proto-oncogene by heat shock cognate protein 70 (Hsc70)

The dbl oncogene product is the defining member of a family of onco-proteins known as Dbl guanine nucleotide exchange factors (GEFs) that facilitate the activation of the small GTP-binding proteins Cdc42, Rac, and Rho. Oncogenic activation of proto-Dbl occurs through loss of the amino-terminal 497 residues, rendering the protein constitutively active. Because both onco- and proto-Dbl contain the structural elements required for GEF activity (i.e. the Dbl homology (DH) and pleckstrin homology (PH) domains), it is thought that the amino terminus of proto-Dbl somehow inhibits the biochemical activity of the protein. To better understand the molecular basis of this regulation, we set forth to identify cellular proteins that preferentially bind the proto-oncogenic form of Dbl. We identified the molecular chaperone heat shock cognate protein (Hsc70) as a binding partner that preferentially interacts with the proto-oncogenic form of Dbl. Dbl is complexed with Hsc70 in transfected cells, as well as in native mouse brain extracts. The interaction between Hsc70 and proto-Dbl is mediated by at least two regions in Dbl, the aminoterminal spectrin homology domain (residues 224-417) and the pleckstrin homology domain (residues 711-808). Overexpression of a dominant negative Hsc70 mutant leads to activation of proto-Dbl GEF activity, indicating that the chaperone negatively regulates proto-Dbl function in vivo. We propose that Hsc70 attenuates Dbl activity by maintaining an inactive conformation in which the amino terminus is "folded over" the catalytic DH-PH domain.     

Heat shock protein 70 is upregulated in the intestine of intrauterine growth retardation piglets

The objective of this study is to investigate the expression and distribution of heat shock protein 70 (Hsp70) in the intestine of intrauterine growth retardation (IUGR) piglets. Samples from the duodenum, prejejunum, distal jejunum, ileum, and colon of IUGR and normal-body-weight (NBW) piglets were collected at birth. The results indicated that the body and intestine weight of IUGR piglets were significantly lower than NBW piglets. The villus height and villus/crypt ratio in jejunum and ileum of IUGR piglets were significantly reduced compared to NBW piglets. These results indicated that IUGR causes abnormal gastrointestinal morphologies and gastrointestinal dysfunction. The mRNA of hsp70 was increased in prejejunum (P < 0.05), distal jejunum (P < 0.05), and colon in IUGR piglets. However, the hsp70 mRNA in ileum of piglets with IUGR was decreased. Similar to hsp70 mRNA, the protein levels of Hsp70 in prejejunum (P < 0.05), distal jejunum, and colon (P < 0.05) in IUGR piglets were higher than those in NBW piglets. These results indicated that the expression of Hsp70 in the intestinal piglets was upregulated by IUGR, and different intestinal sites had different responses to stress. Meanwhile, the localization of Hsp70 in the epithelial cells of the whole villi and intestinal gland rather than in the lamina propria and myenteron suggested that Hsp70 has a cytoprotective role in epithelial cell function and structure.     

Heat shock protein 70: roles in multiple sclerosis

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

Heat shock protein 70 is a potent activator of the human complement system

According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.     

人肝癌细胞BEL-7402中热休克蛋白70相伴甲胎蛋白的实验研究

为研究人肝癌细胞BEL-7402中热休克蛋白70(HSP70)与甲胎蛋白(AFP)的相互作用,采用免疫化学和免疫荧光检测HSP70和AFP在肝癌细胞中的表达和定位.HSP70与AFP的相互关系通过免疫共沉淀和蛋白印迹杂交进行分析.结果免疫化学显示人肝癌细胞BEL-7402中存在高水平的HSP70和AFP共表达,均定位于细胞浆.AFP存在于HSP70单抗的免疫沉淀中,而HSP70则存在于AFP单抗的免疫沉淀中.结果表明人肝癌细胞BEL-7402中HSP70与AFP相伴.两者之间的相互关系研究将成为探讨肝癌的发生和免疫治疗的新途径.     

A critical role of heat shock cognate protein 70 in Apoptin-induced phosphorylation of Akt

Apoptin, a protein from chicken anemia virus, selectively induces apoptosis of transformed or tumor cells, but not in normal cells. However, the mechanism of action of Apoptin is still not well understood. Using yeast two-hybrid and immunoprecipitation approaches, we found that Apoptin interacted with Heat shock cognate protein 70 (Hsc70). In vivo, Apoptin induced the translocation of endogenous Hsc70 from the cytoplasm to the nucleus, and both were co-localized in the nucleus. In addition, Apoptin induced Akt phosphorylation, which was markedly inhibited by Hsc70 knockdown, suggesting that Hsc70 may play a critical role in Apoptin-induced Akt phosphorylation. These findings help to further understand the molecular mechanism of Apoptin.     

Heat shock protein 70 is translocated to lipid droplets in rat adipocytes upon heat stimulation

In mammalian cells, lipid storage droplets contain a triacylglycerol and cholesterol ester core surrounded by a phospholipid monolayer into which a number of proteins are imbedded. These proteins are thought to be involved in modulating the formation and metabolic functions of the lipid droplet. In this study, we show that heat stress upregulates several heat shock proteins (Hsps), including Hsp27, Hsp60, Hsp70, Hsp90, and Grp78, in primary and differentiated adipocytes. Immunostaining and immunoblotting data indicate that among the Hsps examined, only Hsp70 is induced to redirect to the lipid droplet surface in heat-stressed adipocytes. The thermal induction of Hsp70 translocation to lipid droplet does not typically happen in a temperature- or time-dependent manner and occurs abruptly at 30-40 min and rapidly achieves a steady state within 60 min after 40 degrees C stress of adipocytes. Though Hsp70 is co-localized with perilipin on the lipid droplets in stressed adipocytes, immunoprecipitation experiments suggest that Hsp70 does not directly interact with perilipin. Alkaline treatments indicate that Hsp70 associates with the droplet surface through non-hydrophobic interactions. We speculate that Hsp70 might noncovalently associate with monolayer microdomains of the lipid droplet in a manner similar to its interaction with lipid bilayer moieties composed of specific fatty acids. As an acute and specific cellular response to the heat stimulation, accumulation of Hsp70 on adipocytes lipid droplets might be involved in stabilizing the droplet monolayer, transferring nascent proteins to the lipid droplets, or chaperoning denatured proteins on the droplet for subsequent refolding.     

Characterization of rotavirus cell entry

While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-beta-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.     

热休克蛋白60与细胞凋亡

热休克蛋白60(heat shock protein 60, HSP60)是主要存在于线粒体内的分子伴侣蛋白,对于维持线粒体蛋白的正常结构和功能不可或缺.线粒体中的HSP60可作用于凋亡相关因子而抑制线粒体凋亡通路的激活,并且能够减少线粒体产生氧自由基;胞浆中的少量HSP60亦可通过与凋亡相关因子的相互作用等途径抑制细胞凋亡.相反,在某些刺激因素作用下或者HSP60细胞定位异常时,HSP60可产生促凋亡效应.HSP60在细胞凋亡中的双重作用及其对于肿瘤等疾病诊治的意义已引起高度关注.