Heat Shock Protein Hsp70 Family Research Articles

Potential of Plant-Based Extracts to Alleviate Sorbitol-Induced Osmotic Stress in Cabbage Seedlings.

In light of expected climate change, it is important to seek nature-based solutions that can contribute to the protection of our planet as well as to help overcome the emerging adverse changes. In an agricultural context, increasing plant resistance to abiotic stress seems to be crucial. Therefore, the scope of the presented research was focused on the application of botanical extracts that exerted positive effects on model plants growing under controlled laboratory conditions, as well as plants subjected to sorbitol-induced osmotic stress. Foliar spraying increased the length and fresh mass of the shoots (e.g., extracts from Taraxacum officinale, Trifolium pratense, and Pisum sativum) and the roots (e.g., Solidago gigantea, Hypericum perforatum, and Pisum sativum) of cabbage seedlings grown under stressful conditions, as well as their content of photosynthetic pigments (Pisum sativum, Lens culinaris, and Hypericum perforatum) along with total phenolic compounds (Hypericum perforatum, Taraxacum officinale, and Urtica dioica). The antioxidant activity of the shoots measured with the use of DDPH (Pisum sativum, Taraxacum officinale, Urtica dioica, and Hypericum perforatum), ABTS (Trifolium pratense, Symphytum officinale, Valeriana officinalis, Pisum sativum, and Lens culinaris), and FRAP (Symphytum officinale, Valeriana officinalis, Urtica dioica, Hypericum perforatum, and Taraxacum officinale) assays was also enhanced in plants exposed to osmotic stress. Based on these findings, the most promising formulation based on Symphytum officinale was selected and subjected to transcriptomic analysis. The modification of the expression of the following genes was noted: Bol029651 (glutathione S-transferase), Bol027348 (chlorophyll A-B binding protein), Bol015841 (S-adenosylmethionine-dependent methyltransferases), Bol009860 (chlorophyll A-B binding protein), Bol022819 (GDSL lipase/esterase), Bol036512 (heat shock protein 70 family), Bol005916 (DnaJ Chaperone), Bol028754 (pre-mRNA splicing Prp18-interacting factor), Bol009568 (heat shock protein Hsp90 family), Bol039362 (gibberellin regulated protein), Bol007693 (B-box-type zinc finger), Bol034610 (RmlC-like cupin domain superfamily), Bol019811 (myb_SHAQKYF: myb-like DNA-binding domain, SHAQKYF class), Bol028965 (DA1-like Protein). Gene Ontology functional analysis indicated that the application of the extract led to a decrease in the expression of many genes related to the response to stress and photosynthetic systems, which may confirm a reduction in the level of oxidative stress in plants treated with biostimulants. The conducted studies showed that the use of innovative plant-based products exerted positive effects on crops and can be used to supplement current cultivation practices.

Identification of heat shock protein hsp70 family genes from Rana amurensis and its expression profiles upon infection

Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.

Scaffold Hopping and Screening for Potent Small Molecule Agonists for GRP94: Implications to Alleviate ER Stress-Associated Pathogenesis.

