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.     

Increased expression of co-chaperone HOP with HSP90 and HSC70 and complex formation in human colonic carcinoma

Co-chaperone HOP (also called stress-inducible protein 1) is a co-chaperone that interacts with the cytosolic 70-kDa heat shock protein (HSP70) and 90-kDa heat shock protein (HSP90) families using different tetratricopeptide repeat domains. HOP plays crucial roles in the productive folding of substrate proteins by controlling the chaperone activities of HSP70 and HSP90. Here, we examined the levels of HOP, HSC70 (cognate of HSP70, also called HSP73), and HSP90 in the tumor tissues from colon cancer patients, in comparison with the non-tumor tissues from the same patients. Expression level of HOP was significantly increased in the tumor tissues (68% of patients, n = 19). Levels of HSC70 and HSP90 were also increased in the tumor tissues (95% and 74% of patients, respectively), and the HOP level was highly correlated with those of HSP90 (r = 0.77, p < 0.001) and HSC70 (r = 0.68, p < 0.01). Immunoprecipitation experiments indicated that HOP complexes with HSC70 or HSP90 in the tumor tissues. These data are consistent with increased formation of co-chaperone complexes in colon tumor specimens compared to adjacent normal tissue and could reflect a role for HOP in this process.     

Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy

A cell line derived from the tailfin of the marine teleost yellowtail fish Seriola quinqueradiata was established to examine cellular temperature regulation in an ectothermic animal. Three cytosolic members of the HSP70 family, heat-shock cognate proteins HSC70-1, HSC70-2 and heat-shock protein HSP70, were isolated from cultured yellowtail cells as stress-responsive biomarkers. Expression of hsp70 was heat-inducible, in contrast to the hsc70-1 gene product, which was expressed constitutively. In addition, expression of hsc70-2 was only induced under severe heat-shock conditions. Subcellular fractionation and immunocytochemistry showed localization of HSC70/HSP70 in the lysosomes, indicating that chaperone-mediated autophagy is induced by heat shock. Thus, chaperone-mediated autophagy is assisted by HSC70/HSP70, and heat-inducible expression of the genes encoding these proteins may be responsible for survival and adaptation under heat-shock conditions in fish cells.     

大鼠肝大部分切除前热休克对热休克蛋白和磷酸酶的影响

The contribution and content of the continuous heat shock protein 70/induced heat shock protein 68 (HSC70/HSP68), the contribution, variety and activity of acid phosphatases (ACP) and alkaline phosphatases (AKP) had been analysed qualitatively and quantitatively during the liver regeneration after 2/3 hepatectomy (PH) and HS (heat shock at 46 degrees C for 30 min, recovery for 8 h), which were compared with the results only by HS and only by PH. It was shown that the three kinds of treatment all can increase the activity of ACP, AKP and the expression of HSC70/HSP68, but with different change pattern. A further analysis show that after HS-PH the enhanced activity of ACP is related with that of 140 kD phosphatases, the enhanced activity of AKP is associated with that of 140 kD and 160-180 kD phosphatases. It can be reckoned from the results that ACP, AKP and HSC70/HSP68 all act on the heat shock response of hepatocyte and liver regeneration, and may take part in signal transduction in these processes, but ACP may play a dominant role in the start of hepatocyte multiplication, AKP and HSC70/HSP68 may play a dominant role in cytokineses.     

Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes

Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.     

低表达HSP90α和高表达HSP90β HepG2细胞株的建立

目的:建立HSP90α低表达和HSP90β高表达HepG2细胞株。方法:通过电转染方法将质粒pSilencerHSP90α和pSmycHSP90β转染入HepG2细胞中,应用Western-blotting和MTT法分别鉴定转染效果及绘制细胞生长曲线。结果:带有HSP90αsiRNA片段的质粒和带有HSP90β片段的质粒成功转入HepG2中,转染细胞与未转染细胞生长情况无差别。结论:电转染方法可以有效地将质粒转染入HepG2中去,转染细胞的生长情况将不会影响后续实验的结果。     

Phosphorylation of the 105-kDa heat shock proteins, HSP105alpha and HSP105beta, by casein kinase II

