Stress-70 Protein Research Articles

Molecular phylogeny of the stress-70 protein family with reference to algal relationships

The stress-70 protein family has previously been shown to be a useful tool for molecular phylogeny at the kingdom to family levels. Although sequences of many members of the stress-70 family are available, few genes from the Protoctista have been sequenced to date. Phylogenetic analyses of algae based on various molecules have not, as yet, provided clear results concerning relationships between major divisions. We cloned and sequenced several algal stress-70 genes in order to provide additional data and to further analyse phylogenetic relationships among algal divisions. New nuclear sequences were obtained from Guillardia theta (Cryptophyta), Ascophyllum nodosum (Heterokontophyta) and Cyanophora paradoxa (Glaucocystophyta). Phylogenetic trees of the stress-70 protein family calculated using different methods are presented. In our trees, the heterokont alga Ascophyllum nodosum is closely related to the slime mould Dictyostelium discoideum, while the nucleomorph (eukaryotic endosymbiont) of the cryptophyte Rhodomonas salina seems to be related to the chlorobiont lineage. The glaucocystophyte Cyanophora paradoxa and the nuclear sequence (host) of the cryptomonad alga Guillardia theta also seem to be closely related. The Cryptophyta and the heterokont algae have evolved from different secondary endosymbiotic events involving different hosts and probably different endosymbionts. However, until more stress-70 sequences of algal divisions become available no definitive conclusions can be drawn concerning branching of the major divisions.

Molecular phylogeny of the stress-70 protein family with reference to algal relationships

The stress-70 protein family has previously been shown to be a useful tool for molecular phylogeny at the kingdom to family levels. Although sequences of many members of the stress-70 family are available, few genes from the Protoctista have been sequenced to date. Phylogenetic analyses of algae based on various molecules have not, as yet, provided clear results concerning relationships between major divisions. We cloned and sequenced several algal stress-70 genes in order to provide additional data and to further analyse phylogenetic relationships among algal divisions. New nuclear sequences were obtained from Guillardia theta (Cryptophyta), Ascophyllum nodosum (Heterokontophyta) and Cyanophora paradoxa (Glaucocystophyta). Phylogenetic trees of the stress-70 protein family calculated using different methods are presented. In our trees, the heterokont alga Ascophyllum nodosum is closely related to the slime mould Dictyostelium discoideum, while the nucleomorph (eukaryotic endosymbiont) of the cryptophyte Rhodomonas salina seems to be related to the chlorobiont lineage. The glaucocystophyte Cyanophora paradoxa and the nuclear sequence (host) of the cryptomonad alga Guillardia theta also seem to be closely related. The Cryptophyta and the heterokont algae have evolved from different secondary endosymbiotic events involving different hosts and probably different endosymbionts. However, until more stress-70 sequences of algal divisions become available no definitive conclusions can be drawn concerning branching of the major divisions.

Expression of the stress-70 protein family (HSP70) due to heavy metal contamination in the slug, Deroceras reticulatum: An approach to monitor sublethal stress conditions

Adult specimens of the slug, Deroceras reticulatum, were exposed to sublethal concentrations of lead, cadmium, and zinc. After three weeks of exposure, atomic absorption spectrophotometry revealed accumulation of all these metals in the animals. Highest exposure concentrations of lead, cadmium, and zinc resulted in highest contents of the respective metals in the slugs. In accordance with this effect, D. reticulatum showed an increased expression of the 70 kD stress protein (stress-70, hsp70) in response to exposure to all three heavy metals tested. Quantification of hsp70 increase took place with standardized Western-blotting and densitometric imaging after background correction. Multiple investigations revealed different reaction patterns to lead, cadmium, or zinc exposure and showed that the applied technique serves as a versatile tool to assess invertebrate stress reactions in response to environmentally relevant toxicants.

The stress-70 protein family in diplopods: induction and characterization

To induce stress-70 proteins (hsp70), adults of the millipede Julus scandinavius (Diplopoda) were exposed to leaf litter contaminated with different concentrations of Cd2+ (10, 30, 50 and 60 mg·kg-1 as CdCl2). The expression of hsp70 was investigated by semiquantitative and qualitative biochemical methods. After SDS-gel electrophoresis and Western blotting a subsequent digital image analysis showed that increasing dietary concentrations of Cd2+ resulted in elevated levels of hsp70, which in turn indicated proteotoxic condition. Qualitative results were obtained by two-dimensional gel electrophoresis. A stress-70 protein family, similar to that of other arthropods, was detected in Julus scandinavius: at least five different proteins with an approximate molecular weight of 68, 69, 70, 77, and 78 kDa could be distinguished after heat shock as well as after Cd2+ exposure.