Disparity in the activity of Endoplasmic reticulum (ER) leads to degenerative diseases, mainly associated with protein misfolding and aggregation leading to cellular dysfunction and damage, ultimately contributing to ER stress. ER stress activates the complex network of Unfolded Protein Response (UPR) signaling pathways mediated by transmembrane proteins IRE1, ATF6, and PERK. In addition to UPR, many ER chaperones have evolved to optimize the output of properly foldedsecretory and membrane proteins. Glucose-regulated protein 94 (GRP94), an ER chaperone of heat shock protein HSP90 family, directs protein folding through interaction with other components of the ER protein folding machinery and assists in ER-associated degradation (ERAD). Activation of GRP94 would increase the efficacy of protein folding machinery and regulate the UPR pathway toward homeostasis. The present study aims to screen for novel agonists for GRP94 based on Core hopping, pharmacophore hypothesis, 3D-QSAR, and virtual screening with small-molecule compound libraries in order to improve the efficiency of native protein folding by enhancing GRP94 chaperone activity, therefore to reduce protein misfolding and aggregation. In this study, we have employed the strategy of small molecule-dependent ER programming to enhance the chaperone activity of GRP94 through scaffold hopping-based screening approach to identify specific GRP94 agonists. New scaffolds generated by altering the cores of NECA, the known GRP94 agonist, were validated by employing pharmacophore hypothesis testing, 3D-QSAR modeling, and molecular dynamics simulations. This facilitated the identification of small molecules to improve the efficiency of native protein folding by enhancing GRP94 activity. High-throughput virtual screening of the selected pharmacophore hypothesis against Selleckchem and ZINC databases retrieved a total of 2,27,081 compounds. Further analysis on docking and ADMET properties revealed Epimedin A, Narcissoside, Eriocitrin 1,2,3,4,6-O-Pentagalloylglucose, Secoisolariciresinol diglucoside, ZINC92952357, ZINC67650204, and ZINC72457930 as potential lead molecules. The stability and interaction of these small molecules were far better than the known agonist, NECA indicating their efficacy in selectively alleviating ER stress-associated pathogenesis. These results substantiate the fact that small molecule-dependent ER reprogramming would activate the ER chaperones and therefore reduce the protein misfolding as well as aggregation associated with ER stress in order to restore cellular homeostasis.

Functional Characterization of Gomisin N in High-Fat-Induced Drosophila Obesity Models.

Gomisin N (GN) is lignin derived from Schisandra chinensis that has been reported to exhibit hepato-protective, anti-cancer, and anti-inflammatory effects. However, its role in whole-body energetic homeostasis remains unclear. In this study, we employed Drosophila melanogaster as a diet-induced obese model to elucidate the effects of GN on lipid and glucose metabolism by measuring climbing activity, triglyceride levels, and lifespan under a rearing condition of a high-fat diet (HFD) containing 20% coconut oil, with or without GN. Constant exposure of flies to an HFD resulted in increased body weight and decreased climbing activity, along with a shortened life span. Importantly, the administration of GN to HFD groups lowered their body weight and induced a specific upregulation of lipid storage droplet (Lsd)-2 and hormone-sensitive lipase (Hsl), in addition to improved lifespan. Importantly, GN in HFD groups appeared to downregulate heat shock protein Hsp90 family member (dGRP94), a key regulator of the endoplasmic reticulum stress response, which may also contribute to improved life span in the presence of GN. Taken together, these in vivo findings suggest that GN could serve as a useful agent for the prevention and treatment of obesity.

Identification of Heat-Responsive Genes in Guar [Cyamopsis tetragonoloba (L.) Taub

The threat of heat stress on crop production increased dramatically due to global warming leading to the rise on the demand of heat-tolerant crops and understanding their tolerance. The leguminous forage crop Guar [Cyamopsis tetragonoloba (L.) Taub] is a high-temperature tolerant plant with numerous works on its tolerance at morph-physiological levels but lack on molecular thermotolerance level. In the current study, the differential gene expression and the underlying metabolic pathways induced by heat treatment were investigated. An RNA-Seq study on Guar leaves was carried out to estimate gene abundance and identify genes involved in heat tolerance to better understand the response mechanisms to heat stress. The results uncovered 1551 up- and 1466 downregulated genes, from which 200 and 72 genes with unknown function could be considered as new genes specific to guar. The upregulated unigenes were associated with 158 enzymes and 102 KEGG pathways. Blast2GO, InterProScan, and Kyoto Encyclopaedia of Genes and Genomes packages were utilized to search the functional annotation, protein analysis, enzymes, and metabolic pathways and revealed hormone signal transduction were enriched during heat stress tolerance. A total of 301 protein families, 551 domains, 15 repeats, and 3 sites were upregulated and matched to those unigenes. A batch of heat-regulated transcription factor transcripts were identified using the PlantTFDB database, which may play roles in heat response in Guar. Interestingly, several heat shock protein families were expressed in response to exposure to stressful conditions for instance small HSP20, heat shock transcription factor family, heat shock protein Hsp90 family, and heat shock protein 70 family. Our results revealed the expressional changes associated with heat tolerance and identified potential key genes in the regulation of this process. These results will provide a good start to dissect the molecular behaviour of plants induced by heat stress and could identify the key genes in stress response for marker-assisted selection in Guar and reveal their roles in stress adaptation in plants.

