Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).     

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.     

Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in Chinese hamster ovary cells

Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5 degrees C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 microM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c, and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6-9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0-2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3-6 hr after heat shock, whereas that of HSP70b occurred immediately after ARS treatment. The degree of redistribution of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleus.     

Molecular chaperones: proposal of a systematic computer-oriented nomenclature and construction of a centralized database

Molecular chaperones are a wide group of unrelated protein families whose role is to assist others proteins. Comparably, under environmental stress, stress proteins behave as biocatalysts of protein stabilization. Stress proteins include a large class of proteins that were originally termed heat shock proteins (HSPs) due to their initial discovery in tissues exposed to elevated temperatures. Many, but not all, stress proteins and HSPs are molecular chaperones. Moreover, not all HSPs are derivable from stress. HSPs are structurally diversified by the contribution of various domains having specific roles. HSPs have been grouped, mainly on the basis of their molecular masses, into specific families that include small HSPs (sHSPs)/alpha-crystallins, HSP10s, HSP40s, HSP60s, HSP70s, HSP90s, HSP100s and HSP110s. The names of these major families are historical artefacts with limited information content. Using the current databases, names and proteic domains of many molecular chaperones in different species were analyzed. Although traditional names of HSPs are trivial, it is unrealistic to suggest replacing them, because they are preferred and widely used. Here we suggest that these traditional names be chaperoned, in silico, by a systematic nomenclature. Thus, for example, with the same intent of use of [trioxygen: O3] for ozone, we propose here C7HSP70[Ehsa]ER-P11021 for GRP78 (78 kDa endoplasmic Human molecular chaperone in HSP70 superfamily with P11021 as its accession number in the database of the National Center for Biotechnology Information (NCBI)). The proposed systematic computer-oriented naming and classification method is designed for HSPs and also their partners based on the number of amino acids, domain structure, phylogenetic domain, localization in the cell and accession number as stated in the NCBI. Arabidopsis thaliana was analyzed as a model, because it contains a large number of various HSPs localized in several organelles. Overall, this naming system helps in building, optimizing and managing a novel online database entirely devoted to HSPs. The purported taxonomy, coupled with the newly constructed database, can contribute to studies involving large amounts of stored data on HSPs.     

A 70 kDa microtubule-associated protein in NIL8 cells comigrates with the 70 kDa heat shock protein

When eukaryotic cells are exposed to environmental stress such as elevated temperature, the synthesis of heat shock proteins (HSP) is stimulated. We have raised a monoclonal antibody to a 70 kDa cytoskeleton-associated protein; this antibody also appears to recognize HSPs 68, 70 and 90, as well as an additional 40 kDa non-heat shock protein. We have used this monoclonal antibody to study the localization of the 70 kDa protein in the cytoskeletons of NIL8 hamster fibroblasts. By selective sequential solubilization of the components of NIL8 cells and analysis of the resulting cytoskeletal preparations by Western blot technique and indirect immunofluorescence, we have shown that the 70 kDa protein is associated with microtubules in mitotic and interphase cells and comigrates with HSP70 on 2-dimensional gel electrophoretigrams.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

Preferential translation of heat shock mRNAs in HeLa cells deficient in protein synthesis initiation factors eIF-4E and eIF-4 gamma.

Expression of antisense RNA against eukaryotic translation initiation factor 4E (eIF-4E) in HeLa cells causes a reduction in the levels of both eIF-4E and eIF-4 gamma (p220) and a concomitant decrease in the rates of both cell growth and protein synthesis (De Benedetti, A., Joshi-Barve, S., Rinker-Schaffer, C., and Rhoads, R. E. (1991) Mol. Cell Biol. 11, 5435-5445). The synthesis of most proteins in the antisense RNA-expressing cells (AS cells) is decreased, but certain proteins continue to be synthesized. In the present study, we identified many of these as stress-inducible or heat shock proteins (HSPs). By mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by reactivity with monoclonal antibodies generated against human HSPs, four of these were shown to be HSP 90, HSP 70, HSP 65, and HSP 27. The steady-state levels of HSP 90, 70, and 27 were elevated in relation to total protein in AS cells. Pulse labeling and immunoprecipitation indicated that HSP 90 and HSP 70 were synthesized more rapidly in AS cells than in control cells. The accelerated synthesis of HSPs in the AS cells was not due, however, to increased mRNA levels; the levels of HSP 90 and 70 mRNAs either remained the same or decreased after induction of antisense RNA expression. Actin mRNA, a typical cellular mRNA, was found on high polysomes in control cells but shifted to smaller polysomes in AS cells, as expected from the general decrease in translational initiation caused by eIF-4E and eIF-4 gamma depletion. HSP 90 and 70 mRNAs showed the opposite behavior; they were associated with small polysomes in control cells but shifted to higher polysomes in AS cells. These results demonstrate that HSP mRNAs have little or no requirement in vivo for the cap-recognition machinery and suggest that these mRNAs may utilize an alternative, cap-independent mechanism of translational initiation.     

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.     

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

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

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.     

Antibodies specific for heat shock proteins in human and murine malaria

Heat shock proteins (HSPs) are immunodominant antigens recognized by the host immune system in various infectious diseases. We analyzed HSP-specific antibodies, including immunoglobulin G (IgG), IgM and IgA, in sera from malaria patients in Thailand by using an enzyme-linked immunosorbent assay. All of the antibodies to HSP90 were remarkably increased in the patients compared with those in controls, while only IgM to HSP70 or IgA to HSP65 was significantly elevated. Further experiments showed that anti-HSP IgG was significantly increased in C57BL/6 mice infected with a non-lethal strain of Plasmodium yoelii, with anti-HSP90 IgG being the most elevated. These results suggest that the antigenic potential of HSP90 is higher than those of HSP70 and HSP65 in malaria infection.     