The 105-kDa heat shock protein alpha (HSP105alpha) and HSP105beta are mammalian heat shock proteins that belong to the HSP105/HSP110 family. Both HSP105alpha and HSP105beta consist of acidic and basic isoforms. Here we report that the acidic isoforms are serine phosphorylated HSP105alpha or HSP105beta. Furthermore, using an in-gel kinase assay with HSP105alpha or HSP105beta as the substrate, the protein kinase that phosphorylates HSP105alpha and HSP105beta was identified as casein kinase II. Since phosphorylated HSP105alpha is especially prominent in the brain compared to other tissues of mice and rats, the phosphorylation of HSP105alpha by casein kinase II may be biologically significant.     

Structure and expression of the human gene encoding major heat shock protein HSP70.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.     

Differential localization of heat shock proteins 90, 70, 60 and 27 in human decidua and placenta during pregnancy

Little is known about the localization of heat shock proteins (HSPs) in the decidua and placenta during the course of normal pregnancy. In this study, we have examined the localization of the HSPs in decidual and placental tissues obtained from women during the first, second and third trimesters of pregnancy (five in each trimester) by immunohistochemistry using highly specific antisera. HSPs 90, 70, 60 and 27 were detected in decidual stromal cells during each trimester. The intensity of staining did not change during gestation for HSPs 60 and 27, whereas it decreased with advancing gestation for HSPs 90 and 70. HSPs 90 and 60 were localized primarily in the nucleus, whereas HSP 70 was present equally in the nucleus and the cytoplasm; HSP 27 was primarily in the cytoplasm. In the placenta, HSPs 90, 70 and 60 were localized in cytotrophoblast, syncytiotrophoblast, intermediate trophoblast, Hofbauer and endothelial cells. HSPs 90 and 60 were localized primarily in the nucleus, while HSP 70 was in the nucleus and the cytoplasm. In the placenta, HSP 27 was detected only in the intermediate trophoblast and syncytiotrophoblast cells and only in the first two trimesters. These results indicate that there are striking differences in the subcellular localization of HSPs in the decidua and the placenta during normal pregnancy.     

Effects of chronic heat stress on the expressions of heat shock proteins 60, 70, 90, A2, and HSC70 in the rabbit testis

Few studies have focused on the expression of heat shock proteins (HSPs) after chronic heat stress. The objective of this study was to investigate the effect of chronic high temperature–humidity index treatment on the expressions of HSP60, HSP70, HSP90, HSPA2 and HSC70, in the Rex rabbit testis and the expressions of these proteins after recovery from the chronic heat shock. Thirty mature male rabbits of the same age were randomly divided into three groups: control, heat stress, and recovery. The western blot results showed that the expressional levels of HSP60, HSP90, and HSC70 increased significantly and HSPA2 was elevated slightly after a 9-week heat treatment. HSP70 was absent in the control testis and had a high level of expression after heat stress. All of these proteins partially reverted back to normal levels after a 9-week recovery. The immunohistochemical results indicated that the expression patterns of HSP60, HSP90, HSPA2, and HSC70 did not change.     

High expression of heat shock protein 90 alpha and its significance in human acute leukemia cells

This study investigated the expression of heat shock protein 90 alpha (Hsp90α) in acute leukemia cells. The expression of Hsp90α was investigated in leukemia cell lines and human bone marrow mononuclear cells derived from acute leukemia patients and from healthy individuals using polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Compared with cells from healthy individuals, the expression of Hsp90α in the untreated patients was higher. Similarly high levels were observed in remission patients. Significantly higher expression levels were observed in all the tested cell lines, and in cells from refractory and relapsed patients. No obvious relationship was observed between the occurrence of graft versus host disease and the expression of Hsp90α. The untreated patients showing higher expression levels of Hsp90α had lower complete remission rates. During remission of untreated patients, the expression of Hsp90α decreased and reached the lowest level after transplantation, but the expression increased again before relapse. Hsp90α was highly expressed in leukemia cells. The expression level of Hsp90α was associated with leukemia prognosis. However, no obvious relationship was observed between the occurrence of graft versus host disease and the expression of Hsp90α.