A new heat shock protein 70 gene (HSC70) and its expression profiles in response to cadmium stress and after different post-moulting times in Exopalaemon carinicauda (Holthuis, 1950) (Decapoda, Palaemonidae)

In this study, the full-length cDNA sequence (GenBank accession number AGF80339.1) encoding a novel heat shock protein HSP70 family member (Heat shock cognate 70, EcHSC70) was cloned from the ridgetail white prawn,Exopalaemon carinicauda(Holthuis, 1950) [currently also as:Palaemon carinicaudaHolthuis, 1950].EcHSC70full-length cDNA consists of 2452 bp, containing an open reading frame (ORF) of 1935 bp, and it encodes a 650-amino-acid protein with a theoretical size of about 71 kDa and a predicted isoelectric point of 5.32. Phylogenetic analysis showed that EcHSC70 can be categorized together with the known HSP70 family members reported in other crustaceans. Tissue-expression analysis revealed thatEcHSC70was constitutively expressed in all of the tested tissues, with a significantly increased expression in the gill post-moulting. Moreover, the relative mRNA level ofEcHSC70tended to increase in the early stages of post-moulting (from 0 to 5 min), suggesting that EcHSC70 might take part in the recovery ofE. carinicaudaafter moulting. In addition, under different levels of cadmium stress,EcHSC70tended to be significantly expressed only after 24 h of cadmium exposure, and was more inducible by low concentrations of cadmium, as opposed to high concentrations.

The Metacaspase (Mca1p) has a Dual Role in Farnesol-induced Apoptosis in Candida albicans

Manipulating the apoptotic response of Candida albicans may help in the control of this opportunistic pathogen. The metacaspase Mca1p has been described as a key protease for apoptosis in C. albicans but little is known about its cleavage specificity and substrates. We therefore initiated a series of studies to describe its function. We used a strain disrupted for the MCA1 gene (mca1Δ/Δ) and compared its proteome to that of a wild-type isogenic strain, in the presence and absence of a known inducer of apoptosis, the quorum-sensing molecule farnesol. Label-free and TMT labeling quantitative proteomic analyses showed that both mca1 disruption and farnesol treatment significantly affected the proteome of the cells. The combination of both conditions led to an unexpected biological response: the strong overexpression of proteins implicated in the general stress. We studied sites cleaved by Mca1p using native peptidomic techniques, and a bottom-up approach involving GluC endoprotease: there appeared to be a "K/R" substrate specificity in P1 and a "D/E" specificity in P2. We also found 77 potential substrates of Mca1p, 13 of which validated using the most stringent filters, implicated in protein folding, protein aggregate resolubilization, glycolysis, and a number of mitochondrial functions. An immunoblot assay confirmed the cleavage of Ssb1p, a member of the HSP70 family of heat-shock proteins, in conditions where the metacaspase is activated. These various results indicate that Mca1p is involved in a limited and specific proteolysis program triggered by apoptosis. One of the main functions of Mca1p appears to be the degradation of several major heat-shock proteins, thereby contributing to weakening cellular defenses and amplifying the cell death process. Finally, Mca1p appears to contribute significantly to the control of mitochondria biogenesis and degradation. Consequently, Mca1p may be a link between the extrinsic and the intrinsic programmed cell death pathways in C. albicans.

Heat Shock Protein Gp96 as Potential Regulator of Morphostasis after Partial Hepatectomy in Mice

Gp96 (also known as glucose-regulated protein 94, endoplasmin) is the endoplasmic reticulum (ER)-resident protein, which belongs to the heat shock protein HSP90 family. It is upregulated in response to glucose starvation and other stressful stimuli that disrupt protein synthesis in the ER. There, it is acting as a molecular chaperon involved in the correction of unfolded proteins, in the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, and in activation of protein translations that modulate the polypeptide traffic into the ER. In addition, it has been implicated in antigen presentation and MHC class I and II upregulation, in the activation and maturation of dendritic cells and proinflammatory cytokine secretion, as well as in chaperoning of integrins and Toll-like receptors, acting as a "danger signal" to the innate and adaptive immunity. Moreover, owing to its specific function in Ca2+ homeostasis and in the insulin- IGF/signaling pathways, it has been proposed that gp96 might participate in mechanisms that are critical for cell growth, differentiation, and responses to ER stress. Emphasizing that gp96, as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, may also be involved in the maintenance of morphostasis and self tolerance, in this survey we show that high levels of upregulation of gp96 in regenerating liver and thymus are followed by signs of transient autoimmunity, augmented apoptosis in thymus, and the presence of autoreactive NKT and regulatory T cells that might be involved in the control of rapid liver growth induced by partial hepatectomy.

