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.     

HSP70-related proteins in bovine skeletal muscle

Constitutive expression of HSP70-related proteins was detected in a variety of bovine tissues using a specific antibody. All tissues contained a 73 kilodalton protein. A lower molecular weight form (72 kilodaltons) that co-migrated on two-dimensional gels with the stressed-induced HSP70 was present in high levels in bovine skeletal muscle, but absent from rat skeletal muscle. Two-dimensional gel analysis revealed several isoforms for both the 73 and 72 kilodalton forms. Purification of HSP70-related proteins from bovine skeletal muscle, thymus gland and rat skeletal muscle demonstrated that the antibody recognized all the forms present in the tissue homogenates. The two proteins are similar but distinct as detected by one-dimensional peptide mapping. The lower molecular form was not present in fetal tissue but was detectable in newborn animals, suggesting that the levels are regulated during development.     

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B'') and isolation of its cDNA and genomic DNA.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.     

Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific oyster (Crassostrea gigas): implications for thermal limits and induction of thermal tolerance

Pacific oysters, Crassostrea gigas, living at a range of tidal heights, routinely encounter large seasonal fluctuations in temperature. We demonstrate that the thermal limits of oysters are relatively plastic, and that these limits are correlated with changes in the expression of one family of heat-shock proteins (HSP70). Oysters were cultured in the intertidal zone, at two tidal heights, and monitored for changes in expression of cognate (HSC) and inducible (HSP) heat-shock proteins during the progression from spring through winter. We found that the "control" levels (i.e., prior to laboratory heat shock) of HSC77 and HSC72 are positively correlated with increases in ambient temperature and were significantly higher in August than in January. The elevated level of HSCs during the summer was associated with moderate, 2-3 degrees C, increases in the upper thermal limits for survival. We measured concomitant increases in the threshold temperatures (T(on)) required for induction of HSP70. Total hsp70 mRNA expression reflected the seasonal changes in the expression of inducible but not cognate members of the HSP70 family of proteins. A potential cost of increased T(on) in the summer is that there was no extension of the upper thermal limits for survival (i.e., induction of thermotolerance) after sublethal heat shock at temperatures that were sufficient to induce thermotolerance during the winter months.     

Quantitative RT-PCR analysis and immunohistochemical localization of HSP70 in sea bass Dicentrarchus labrax exposed to transport stress

In aquaculture, fish are exposed to stressful conditions, which cause an increased synthesis of heat shock proteins (HSPs) at the cellular level. In this work we considered the expression of the constitutive and inducible forms of HSP70 as an indicator of stress caused by transport, during development of the sea bass (Dicentrarchus labrax), a teleost fish of high value for aquaculture. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals. In all stressed animals, regardless of their developmental stage, cortisol levels were higher than in control animals.     

Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation.

The 70-kD heat-shock proteins (HSP70s) are encoded by a multigene family in eukaryotes. In plants, the 70-kD heat-shock cognate (HSC70) proteins are located in organellar and cytosolic compartments of cells in most tissues. Previous work has indicated that HSC70 proteins of spinach (Spinacia oleracea) are actively synthesized during cold-acclimating conditions. We have isolated, sequenced, and characterized cDNA and genomic clones for the endoplasmic reticulum (ER) luminal HSC70 protein (immunoglobulin heavy chain-binding protein; BiP) of spinach. The spinach ER-luminal HSC70 is a constitutively expressed gene consisting of eight exons. Spinach BiP mRNA appears to be up-regulated during cold acclimation but is not expressed during water stress or heat shock. In contrast to the differential regulation of mRNA, the ER-luminal HSC70 protein levels remain constant in response to various environmental stresses. Two other members of the spinach 70-kD heat-shock (HS70) multigene family also show differential expression in response to a variety of environmental stresses. A constitutively expressed cytosolic HSC70 protein in spinach appears also to be up-regulated in response to both cold-acclimating and heat-shock treatments. Spinach also contains a cold-shock-induced HS70 gene that is not expressed during heat shock or water stress. Since HSP70s are considered to be involved with the chaperoning and folding of proteins, the data further support the concept that they may be important for maintaining cellular homeostasis and proper protein biogenesis during cold acclimation of spinach.     

Investigating Apoptozole as a Chemical Probe for HSP70 Inhibition

The use of chemical tools to validate clinical targets has gained in popularity over recent years and the importance of understanding the activity, selectivity and mechanism of action of these compounds is well recognized. Dysregulation of the HSP70 protein family has been linked to multiple cancer types and drug resistance, highlighting their importance as popular targets for anti-cancer drug development. Apoptozole is a recently identified small molecule, which has been reported to possess strong affinity for the HSP70 isoforms HSP72 and HSC70. We investigated apoptozole as a potential chemical tool for HSP70 inhibition. Unfortunately, using both biochemical and biophysical techniques, we were unable to find any experimental evidence that apoptozole binds to HSP70 in a specific and developable way. Instead, we provide experimental evidence that apoptozole forms aggregates under aqueous conditions that could interact with HSP70 proteins in a non-specific manner.     

