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.     

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.     

Spinach leaf 70-kilodalton heat-shock cognate stabilizes bovine adrenal glucose-6-phosphate dehydrogenase in vitro without apparent stable binding

Spinach (Spinacia oleracea L.) leaf tissue 70-kilodalton heat-shock cognate was purified by ATP-agarose affinity and gel filtration. Gel filtration of the affinity-purified protein resolved it into three forms: monomer, dimer, and oligomer. In the absence of ATP, the majority of the heat-shock cognate existed as a monomeric form with lesser amounts of dimer and oligomer. Addition of 3 mM ATP to the purified protein, containing all three forms, converted the dimeric and monomeric forms to a high-molecular-weight complex. Removal of ATP from the complex by dialysis resulted in the reappearance of the dimeric and monomeric forms. Addition of ATP to the highly purified monomer had no effect on its gel-filtration migration. Neither purified monomeric or dimeric forms showed stable binding to denatured proteins; however, both forms of the purified heat-shock cognate were able to stabilize the enzymatic activity of bovine adrenal glucose-6-phosphate dehydrogenase over a 48-h period at 25° C. In addition, the activity of glucose-6-phosphate dehydrogenase in the presence of purified heat-shock cognate dimer or monomer could be rapidly decreased in an ATP-dependent fashion depending on the order of the substrate addition to the reaction mixture. Circular-dichroism studies indicated that addition of ATP to the spinach 70-kDa heat-shock cognate caused a conformation change from -helical to a greater -sheet content. How conformational character may influence the stabilizing activity of the heat-shock cognate in a mechanism which does not require stable peptide binding is discussed.Abbreviations BiP immunoglobulin binding protein - BSA bovine serum albumin - CA carbonic anhydrase - CD circular dichroism - G6PDH glucose-6-phosphate dehydrogenase - HSP heat shock protein - HSP70 70-kilodalton heat shock protein - HSC70 70-kilodalton heat-shock cognate - MDH malate dehydrogenase - PVDF polyvinylidene difluoride The authors wish to thank C. Kaye and R. Henry for their critical review and discussion of this paper. The antibody for tobacco BiP was the generous gift of J. Denecke. Financial support for this work was provided by the National Science Foundation DCB 9017625. This is Florida Agricultural Experiment Station Journal Series No. R-04110.     

Characterization of multiple members of the HSP70 family in platyfish culture cells: molecular evolution of stress protein HSP70 in vertebrates

A shift from 28 to 37 degrees C in the incubation temperature of a culture of the platyfish fibroblast cell line, EHS cells (platyfish fibroblast cell line), induced a set of stress proteins. A two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed that the cells expressed three genetically distinct forms of heat-shock protein 70 (HSP70) family proteins: heat-inducible forms of HSP70, the constitutively expressed heat-shock cognate protein 70 (HSC70) and its phosphorylated isoform, and the glucose-regulated protein 78 (GRP78). Three different clones encoding two major isoforms of heat-inducible HSP70, platyfish HSP70-1 and HSP70-2, and of the HSC70 were isolated from a platyfish cDNA library. We compared the deduced amino acid sequences of the platyfish HSP70 and HSC70 proteins with those of other vertebrates. Phylogenetic analysis showed that vertebrate HSP70 could be classified into four cluster groups: (a) fish HSP70, with two isoforms of heat-inducible HSP70 in fish, fish HSP70-1 and HSP70-2; (b) the mammalian testis-specific HSP70-related protein HST70; (c) the mammalian heat-inducible HSP70B'; and (d) the mammalian major histocompatibility complex (MHC)-linked HSP70, including the MHC-linked heat-inducible HSP70 and the testis-specific HSP70-related protein. These findings suggest that vertebrate HSP70 was derived from a single ancestral HSP70 gene during vertebrate evolution and that multiple copies of heat-inducible HSP70 were probably evolved during genetic divergence in fish and higher vertebrates.     

Identification of two molecular chaperons (HSX70, HSC70) in mature human erythrocytes

Two-dimensional gel electrophoresis of cytosolic proteins from mature human erythrocytes combined with immunoblotting revealed the presence of a group of heat shock proteins (HSPs) that included two molecular chaperons of the HSP70 family (HSX70, inducible; HSC70, constitutively expressed) and HSP90. As expected for cells devoid of organelles, erythrocytes do not contain stress proteins that are localized either in the mitochondria (HSP60, glucose-regulated protein (GRP 75) or in the endoplasmic reticulum (GRP78 or Ig heavy chain-binding protein, endoplasmin). Since red cells are unable to replace proteins whose structure has been damaged by environmental changes the results are taken to imply a role for chaperons in monitoring, protecting, and maintaining the structure and stability of erythrocyte proteins.     