Divergence between two Antarctic species of the ciliate Euplotes, E. focardii and E. nobilii, in the expression of heat‐shock protein 70 genes

Most organisms oppose many environmental stresses by rapidly enhancing synthesis of the highly conserved Hsp70 family of heat-shock proteins. Two ciliates which are endemic in Antarctic coastal seawater, Euplotes focardii and E. nobilii, and behave as psychrophile and psychrotroph micro-organisms, respectively, revealed a divergence in the capacity to respond to thermal stress with an activation of the transcription of their hsp70 genes. In both species, these genes were shown to be represented by thousands of copies in the cell's somatic functional nucleus (macronucleus). However, while a strong transcriptional activity of hsp70 genes was induced in E. nobilii cells transferred from 4 to 20 degrees C, a much smaller increase was revealed in heat-shocked cells of E. focardii. These findings suggest a closer adaptation to the stably cold Antarctic waters in the genetic response of E. focardii to thermal stress.

Overexpression of the Heat Shock Protein HSP70 Family and p53 Protein and Prognosis for Patients with Gastric Cancer

The cell synthesis of heat shock proteins is increased by a variety of environmental and pathophysiological stressful conditions. The 70-kD heat shock protein family (HSP70 family) which constitutively expresses hsc70 and heat-inducible hsp70 is thought to be involved in protein-protein interactions, including oncogene products. We investigated the HSP70 family expression and biological behavior of gastric cancer, and its relation to p53 overexpression. Expressions of HSP70 and p53 in 164 primary gastric tumors were determined immunohistochemically. Exploratory data were analyzed on a set of 164 primary gastric cancers, and we constructed in prognostic significance of the HSP70 expression level and the relation to p53 overexpression. Expression of HSP70 (hsc70 and hsp70) were detected in nuclei and/or cytoplasm of cancer cells. Western blotting analysis showed that hsc70 and hsp70 were both expressed in five gastric cancer cell lines. Immunohistochemically stained positive cells of HSP70 varied from 0 (very weak) to 100%, in each case. The median level of positive cell rate was 19.0%. A HSP70 expression of over 19.0% was related to the differentiated tissue type of gastric cancer, but not to other clinicopathological factors. There was no difference in survival rates in subjects with higher and lower groups of HSP70 expression. HSP70 expression was also not related to p53 overexpression in the nuclei and p53 overexpression-related poorer prognosis. Our findings show that the expression of HSP70 is not associated with tumor advance-related characteristics or with the prognosis of gastric cancer. Measurements of HSP70 expression do not appear to be a useful prognostic marker.

Fibroblast growth factor-1 interacts with the glucose-regulated protein GRP75/mortalin

Fibroblast growth factor-1 (FGF-1), which lacks a signal peptide and is intracellularly localized as a result of endogenous expression or endocytosis, is thought to be involved in regulating cell growth and differentiation. In the study reported here, we purified proteins that bind intracellular FGF-1. Affinity adsorption was used to purify FGF-1-binding proteins from rat L6 cells expressing FGF-1. One of the isolated proteins was identified as the glucose-regulated protein GRP75/mortalin/PBP-74/mthsp70, a member of the hsp70 family of heat-shock proteins known to be involved in regulating glucose responses, antigen processing and cell mortality. The interaction of FGF-1 and GRP75/mortalin in vivo was confirmed by co-immunoprecipitation, immunohistochemical co-localization in Rat-1 fibroblasts and by using the yeast two-hybrid system. Moreover, a binding assay in vitro with the use of recombinant FGF-1 and mortalin demonstrated a direct physical interaction between the two proteins. These results reveal that GRP75/mortalin is an intracellular FGF-1-binding protein in cells and suggest that GRP75/mortalin is involved in the trafficking of and/or signalling by FGF-1.