Localization of STCH to Human Chromosome 21q11.1

STCH is a member of the stress70 chaperone family, which plays a major role in the processing of cytosolic and secretory proteins. Members of the stress70 protein chaperone family participate in protein processing events by binding denatured or misfolded peptide sequences and then releasing these polypeptide chains by an ATP-dependent mechanism. Members of this protein family possess an ATPase activity located in the highly conserved amino-terminal domain and a less well-conserved carboxy-terminal domain necessary for peptide binding. The Stch gene encodes a 60-kDa peptide that is constitutively expressed in all human cell types and shares a high degree of amino acid identity with HSP70 and BiP. The protein differs from previously identified stress70 gene products by the presence of a unique hydrophobic signal sequence and the absence of a carboxy-terminal peptide-binding domain. This results in the protein being highly enriched in the lumen of a crude cellular microsome fraction and possessing an ATPase activity that, unlike other HSP70-like proteins, is not peptide inducible. Analysis of the rat Stch cDNA demonstrates that the unique properties of this C-terminal truncated HSP70-like molecule are conserved through mammalian evolution. As with the human Stch gene, the rat cDNA encodes a hydrophobic leader sequence, andmore » the putative translation product lacks a C-terminal peptide-binding domain. 7 refs., 2 figs.« less

The simultaneous immunological detection of four stress-70 protein isoforms in Mytilus edulis

Organisms exposed to adverse environmental conditions exhibit a stress-70 protein response which is highly conserved from bacteria to man. Immunological analysis of the stress-70 response in mussels is of interest for its potential use in marine biomonitoring. We have analysed the gill tissues of Mytilus edulis individuals for changes in stress-70 complement after exposure to 48 h of temperature stress. Using a commercially available monoclonal antibody raised against human hsp70 and epitope mapped to a conserved ATP binding region, the authors were able to detect by western analysis constitutively-expressed 72kDa and 78kDa isoforms in control animals acclimated at normal temperatures. After 48 h of heat stress, induction of a 70kDa protein was clearly seen along with an increase in the level of the 72kDa protein. Western analysis using two-dimensional gel electrophoresis indicated that the antibody recognised four stress-70 isoforms (70, 72, 72 and 78kDa). This simultaneous detection of four members of the stress-70 family will permit studies on the differential regulation of each of the four members of the multigene family in response to a variety of environmental Stressors.

Phylogenetic analysis of the stress-70 protein family

The eukaryotic cyto-/nucleoplasmatic 70-kDa heat-shock protein (HSP70) has homologues in the endoplasmic reticulum as well as in bacteria, mitochondria, and plastids. We selected a representative subset from the large number of sequenced stress-70 family members which covers all known branches of the protein family and calculated and manually improved an alignment. Here we present the consensus sequence of the aligned proteins and putative nuclear localization signals (NLS) in the eukaryotic HSP70 homologues. The phylogenetic relationships of the stress-70 group family members were estimated by use of different computation methods. We present a phylogenetic tree containing all known stress-70 subfamilies and demonstrate the usefulness of stress-70 protein sequences for the estimation of intertaxonic phylogeny.

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.

Structure and organization of the gene encoding a mouse mitochondrial stress-70 protein

We have previously found that an antigenic protein specific for C3H strain mouse (C3H strain-specific antigen, CSA) is identical to peptide-binding protein 74 (PBP74). PBP74/CSA is a novel member of the stress-70 protein family in mitochondria. In this study, mouse genomic clones encoding PBP74/CSA, including the 5'- and 3'-flanking regions of the gene, have been isolated and sequenced. The PBP74/CSA gene contained 17 exons interrupted by 16 introns. Two dimeric repeats of the consensus sequence of the heat-shock element are present in the 5'-flanking region of the PBP74/CSA gene. Moreover, the first intron is interrupted within the amino-terminal leader sequence, the pattern of which is similar to that of cytochrome c 1, located in the mitochondria.

Antigenic Protein Specific for C3H Strain Mouse Is a Mitochondrial Stress-70 Protein

Cells derived from C3H strain mouse produce an antigenic protein (CSA) specific for this strain [Kusakabe, M., et al. (1988) J. Cell Biol. 107, 257-265]. To examine the molecular basis of CSA, eDNA cloning of the antigenic protein was carried out. The deduced amino acid sequence demonstrates that CSA is the same protein as peptide-binding protein74 (PBP74), a novel member of the stress-70 family. However, comparison of the PBP74/CSA sequences between C3H/HeN and BALB/c strain mice reveals the substitution of two amino acids in the substrate-recognition domain of the stress-70 protein. Western blot analysis indicates that one out of these two residues, arginine at residue 578 in the PBP74/CSA sequence of C3H mouse, contributes to the immunogenecity of CSA. Moreover, the subcellular localization of PBP74/CSA in mitochondria is also demonstrated by immunohistochemical analysis using anti-CSA monoclonal antibody. Thus, it is interesting that a genetic marker sequence in mice is located on the gene encoding a mitochondrial stress-70 protein.

Structure and Mechanism of 70-kDa Heat-Shock-Related Proteins

This chapter focuses on the protein chemistry and mechanistic enzymology of the 70-kDa heat-shock-related protein family. Members of this family of proteins are present in cells under normal conditions. Induction of expression of some members in response to heat shock or other forms of cell stress is only a manifestation of an additional facet of the collective functions of the larger family of proteins. A stress-70 protein is localized in the endoplasmic reticulum and is referred as the binding protein. The stress-70 proteins are demonstrated to participate in diverse and seemingly unrelated activities: (1) disassembly of specific macromolecular aggregates, (2) interaction with extended and presumably unfolded polypeptides, and (3) renaturation or reactivation of certain proteins under some conditions. The stress-70 proteins have a common mechanism for transducing free energy of nucleotide hydrolysis into interactions with the protein/polypeptide substrates. The functional organization of stress-70 proteins within their primary, tertiary, and temperature-dependent structural transitions are provided. The enzymatic mechanism of stress-70 proteins involves ATPas mechanism, peptide recognition, coupling of peptide-binding and ATPase activities, and clathrin uncoating reaction.