Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.     

Temperature-dependent binding to the thylakoid membranes of nuclear-coded chloroplast heat-shock proteins

Two nuclear-coded heat-shock proteins (HSP) of pea (Pisum sativum) are synthesized as larger precursors of 26 kDa and 30 kDa in vitro. They are transported post-translationally into isolated, homologous chloroplasts where they are processed to mature proteins of 22 kDa and 25 kDa, respectively. When the chloroplasts used for the transport are isolated from control plants grown at 25 degrees C the 22-kDa and 25-kDa HSPs are located in the stroma of the chloroplasts. However, when chloroplasts are prepared from heat-shocked plants both proteins are found bound to the thylakoid membranes. The transition of the non-binding to the binding status is comparatively sharp and occurs between 36 degrees C and 40 degrees C in the variety 'Rosa Krone'. The transition temperature has been determined at 38 degrees C for 'Rosa Krone' and at 40 degrees C for the variety 'Golf'. At 42 degrees C, 15-min treatment of the plants is sufficient to induce membrane binding, which persists for at least 4-6 h (but not for 24 h) after return to the ambient temperature. Once lost, membrane binding can be reinduced by a second heat-shock treatment in vivo. High light intensities during the heat shock interfere with the binding capacity for heat-shock proteins.     

Thermotolerance and the heat-shock response in Candida albicans

At elevated temperatures, yeast cells of Candida albicans synthesized nine heat-shock proteins (HSPs) with apparent molecular masses of 98, 85, 81, 76, 72, 54, 34, 26 and 18 kDa. The optimum temperature for the heat-shock response was 45 degrees C although HSPs were detected throughout the range 41-46 degrees C. Protein synthesis was not observed in cells kept at 48 degrees C. Yeast cells survived exposure to an otherwise lethal temperature of 55 degrees C when they had previously been exposed to 45 degrees C. The thermotolerance induced during incubation at 45 degrees C required protein synthesis, since protection was markedly reduced by trichodermin. Mercury ions induced a set of three stress proteins, one of which corresponded in size to an HSP, and cadmium ions evoked one stress protein seemingly unrelated to the HSPs observed after temperature shift.     

The 23-kDa light-stress-regulated heat-shock protein of Chenopodium rubrum L. is located in the mitochondria

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et␣al. (1995, Biochem J 311:805–813) and LaFayette et␣al. (1996, Plant Mol Biol 30:159–169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions. Received: 11 July 1996 / Accepted: 24 August 1996     

Complexity and Genetic Variability of Heat-Shock Protein Expression in Isolated Maize Microspores

The expression of heat-shock proteins (HSPs) in isolated maize (Zea mays L.) microspores has been investigated using high-resolution two-dimensional electrophoresis coupled to immunodetection and fluorography of in vivo synthesized proteins. To this end, homogeneous and viable populations of microspores have been purified in sufficient amounts for molecular analysis from plants grown in controlled conditions. Appropriate conditions for thermal stress application have been defined. The analysis revealed that isolated microspores from maize display a classical heat-shock response characterized by the repression of the normal protein synthesis and the expression of a set of HSPs. A high complexity of the response was demonstrated, with numerous different HSPs being resolved in each known major HSP molecular weight class. However, the extent of this heat-shock response is limited in that some of these HSPs do not accumulate at high levels following temperature elevation. Comparative analysis of the heat-shock responses of microspores isolated from five genotypes demonstrated high levels of genetic variability. Furthermore, many HSPs were detected in microspores at control temperature, indicating a possible involvement of these proteins in pollen development at stages close to first pollen mitosis.     