A predicted consensus structure for the N-Terminal fragment of the heat shock protein HSP90 family

A secondary structure has been predicted for the heat shock protein HSP90 family from an aligned set of homologous protein sequences by using a transparent method in both manual and automated implementation that extracts conformational information from patterns of variation and conservation within the family. No statistically significant sequence similarity relates this family to any protein with known crystal structure. However, the secondary structure prediction, together with the assignment of active site positions and possible biochemical properties, suggest that the fold is similar to that seen in N-terminal domain of DNA gyrase B (the ATPase fragment).

A predicted consensus structure for the N‐Terminal fragment of the heat shock protein HSP90 family

A secondary structure has been predicted for the heat shock protein HSP90 family from an aligned set of homologous protein sequences by using a transparent method in both manual and automated implementation that extracts conformational information from patterns of variation and conservation within the family. No statistically significant sequence similarity relates this family to any protein with known crystal structure. However, the secondary structure prediction, together with the assignment of active site positions and possible biochemical properties, suggest that the fold is similar to that seen in N-terminal domain of DNA gyrase B (the ATPase fragment). Proteins 27:450–458, 1997. © 1997 Wiley-Liss, Inc.

HSP70-2 is part of the synaptonemal complex in mouse and hamster spermatocytes

Mouse spermatogenic cells are known to express HSP70-2, a member of the HSP70 family of heatshock proteins. The purpose of the present study was to characterize further the expression and localization of HSP70-2 in meiotic cells of mice and hamsters. After separating mouse spermatogenic cells into cytoplasmic and nuclear fractions, proteins were separated by two-dimensional gel electrophoresis and detected with HSP-specific antibodies. Of several HSP70 proteins identified in the cytoplasm, only HSC70 and HSP70-2 were also detected in the nucleus. Immunocytological analyses of spermatocyte prophase cells revealed that HSP70-2 was associated with the synaptonemal complex. Surfacespread synaptonemal complexes at pachytene and diplotene stages labeled distinctly with the antiserum to HSP70-2. Synaptonemal complexes from fetal mouse oocytes failed to show any evidence of HSP70-2. Reverse-transcriptase-polymerase chain reaction (RT-PCR) analyses of gene expression confirmed this sex specificity; Hsp70-2 mRNA was detected in mouse testes, but not ovaries. These findings are suggestive of a previously unsuspected sexual dimorphism in structure and/or function of the synaptonemal complex.

HSP70-2 is part of the synaptonemal complex in mouse and hamster spermatocytes

Mouse spermatogenic cells are known to express HSP70-2, a member of the HSP70 family of heat-shock proteins. The purpose of the present study was to characterize further the expression and localization of HSP70-2 in meiotic cells of mice and hamsters. After separating mouse spermatogenic cells into cytoplasmic and nuclear fractions, proteins were separated by two-dimensional gel electrophoresis and detected with HSP-specific antibodies. Of several HSP70 proteins identified in the cytoplasm, only HSC70 and HSP70-2 were also detected in the nucleus. Immunocytological analyses of spermatocyte prophase cells revealed that HSP70-2 was associated with the synaptonemal complex. Surface-spread synaptonemal complexes at pachytene and diplotene stages labeled distinctly with the antiserum to HSP70-2. Synaptonemal complexes from fetal mouse oocytes failed to show any evidence of HSP70-2. Reverse-transcriptase-polymerase chain reaction (RT-PCR) analyses of gene expression confirmed this sex specificity; Hsp70-2 mRNA was detected in mouse testes, but not ovaries. These findings are suggestive of a previously unsuspected sexual dimorphism in structure and/or function of the synaptonemal complex.

Electrophoretic analysis of yeast proteins.

