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.     

Diminished reproductive potential of male mice in response to selenium‐induced oxidative stress: Involvement of HSP70, HSP70‐2, and MSJ‐1

The oxidative stress imposed by nutritional variations in selenium (Se) has plausible role in reproductive toxicology and affects the reproductive potential. Also, the expression of heat shock proteins (HSPs) is a highly regulated event throughout the process of spermatogenesis and is modulated by stressful stimuli. This prompted us to investigate the possibility that Se‐induced oxidative stress may affect the fertility status by altering the expressions of the constitutive and inducible HSP70 proteins, having crucial role in spermatogenesis. Different Se status‐deficient, adequate, and excess, male Balb/c mice were created by feeding yeast‐based Se‐deficient diet (group I) and deficient diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III) for a period of 8 weeks. After completion of the diet‐feeding schedule, a significant decrease in the Se and glutathione peroxidase (GSH‐Px) levels was observed in the Se‐deficient group (I), whereas Se‐excess group (III) demonstrated an increase. Increased levels of reactive oxygen species, malondialdehyde, and alterations in the redox status in both groups I and III indicated oxidative‐stressed conditions. There was an overall reduced fertility status in mice supplemented with Se‐deficient and Se‐excess diet. The mRNA and protein expression of HSP70 was found to be elevated in these two groups, whereas the expression patterns of HSP70‐2 and MSJ‐1 demonstrated a reverse trend. In vitro CDC2 kinase assay showed reduced kinase activity in group I and group III. These findings suggest that Se‐induced oxidative stress by differentially regulating various HSP70s can affect its downstream factors having crucially important role in differentiation of germ cells and completion of spermatogenesis. Therefore, it can provide an insight into the mechanism(s) by which the oxidative stress–induced reproductive toxicity can lead to increased apoptosis/growth arrest and infertility. This will thus add new dimensions to the molecular mechanism underlying the human male infertility and open new vistas in the development of various chemo‐preventive methods. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:125–136, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20276     

Heat shock proteins of chicken lens

The presence of heat shock proteins HSP-40, HSP-70, and HSc-70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP-40, HSP-70, and HSc-70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP-40, HSP-70, and HSc-70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunofluorescence technique. Data obtained from these experiments revealed that HSP-40, HSP-70, and HSc-70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP-40, HSP-70, and HSc-70 can interact with protein of these areas and protect them from stress induced denaturation.     

The interaction of heat shock proteins with cellular membranes: a historical perspective

The interaction of heat shock proteins (HSP) with cellular membranes has been an enigmatic process, initially observed by morphological studies, inferred during the purification of HSP70s, and confirmed after the detection of these proteins on the surface of cancer cells and their insertion into artificial lipid bilayers. Today, the association of several HSP with lipid membranes is well established. However, the mechanisms for membrane insertion have been elusive. There is conclusive evidence indicating that HSP70s have a great selectivity for negatively charged phospholipids, whereas other HSP have a broader spectrum of lipid specificity. HSP70 also oligomerizes upon membrane insertion, forming ion conductance channels. The functional role of HSP70 lipid interactions appears related to membrane stabilization that may play a role during cell membrane biogenesis. They could also play a role as membrane chaperones as well as during endocytosis, microautophagy, and signal transduction. Moreover, HSP membrane association is a key component in the extracellular export of these proteins. The presence of HSP70 on the surface of cancer cells and its interaction with lysosome membranes have been envisioned as potential therapeutic targets. Thus, the biology and function of HSP membrane association are reaching a new level of excitement. This review is an attempt to preserve the recollection of the pioneering contributions of many investigators that have participated in this endeavor.     

盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70).为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C.A.Mey.)O.E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70.实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导.Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性.     