Induction of heat-shock (stress) protein gene expression by selected natural and anthropogenic disturbances in the octocoral Dendronephthya klunzingeri

Previously it was found that the expression of selected heat-shock proteins is upregulated in corals after exposure to elevated temperature. We published that HSPs are suitable markers in sponges to monitor the degree of environmental stress on these animals. In the present study the heat-shock proteins (HSPs) with a molecular weight of 90 kDa have been selected to prove their potential usefulness as biomarkers under controlled laboratory conditions and in the field. The studies have been performed with the octocoral Dendronephthya klunzingeri4.5-fold higher steady-state level of the respective mRNA. Also animals taken from stressed locations in the field showed an increased expression. The amount of HSP90 protein in D. klunzingeri was found to be strongly increased under thermal stress, or exposure to polychlorinated biphenyl (congener 118), but not after treatment with cadmium. Field studies revealed that samples taken from a nonstressed area have a low level of HSP90, but those collected from locations at which the corals are under physical stress (sedimentation through landfilling) show a high expression of HSP90. It is concluded that the chaperone HSP90 might become a suitable biomarker to monitor environmental stress on corals.     

Activation of Heat-Shock Genes by Digitonin is Selectively Repressed in Preheated Drosophila Salivary Glands

Treatment of Drosophila salivary glands with a mild detergent, digitonin, activates puffing at 35 chromosome loci. These digitonin-activated puffs include all of the nine heat-shock puffs known in D. melanogaster . Here we show that the activation of heat-shock genes, but not of other digitoninstimulated puffs, is repressed in salivary glands which have been subjected to and have recovered from heat shock before being treated with digitonin. The findings indicate that, (a) the activation of heat-shock genes by digitonin, as that by temperature elevation, is self-regulated by the heat-shock proteins (HSPs). (b) the gene repressive activity of HSPs is heat-shock-gene specific, and (c) the repression mechanism of heat-shock genes by HSPs is resistant to digitonin, in contrast to that the suppression of heat-shock genes is prevented by the detergent in non-heat-shocked salivary glands. The selective repression of heat-shock genes in preheated salivary glands suggests that the heat-shock genes and other digitonin-activated genes may be controlled by a different mechanism(s).     

ATP causes small heat shock proteins to release denatured protein.

Small heat-shock proteins (sHSPs) are a ubiquitous family of low molecular mass (15-30 kDa) stress proteins that have been found in all organisms. Under stress, sHSPs such as alpha-crystallin can act as chaperones binding partially denatured proteins and preventing further denaturation and aggregation. Recently, it has been proposed that the function of sHSPs is to stabilize stress-denatured protein and then act cooperatively with other HSPs to renature the partially denatured protein in an ATP-dependent manner. However, the process by which this occurs is obscure. As no significant phosphorylation of alpha-crystallin was observed during the renaturation, the role of ATP is not clear. It is now shown that ATP at normal physiological concentrations causes sHSPs to change their confirmation and release denatured protein, allowing other molecular chaperones such as HSP70 to renature the protein and renew its biological activity. In the absence of ATP, sHSPs such as alpha-crystallin are more efficient than HSP70 in preventing stress-induced protein aggregation. This work also indicates that in mammalian systems at normal cellular ATP concentrations, sHSPs are not effective chaperones.     

Chilling tolerance of cucumber (Cucumis sativus) seedling radicles is affected by radicle length, seedling vigor, and induced osmotic- and heat-shock proteins

Cucumber seedling radicles decrease in chilling tolerance as they increase in length or decrease in vigor. The protein content of the apical 5 mm of the radicle decreased with decreases in chilling tolerance ( R ² = 0.92). This general reduction in protein content was reflected in a decrease of six dehydrin-like proteins with apparent molecular weights of 13.0, 15.0, 16.8, 23.0, 26.8, and 33.5 kDa. The disappearance of naturally occurring dehydrin-like proteins in cucumber seedling radicles as they elongate or lose vigor was correlated with a loss of chilling tolerance. Exposure to an osmotic (0.6 M mannitol) or heat (2 min at 45°C) stress enhanced chilling tolerance. The osmotic-shock treatment induced both chilling tolerance and the appearance or strengthening of dehydrin-like proteins previously present in radicles. The heat-shock treatment also induced high levels of chilling tolerance and protein(s) that reacted with a 23 and 70 kDa antibody. However, these heat-shock protein (HSPs) did not cross react with the probe for dehydrin-like proteins. When organized into high, medium, and low chilling tolerance groups, radicle that were chilling tolerant contained either the 13.0 and 16.8 kDa dehydrin-like proteins, or the 15.0 and 23.0 kDa dehydrin-like proteins, or the 23 or 70 kDa HSP.     

Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB

We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (相似文献   

Heat-shock proteins induce T-cell regulation of chronic inflammation

Immune responses to certain heat-shock proteins (HSPs) develop in almost all inflammatory diseases; however, the significance of such responses is only now becoming clear. In experimental disease models, HSPs can prevent or arrest inflammatory damage, and in initial clinical trials in patients with chronic inflammatory disease, HSP-derived peptides have been shown to promote the production of anti-inflammatory cytokines, indicating that HSPs have immunoregulatory potential. In this Review, we discuss the unique characteristics of HSPs that endow them with these immunoregulatory qualities.     

Interferon pretreatment lowers the threshold for maximal heat-shock response in mouse cells

Interferons (IFNs) are proteins which have antiviral and antiproliferative properties and are known to affect various immunological processes. Some of these activities have been shown to be potentiated by increased temperatures. When cells are subjected to a rise in temperature, the synthesis of the heat-shock proteins (HSPs) is 'switched on.' In this report we demonstrate a synergistic effect of IFN and stress (arsenite treatment or elevated temperature) on the heat-shock response. On the one hand, IFN pretreatment enhances the accumulation of HSP mRNAs and the corresponding protein synthesis after a mild stress and, on the other hand, it amplifies the decrease of the total protein synthesis after a severe stress. Thus in IFN pretreated cells the range of temperatures leading to the heat-shock response is shifted towards common physiological values.     

Heat shock induced proteins in plant cells

Tobacco (Nicotiana tabacum) and soybean (Glycine max) tissue culture cells were exposed to a heat shock and protein synthesis studied by SDS-polyacrylamide gel electrophoresis after labeling with radioactive amino acids. A new pattern of protein synthesis is observed in heat-shocked cells compared to that in control cells. About 12 protein bands, some newly appearing, others synthesized in greatly increased quantities in heat-shock cells, are seen. Several of the heat-shock proteins (HSPs) in both tobacco and soybean are similar in size. One of the HSPs in soybean (76K) shares peptide homology with its presumptive 25°C counterpart, indicating that the synthesis of at least some HSPs may not be due to activation of new genes. The optimum temperature for maximal induction of most HSPs is 39–40°C. Total protein synthesis decreases as heat-shock temperature is increased and is barely detectable at 45°C. The heat-shock response is maintained for a relatively short time in tobacco cells. After 3 hr at 39°C, a decrease is seen in the synthesis of the HSPs, and after 4 hr practically no HSPs are synthesized. After exposure to 39°C for 1 hr, followed by a return of tobacco cells to 26°C, recovery to the control pattern of synthesis requires greater than 6 hours. These results indicate that cells of flowering plants exhibit a heat-shock response similar to that observed in animal cells.     

Analytical assays of human HSP27 and thermal-stress survival of Escherichia coli cells that overexpress it

HSP27 is a small heat-shock protein (sHSP). Such proteins are produced in all organisms. These small HSPs exhibit chaperone-like activity that can bind to unfolded polypeptides and prevent uncontrolled protein aggregation in vitro. Cellular anti-apoptosis function and enhanced cell survival are correlated with increased expression of HSPs. This study presents a thermal-stress survival model for cells using the Escherichia coli expression system for which human HSP27, a recombinant protein, is inducible. Results show that E. coli cells overexpressing human HSP27 have enhanced tolerance to 50 degrees C thermal stress.     