Developmental changes of heat-shock proteins in porcine testis by a proteomic analysis

Heat-shock proteins (HSPs) are important in spermatogenesis. This study investigated developmental changes in the expression of major HSPs in porcine testis. The testis from five immature (mean age 2.9+/-0.1 months) and five mature boars (35.7+/-14.0 months) were examined. Two-dimensional polyacrylamide gel electrophoresis was conducted and proteins were identified by Western blotting and/or matrix-assisted laser desorption/ionization mass spectrometry. Moreover, the 90, 70, and 60 kDa HSPs, 70 kDa heat-shock cognate protein (HSC 70), tubulin, and actin were quantified on two-dimensional gels. Protein spots were quantified by densitometry, combined with a computer-assisted image analysis system. Immunohistochemistry was performed to analyze the expression pattern of major HSPs and beta-tubulin in testis. One isoform of HSP 90 (HSP 90 alpha), two isoforms of HSC 70 (HSC 70a and HSC 70c), one isoform of HSP70 (HSP 70e), and tubulin increased after sexual maturation (P<0.05). A testis-specific HSP70 (P70t) was markedly increased in the testes of sexually mature boars. Meanwhile, levels of actin and some isoforms of HSPs including 60 kDa HSP remained similar in both groups. These observations were further confirmed by immunohistochemistry; therefore, the upregulation of protein expression in the adult testis could be attributed to a higher level of protein expression and the number of cells that were HSPs-positive already resided in the immature testis. The differential expression of major HSPs suggested that they may be important in porcine spermatogenesis.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

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.     

The Constitutive Heat Shock Protein-70 Is Required for Optimal Expression of Myelin Basic Protein During Differentiation of Oligodendrocytes

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.     

The cytosolic/nuclear HSC70 and HSP90 molecular chaperones are important for stomatal closure and modulate abscisic acid-dependent physiological responses in Arabidopsis

Cytosolic/nuclear molecular chaperones of the heat shock protein families HSP90 and HSC70 are conserved and essential proteins in eukaryotes. These proteins have essentially been implicated in the innate immunity and abiotic stress tolerance in higher plants. Here, we demonstrate that both chaperones are recruited in Arabidopsis (Arabidopsis thaliana) for stomatal closure induced by several environmental signals. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are compromised in the dark-, CO(2)-, flagellin 22 peptide-, and abscisic acid (ABA)-induced stomatal closure. HSC70-1 and HSP90 proteins are needed to establish basal expression levels of several ABA-responsive genes, suggesting that these chaperones might also be involved in ABA signaling events. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are hypersensitive to ABA in seed germination assays, suggesting that several chaperone complexes with distinct substrates might tune tissue-specific responses to ABA and the other biotic and abiotic stimuli studied. This study demonstrates that the HSC70/HSP90 machinery is important for stomatal closure and serves essential functions in plants to integrate signals from their biotic and abiotic environments.     

Adenovirus capsid proteins interact with HSP70 proteins after penetration in human or rodent cells

Soon after penetration of adenovirus serotype 2 in BHK-21 and HeLa cells, HSP70 and HSC70 proteins become associated with the viral capsid. By analysis with a polyclonal antibody derived from a fusion protein containing the C-terminal domain, 290 amino acids of HSP70, and using both immunological methods and infected cells fractionation we observed that a significant amount of HSP70 proteins moved to the nucleus and colocalized with the adenovirus particles. HSP70 proteins of infected cells were isolated as a complex cross-linked with intracytoplasmic adenovirus type 2. By coprecipitation, using a polyclonal-specific antiserum derived from the fusion protein, or two different monoclonal-specific antisera, we showed that HSP70 and HSC70 proteins were associated with hexon, the major adenovirus capsid protein.     

Polymorphic analysis of the three MHC-linked HSP70 genes

Three genes encoding members of the M _r 70 000 heat shock protein family (HSP70) are known to lie in the class III region of the human major histocompatibility complex. IN order to determine whether these genes or their protein products exhibit any polymorphism the three genes have been specifically amplified from genomic DNA and sequenced. The HSP70-1 and HSP70-2 genes encode the major heat-inducible HSP70. A comparison of the nucleotide sequences of these genes from B8, SC01, DR3, B18, F1C30, DR3, and B7, SC30, DR2 haplotypes has revelad only very limited sequence variation which is not associated with any amino acid polymorphism. The HSP70-Hom gene encodes a protein that is highly related to HSP70-1, but which is not heat-inducible. Nucleotide sequence analysis of this gene from different haplotypes has revealed a Met Thr amino acid substitution at residue 493 in a number of the haplotypes tested. This variable amino acid lies in the proposed peptide-binding site of the HSP70-Hom protein. Address correspondence and offprint requests to: R. D. Campbell.     

Antarctic marine molluscs do have an HSP70 heat shock response

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.