Tissue-specific expression of heat shock proteins of the mouse in the absence of stress

The steady-state levels of four members of the heat shock proteins families (HSP84, HSC73, HSP71, and HSP25) were examined by immunoblot analysis of several different tissues of young and adult mice in the absence of stress. These hsps were detected in all tissues but their level was variable. The levels of HSC73 and HSP84 varied only slightly between different tissues in either young or adult mice, with the exception of skin where these hsps were found in reduced amounts. In contrast, the stress-inducible member of the HSP70 family, HSP71, was found to be expressed in all tissues but in amounts which differed by as much as two orders of magnitude between tissues. In general, the levels of both HSP71 and HSP25 were found to be tissue dependent, with higher levels found in tissues such as stomach, intestine, colon and bladder, tissues which are exposed to toxic environmental or metabolic products, and which may concentrate these substances by water resorption and/or be exposed to them for longer periods. The levels of HSP71 and HSP25 were generally positively correlated both in young and adult mice although this correlation was not found in certain tissues such as kidney, testes, and bone. Tissues of young mice contained lower amounts of HSP25 and HSP71 than were found in the same tissues from adults. We conclude that hsps are expressed in all tissues of the mouse in the absence of stress and that some organs, particularly those exposed to potentially toxic metabolites, show a higher level of expression of HSP71 and HSP25. © 1993Wiley-Liss, Inc.     

盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定(英文)

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70)。为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C. A. Mey. )O. E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70。实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导。Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性。     

Identification of new subgroup of HSP70 in Bythograeidae (hydrothermal crabs) and Xanthidae

Crabs of the Bythograeidae family (Crustacea: Brachyura: Bythogreoidea) are the only endemic crab family living in hydrothermal fields. The hydrothermal environment is characterized by unique ecological parameters, such as the high temperature gradient around the hydrothermal chimney (2-350 degrees C), a fluid environment containing high levels of metals and numerous gases. The 70-kDa Heat Shock Protein (HSP70) group is the most-studied HSP, because it is ubiquitous, and a strong positive correlation has been found between the amounts of HSP70 produced in response to stress, and the ability of the organism to withstand stressful conditions. The 70-kDa heat shock protein genes from Bythograeids (species analyzed: Bythograea thermydron, Cyanagraea praedator and Segonzacia mesatlantica) were characterized. Our results revealed that Bythograeidae possess genes which are similar with those present in Xanthids (coastal crabs). The deduced protein sequences displayed motifs distinct from those in the other crustacean HSC70/HSP70s available in the databases. Phylogenetic analysis showed that these members of HSP70 family identified in Bythograeidae and Xanthidae constitute a new subgroup within this family.     

Genomewide identification and analysis of heat‐shock proteins 70/110 to reveal their potential functions in Chinese soft‐shelled turtle Pelodiscus sinensis

Heat‐shock proteins 70/110 (Hsp70/110) are vital molecular chaperones and stress proteins whose expression and production are generally induced by extreme temperatures or external stresses. The Hsp70/110 family is largely conserved in diverse animals. Although many reports have studied and elaborated on the characteristics of Hsp70/110 in various species, the systematic identification and analysis of Hsp70/110 are still poor in turtles. In this study, a genomewide search was performed, and 18 candidate PsHSP70/110 family genes were identified in Chinese soft‐shelled turtle, Pelodiscus sinensis. These PsHSP70/110 proteins contained the conserved “heat shock protein 70” domain. Phylogenetic analysis of PsHSP70/110 and their homologs revealed evolutionary conservation of Hsp70/110 across different species. Tissue‐specific expression analysis showed that these PsHSP70/110 genes were differentially expressed in different tissues of P. sinensis. Furthermore, to examine the putative biological functions of PsHSP70/110, the dynamic expression of PsHSP70/110 genes was analyzed in the testis of P. sinensis during seasonal spermatogenesis following germ cell apoptosis. Notably, genes such as PsHSPA1B‐L, PsHSPA2, and PsHSPA8 were significantly upregulated in P. sinensis testes along with a seasonal decrease in apoptosis. Protein interaction prediction revealed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 may interact with each other and participate in the MAPK signaling pathway. Moreover, immunohistochemical analysis showed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 protein expression was associated with seasonal temperature variation. The expression profiling and interaction relationships of the PsHSPA1B‐L, PsHSPA2, and PsHSPA8 proteins implied their potential roles in inhibiting the apoptosis of germ cells in P. sinensis. These results provide insights into PsHSP70/110 functions and will serve as a rich resource for further investigation of HSP70/110 family genes in P. sinensis and other turtles.     