AbstractPolyacrylamide gel electrophoresis (PAGE) of proteins provides a powerful qualitative and quantitative tool for studying yeast protein synthesis. Complex mixtures of proteins can be separated purely on the basis of molecular weight by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1), or alternatively on the basis of a combination of both molecular weight and electric charge using two-dimensional electrophoresis (2D SDS-PAGE). Two methods of 2D SDS-PAGE have been described, namely isoelectric focusing (IEF) gels (2) and nonequilibrium pH gradient (NEPHGE) gels (3). NEPHGE gels give better resolution of basic proteins compared to IEF gels (Fig. 1), and this is particularly useful for analyzing yeast proteins that contain many basic amino acids, e.g., ribosomal proteins and histones. Two-dimensional SDS-PAGE analysis of proteins synthesized in vivo in the yeast Saccharomyces cerevisiae. (A) Exponentially growing cells pulse-labeled for 10 min with [35S]-methionine, then separated in the first dimension by a nonequilibrium pH gradient gel (NEPHGE), and in the second dimension by 15% SDS-PAGE (SDS). (B) As for (A), except labeled with a [14C]-amino acid mixture. Open arrow heads indicate proteins not labeled with [35S]-methionine. (C) As for (A), except protein samples were run in a first dimension isoelectric focusing gel (IEF). The proteins indicated are: 1, elongation factor EF-1α; 2, the heat shock protein Hsp70 family of proteins; 3, glyceralde-hyde-3-phosphate dehydrogenase. KeywordsSodium Dodecyl SulfateAmmonium PersulfateYeast ProteinAmino Acid MixtureLabel Amino AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A member of the Hsp70 family of heat-shock proteins is a putative target for the immunosuppressant 15-deoxyspergualin.

Annals of the New York Academy of SciencesVolume 685, Issue 1 p. 202-204 A Member of the Hsp70 Family of Heat-Shock Proteins Is a Putative Target for the Immunosuppressant 15-Deoxyspergualin. Charles E. Mazzucco, Charles E. MazzuccoSearch for more papers by this authorSteven G. Nadler, Steven G. NadlerSearch for more papers by this author Charles E. Mazzucco, Charles E. MazzuccoSearch for more papers by this authorSteven G. Nadler, Steven G. NadlerSearch for more papers by this author First published: June 1993 https://doi.org/10.1111/j.1749-6632.1993.tb35866.xCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume685, Issue1Immunomodulating DrugsJune 1993Pages 202-204 RelatedInformation

A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria.

ABF2 (ARS-binding factor 2), a small, basic DNA-binding protein that binds specifically to the autonomously replicating sequence ARS1, is located primarily in the mitochondria of the yeast Saccharomyces cerevisiae. The abundance of ABF2 and the phenotype of abf2- null mutants argue that this protein plays a key role in the structure, maintenance, and expression of the yeast mitochondrial genome. The predicted amino acid sequence of ABF2 is closely related to the high-mobility group proteins HMG1 and HMG2 from vertebrate cell nuclei and to several other DNA-binding proteins. Additionally, ABF2 and the other HMG-related proteins are related to a globular domain from the heat shock protein hsp70 family. ABF2 interacts with DNA both nonspecifically and in a specific manner within regulatory regions, suggesting a mechanism whereby it may aid in compacting the mitochondrial genome without interfering with expression.

Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family.

Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site.

Identification of heat shock protein hsp70 homologues in chloroplasts.

Cytoplasmic members of the heat shock protein hsp70 family have recently been implicated in the transport of proteins to the endoplasmic reticulum and mitochondria. In addition, other hsp70 homologues have been found in the endoplasmic reticulum and mitochondria and, at least for the endoplasmic reticulum hsp70 homologue, may be involved in the proper folding and assembly of newly transported proteins. Since chloroplasts are an important site of protein transport in plant cells, we were interested in determining whether hsp70 proteins might be located in this organelle. By using immunoblotting techniques and two antibody preparations against hsp70 proteins, we have identified three chloroplastic proteins of approximately 70 kDa that are related to hsp70 proteins. One of these proteins was tightly associated with the outer envelope membrane and was not exposed at the outer surface of the chloroplasts. The other two were soluble proteins located in the stroma. Steady-state levels of the chloroplastic hsp70 homologues did not change after heat stress nor were any additional hsp70 homologues detected in chloroplasts isolated from heat-stressed plants. We discuss the possible functions of these hsp70 homologues in the transport of proteins into and within chloroplasts.