Changes in protein composition ofSaccharomyces brewing strains in response to heat shock and ethanol stress

Summary Heat shock and ethanol stress of brewing yeast strains resulted in the induction of a set of proteins referred to as heat shock proteins (HSPs). At least six strongly induced HSPs were identified in a lager brewing strain and four HSPs in an ale brewing strain. Four of these HSPs with molecular masses of approximately 70, 38, 26 and 23 kDa were also identified in two laboratory strains ofSaccharomyces cerevisiae. The appearance of HSPs correlated with increased survival of strains at elevated temperatures and high concentrations of ethanol. These results suggest that HSPs may play a role in the ethanol and thermotolerance of yeasts. The properties of these proteins and membrane fatty acids in relation to heat and ethanol shock are being investigated.     

Heat shock induced changes in the gene expression of terminally differentiating avian red blood cells

Reticulocytes, purified from the blood of quail and chickens recovering from anaemia, respond to heat shock by the new and (or) enhanced synthesis of heat-shock protein (HSPs) with relative molecular masses of greater than 400,000, 90,000, 70,000, and 26,000 (quail) or 24,000 (chicken) and the depressed synthesis of many proteins normally produced at a control temperature. The synthesis of these HSPs is noncoordinate since the expression of each protein depends upon the particular temperature and duration of the time at that temperature. Separation of proteins from quail reticulocytes into Triton X-100 soluble and insoluble fractions demonstrates that the 70,000 and 26,000 Da HSPs are found in both fractions, whereas the greater than 400,000 and 90,000 Da HSPs are located only in the detergent-soluble fraction. Triton X-100 fractionation also reveals that there are three isoelectric variants of the 70,000 Da HSP and that they are constitutively synthesized and selectively partitioned between cellular compartments. Heat shock induced synthesis of the 90,000, 70,000, and 26,000 Da quail HSPs is prevented by actinomycin D, while enhanced synthesis of the greater than 400,000 Da HSP is unaffected by this inhibitor. These results demonstrate that nucleated, terminally differentiating avian red blood cells are capable of responding to heat stress by rapid changes in their highly restricted "program" of gene expression.     

Heat stress facilitates stretch-induced hypertrophy of cultured muscle cells

Increased mechanical stress induced by stretch is an important growth stimulus in skeletal muscle. Heat shock proteins (HSPs) are an important family of endogenous, protective proteins. HSP90 and HSP70 families show elevated levels under beat stress. Mechanical stress, such as physical exercise, is known to induce not only muscular hypertrophy but also the elevation of HSPs expression in skeletal muscle. The purpose of this study was to determine whether heat stress facilitates the stretch-induced hypertrophy of skeletal muscle cells. Cultured rat myotubes (L6) were plated on collagenized Silastic membranes and incubated at 41 degrees C for 60 and 75 minutes (heat shock). Following the incubation, the cells were subjected two-second stretching and four-second releasing for 4 days at 37 degrees C. Protein concentrations in the homogenates and pellets of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. The protein concentration of cells following mechanical stretching following heat shock was significantly higher than that following either heat shock or mechanical stretching alone. HSP72 in supernatants and HSP90 in pellets increased under heat shock and/or mechanical stretching. HSP90 in supernatants decreased following heat shock and/or mechanical stretching. Changes in HSPs and cellular protein concentrations in stressed cells suggest that the expression of HSPs may be closely related with muscular hypertrophy.     

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)     

Synthesis of heat shock proteins in quail red blood cells following brief, physiologically relevant increases in whole body temperature

Cultured RBCs from quail respond to thermal stress (heat shock) by a rapid and dramatic change in gene expression. This change in gene expression includes the new and/or enhanced non-coordinate synthesis of a small group of heat shock polypeptides (HSPs) having molecular masses of 90,000, 70,000 and 26,000. RBCs obtained from hyperthermic quail exhibit a change in gene expression similar to that observed in RBCs heat-shocked in vitro. Since in vitro studies have linked the synthesis of HSPs in heat-stressed cells with thermotolerance, the similar change in gene expression in RBCs from hyperthermic quail suggests that, here too, this cellular response may be an important homeostatic mechanism by which avian RBCs cope with and/or survive hyperthermic conditions.     

A postgenomic view of the heat shock proteins in kinetoplastids

The kinetoplastids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi are causative agents of a diverse spectrum of human diseases: leishmaniasis, sleeping sickness and Chagas' disease, respectively. These protozoa possess digenetic life cycles that involve development in mammalian and insect hosts. It is generally accepted that temperature is a triggering factor of the developmental programme allowing the adaptation of the parasite to the mammalian conditions. The heat shock response is a general homeostatic mechanism that protects cells from the deleterious effects of environmental stresses, such as heat. This response is universal and includes the synthesis of the heat-shock proteins (HSPs). In this review, we summarize the salient features of the different HSP families and describe their main cellular functions. In parallel, we analyse the composition of these families in kinetoplastids according to literature data and our understanding of genome sequence data. The genome sequences of these parasites have been recently completed. The HSP families described here are: HSP110, HSP104, group I chaperonins, HSP90, HSP70, HSP40 and small HSPs. All these families are widely represented in these parasites. In particular, kinetoplastids possess an unprecedented number of members of the HSP70, HSP60 and HSP40 families, suggesting key roles for these HSPs in their biology.     

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.