Crossreactivity of SLE autoantibodies with 70 kDa heat shock proteins of Mycobacterium tuberculosis

Heat shock proteins (hsp) may be involved in the initiation and perpetuation of autoimmune diseases. In order to investigate the possible role of hsp and other intracellular proteins of Mycobacterium tuberculosis in the autoantibody production in SLE, the immuno-crossreactivity of SLE autoantibodies with Mycobacterium tuberculosis sonic extract and hsp-70 kDa was investigated. These proteins showed significant binding with Protein A-Sepharose isolated SLE IgG. Western blotting of hsp-70 with SLE IgG showed strong recognition, suggesting possible involvement of hsp and other intracellular proteins of Mycobacterium tuberculosis in the autoantibody induction in SLE.     

Heat shock protein expression in fish

Heat shock proteins (HSP) are a family of proteins expressed in response to a wide range of biotic and abiotic stressors. They are thus also referred to as stress proteins. Their extraordinarily high degree of identity at the amino acid sequence level and the fact that this cellular stress response has been described in nearly all organisms studied, make this group of proteins unique. We provide a brief historical overview of HSP research, as a background to summarizing what is known about HSP expression in fish. The expression of HSPs in fish has been described in cell lines, primary cultures of various cells, and in the tissues of whole organisms. Collectively, the data show that the expression of HSPs are affected in a wide variety of fish cells and tissues, in response both to biological stressors such as infectious pathogens, as well as to abiotic stressors such as heat and cold shock, and environmental contaminants. HSP research in fish is in its early stages and many studies are describing the expression of proteins in response to various stressors. Several studies have contributed to our understanding of the molecular nature and the molecular biology of HSPs in fish. Recent studies have shown a relationship between HSP expression and the generalized stress response in fish, but further research is needed to clarify the complex relationships between stress hormones and the cellular HSP response. In general, the HSP response seems to be related to the sensing of the stressor and the subsequent cellular effects which may adapt the cells to cope with the stressors. Consequently, such data may be of central importance in understanding the significance of HSP expression to the whole organism. We conclude with sections on laboratory methods used in HSP research and on potential applications of this knowledge in biomonitoring.     

The effect of 100 mT SMF on activation of the hsp70 promoter in a heat shock/luciferase reporter system

Human exposure to magnetic fields, increased through use of new technologies like magnetic resonance imaging (MRI), has prompted investigations into possible effects of static magnetic fields (SMFs) on cellular processes. However, controversy still remains between many studies, which likely results from a lack of uniformity across experimental parameters, including the length of magnetic field exposure, the strength of the magnetic field, and the cell type or organism under investigation. The purpose of this research was to monitor effects of SMF exposure using real‐time luminescence photometry. The study investigated the potential interaction of a 100 mT SMF on a heat shock protein (hsp70)/luciferase reporter construct in stably transfected NIH3T3 cells. Changes in heat shock promoter activation following 100 mT SMF exposure were analyzed and detected as bioluminescence in real‐time. Two heat parameters were considered in combination with sham‐ and 100 mT‐exposed experiments: no heat or 1,800 s heat. As expected, there was a significant increase in bioluminescence in response to 1,800 s of heat alone. However, no significant difference in average hsp70 promoter activation between sham and 100 mT experiments was observed for no heat or 1,800 s heat experiments. Therefore, a 100 mT SMF was shown to have no effect on the activation of the heat shock protein promoter during SMF exposure or when SMF exposure was combined with a heat insult. J. Cell. Biochem. 108: 956–962, 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025-10 μg ml^-1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.     

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025–10 μg?ml^?1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.