Transient increase in vimentin phosphorylation and vimentin-HSC70 association in 9L rat brain tumor cells experiencing heat-shock

Characteristic changes in vimentin were studied in 9L rat brain tumor cells treated at 45°C. During heat-shock treatment, vimentin molecules were rapidly phosphorylated and reorganized from a filamentous form into a perinuclear higher-order structure that was less extractable by nonionic detergent. These effects were found to be highly transient, peaked at 30 min after the onset of heat-shock treatment, and subsided thereafter. Simultaneously, the solubility of the constitutively expressed heat-shock protein70 (HSC70) was also temporarily decreased and the kinetics was identical to that of vimentin. The results indicated that HSC70 and vimentin were co-insolubilized during the heat-shock treatment. We propose that the reorganization of the intermediate filaments resulted from enhanced phosphorylation of vimentin leads to the concurrent association of HSC70 to the intermediate filaments. This process may play an essential role in regulating heat-shock genes.     

Localization of a human heat-shock HSP 70 gene sequence to chromosome 6 and detection of two other loci by somatic-cell hybrid and restriction fragment length polymorphism analysis

Summary The human 70 kdalton heat-shock protein (HSP 70) is a member of a multigene family which is expressed in response to various physiological stresses including elevated temperatures. Using a cloned genomic HSP 70 DNA sequence we demonstrate by somatic cell hybrid and restriction fragment length polymorphism (RFLP) analyses that there are a minimum of three distinct HSP 70 loci in the human genome, one of which is located on chromosome 6.     

大菱鲆组成型热休克蛋白70的全长cDNA克隆及表达分析

根据感染哈维氏弧菌(Vibrio harveyi)的大菱鲆(Scophthalmus maximus)的差减cDNA文库中hsp70EST序列设计引物,用RACE方法首次克隆到长2188bp的大菱鲆HSC70(Heat-shock cognate protein 70)全长cDNA序列,包括1956bp的开放阅读框及5′和3′非翻译区。在预测的651个氨基酸序列中发现了Dnak特征性基序,胞质HSP70特征基序以及四肽简并重复序列。与其他真核生物HSP70家族成员进行同源性比较,发现大菱鲆HSC70与牙鲆(Paralichthys olivaceus)HSC71、虹鳟(Oncorhynchus mykiss)HSC71、人(Homo sapiens)HSC70、家鼠(Mus musculus)HSC70、烟草天蛾(Manduca sexta)HSC70的氨基酸相似性分别是97%,95%,94%,93%,86%,表现出较高的保守性。表达分析显示,hsc70mRNA在大菱鲆正常肝脏、鳃、肠、脾脏、头肾、肾等组织中以不同的水平存在,呈组成型表达;被哈维氏弧菌感染后,大菱鲆肝脏和脾脏组织hsc70mRNA表达水平分别在24h和12h出现上调(2.5-fold和1.6-fold);注射生理盐水组与未注射组之间差异不显著。       

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.     

Seasonal patterns of dehydrins and 70-kDa heat-shock proteins in bark tissues of eight species of woody plants

Although considerable effort has been directed at identifying and understanding the function and regulation of stress-induced proteins in herbaceous plants, reports concerning woody plants are limited. Studies with herbaceous crops have revealed similarities in the types of proteins that accumulate in response to a wide array of abiotic stresses and hormonal cues such as the accumulation of abscisic acid. Many of the identified proteins appear to be related to dehydrins (the D-11 subgroup of late-embryogenesis-abundant proteins). The objective of the present study was to determine if seasonal induction of dehydrins is a common feature in woody plants and to see if seasonal patterns existed for other stress-induced proteins. Bark tissues from eight species of woody plants were collected monthly for a period of 1.5 years. The species included: peach (Prunus persica) cv. Loring; apple (Malus domestica) cv. Golden Delicious; thornless blackberry (Rubus sp.) cv. Chester; hybrid poplar (Populus nigra); weeping willow (Salix babylonica); flowering dogwood (Cornus florida); sassafras (Sassafras albidum); and black locust (Robinia pseudo-acacia). Immunoblots of bark proteins were probed with a polyclonal antibody recognizing a conserved region of dehydrin proteins, and monoclonal antibodies directed against members of the HS70 family of heat-shock proteins. Some proteins, immunologically related to dehydrins, appeared to be constitutive; however, distinct seasonal patterns associated with winter acclimation were also observed in all species. The molecular masses of these proteins varied widely, although similarities were observed in related species (willow and poplar). Identification of proteins using the monoclonal antibodies (HSP70, HSC70, BiP) was more definitive because of their inherent specificity, but seasonal patterns were more variable among the eight species examined. This study represents only a precursory examination of several proteins reported to be stress related in herbaceous plants, but the results indicate that these proteins are also common to woody plants and that further research to characterize their regulation and function in relation to stress adaptation and the perennial life cycle of woody plants is warranted.     

Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70

We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.     

Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton rubrum

Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. To characterize T. rubrum proteins at the molecular level, we established a cDNA library of this pathogen. Here we describe a recombinant cDNA clone identical to eukaryotic 70kDa heat-shock proteins (HSPs). Western blot analysis using an anti HSP70 monoclonal antibody detected a recombinant fusion protein in Escherichia coli transformed with the expression vector containing the cloned cDNA insert. Southern blot analysis of T. rubrum genomic DNA detected no other members of the HSP70 gene family. Further analysis revealed the presence of two introns within the ORF of the HSP70 gene. In Northern blot analysis, the cDNA clone was hybridized to a RNA species of about 3.5kb which was constitutively expressed by cells cultured at 27 degrees C and was strongly up-regulated after culture at 37 degrees C. In summary, we have cloned the first member of the HSP family of dermatophytes and characterized it as a member of the Dnak subfamily of 70kDa HSPs.     

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.     

Molecular identification and expression of heat shock cognate 70 (HSC70) in the Pacific white shrimp Litopenaeus vannamei

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80-99.6% homology with 12 diverse species' HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multi-tissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of beta-actin. Furthermore, quantitative real-time experiments showed that HSC70 was up-regulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps' cytoprotection.     

The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum - a quantitative approach

Traditionally, the canine pancreatic endoplasmic reticulum (ER) has been the workhorse for cell-free studies on protein transport into the mammalian ER. These studies have revealed multiple roles for the major ER-luminal heat shock protein (Hsp) 70, IgG heavy chain-binding protein (BiP), at least one of which also involves the second ER-luminal Hsp70, glucose-regulated protein (Grp) 170. In addition, at least one of these BiP activities depends on Hsp40. Up to now, five Hsp40s and two nucleotide exchange factors, Sil1 and Grp170, have been identified in the ER of different mammalian cell types. Here we quantified the various proteins of this chaperone network in canine pancreatic rough microsomes. We also characterized the various purified proteins with respect to their affinities for BiP and their effect on the ATPase activity of BiP. The results identify Grp170 as the major nucleotide exchange factor for BiP, and the resident ER-membrane proteins ER-resident J-domain protein 1 plus ER-resident J-domain protein 2/Sec63 as prime candidates for cochaperones of BiP in protein transport in the pancreatic ER. Thus, these data represent a comprehensive analysis of the BiP chaperone network that was recently linked to two human inherited diseases, polycystic liver disease and Marinesco-Sj?gren syndrome.     

Stch encodes the 'ATPase core' of a microsomal stress 70 protein.

The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome-associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP-binding domains and truncates the carboxyl terminal peptide-binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane-bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium-inducible, microsome-associated ATPase activity with properties similar to a proteolytically cleaved N-terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements.     

Nonlinear relationships among evolutionary rates identify regions of functional divergence in heat-shock protein 70 genes

The neutral theory predicts that, in comparisons among related genes, the number of amino acid replacements per site in a given gene region should be a linear function of that in another region of the same gene, unless the genes have diverged functionally in one region. Therefore, nonlinearity of this relationship can be used to identify regions of possible functional divergence among members of a multigene family. This method of analysis was applied to members of the heat-shock protein 70 (HSP70) gene family, which encode highly conserved ATP- dependent chaperone proteins found in all organisms. A nonlinear relationship was found between the rate of amino acid replacement in the conserved IA domain of the ATPase portion of the molecule and that in other ATPase domains and the peptide-binding domain. These results suggest that genes in the HSP70 subfamily C (dnaK of bacteria and SSC1 of yeast) may have diverged functionally from other subfamilies in the ATPase domains, especially IIB, whereas SSB1 of yeast has diverged markedly in the peptide-binding domain. Functional divergence within these regions is consistent with what is known about functional differences between the HSP70 subfamilies in yeast.      

Interaction between SGT1 and cytosolic/nuclear HSC70 chaperones regulates Arabidopsis immune responses

The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene-triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70-SGT1 association.     

HSPA8/HSC70 chaperone protein

HSPA8/HSC70 protein is a fascinating chaperone protein. It represents a constitutively expressed, cognate protein of the HSP70 family, which is central in many cellular processes. In particular, its regulatory role in autophagy is decisive. We focused this review on HSC70 structure-function considerations and based on this, we put a particular emphasis on HSC70 targeting by small molecules and peptides in order to develop intervention strategies that deviate some of HSC70 properties for therapeutic purposes. Generating active biomolecules regulating autophagy via its effect on HSC70 can effectively be designed only if we understand the fine relationships between HSC70 structure and functions.