The synergistic effect of hyperthermia and ethanol on changing gene expression of mouse lymphocytes

Cultured mouse lymphocytes respond to a brief incubation at an elevated temperature (41-43 degrees C) with the new and (or) enhanced synthesis of a select group of polypeptides (known as heat-shock proteins, HSPs) having relative molecular masses of 110, 100, 90, 70, and 65 kilodaltons (kDa). Expression of these HSPs is dependent on new RNA synthesis. Because the synthesis of any particular HSP is dependent on the temperature and the length of time cells remain at a particular elevated temperature, synthesis of each HSP is not necessarily coordinated with the synthesis of the other HSPs. Cultured mouse lymphocytes treated with arsenite or ethanol exhibit new and (or) enhanced synthesis of HSPs with molecular masses of 110, 90, 70, and 65 kDa but do not exhibit enhanced synthesis of the 100-kDa HSP. Short-term concurrent exposure of mouse lymphocytes to an elevated temperature and a level of ethanol, which individually do not induce detectable HSP synthesis, results in the pronounced synthesis of HSPs similar to those seen following exposure to higher levels of either stress applied separately. Thus, in this study we demonstrate that hyperthermia and ethanol stress can act synergistically to affect a dramatic change in the gene expression of mouse lymphocytes.     

The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.     

Protection of Chinese hamster ovary cells from heat killing by treatment with cycloheximide or puromycin: involvement of HSPs?

Cycloheximide (CHM) or puromycin (PUR) added for 2 h before heating at 43 degrees C followed by either PUR or CHM during heat greatly protected cells from heat killing. This protection increased with inhibition of protein synthesis. Since treatment with a drug both before and during heating was required for heat protection, and since one drug could be exchanged for the other after the 2-h pretreatment without affecting the heat protection, a common mode of action involving inhibition of protein synthesis is suggested for the two drugs. Drug treatment reduced the synthesis of heat-shock proteins (HSPs) as studied by one-dimensional gel electrophoresis by 80-98% relative to 37 degrees C untreated controls. Synthesis of large molecules (greater than 30 kDa) was preferentially inhibited by PUR but not by CHM. Also for CHM, but not for PUR treatment, a 42 kDa band appeared along with a great reduction in the 43 kDa actin band during CHM treatment at both 37 and 43 degrees C. Furthermore, during CHM or PUR treatment, incorporation of [35S]methionine into HSP families 70, 87, or 110 was not increased relative to incorporation into total protein. However, synthesis of the 70 kDa HSP family was selectively suppressed when cells were incubated at 37 degrees C after CHM treatment, but when cells were incubated at 37 degrees C after treatment at 43 degrees C with CHM, synthesis of the 70 kDa HSP family resumed. When cells were labeled for 3 days, there was no preferential accumulation or turnover of HSP families during heating with or without CHM. Therefore, heat protection caused by treatment with CHM or PUR apparently involves a common mode of action not associated with changes in either total levels or synthesis of HSP families during drug treatment before and during heating. The significance of the changes observed in the synthesis of the HSP 70 family after heat is unknown. As thermotolerance developed during 5 h at 42 degrees C without drugs, synthesis of HSP families 70, 87, and 110, as studied with one-dimensional gels, increased 1.4-fold relative to synthesis of total protein, but compared to HSP families in cells labeled for 5 h at 37 degrees C incorporation was reduced by 40%. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Heat shock proteins in oncology: Diagnostic biomarkers or therapeutic targets?

Heat shock proteins (HSP) are a family of proteins induced in cells exposed to different insults. This induction of HSPs allows cells to survive stress conditions. Mammalian HSPs have been classified into six families according to their molecular size: HSP100, HSP90, HSP70, HSP60, HSP40 and small HSPs (15 to 30kDa) including HSP27. These proteins act as molecular chaperones either helping in the refolding of misfolded proteins or assisting in their elimination if they become irreversibly damaged. In recent years, proteomic studies have characterized several different HSPs in various tumor types which may be putative clinical biomarkers or molecular targets for cancer therapy. This has led to the development of a series of molecules capable of inhibiting HSPs. Numerous studies speculated that over-expression of HSP is in part responsible for resistance to many anti-tumor agents and chemotherapeutics. Hence, from a pharmacological point of view, the co-administration of HSP inhibitors together with other anti-tumor agents is of major importance in overcoming therapeutic resistance. In this review, we provide an overview of the current status of HSPs in autoimmune, cardiovascular, and neurodegenerative diseases with special emphasis on cancer.     

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.