Heat shock inhibits and activates different protein degradation pathways and proteinase activities in Neurospora crassa

Abstract In Neurospora crassa , heat shock treatment inhibits proteolytic activity. ATP-independent proteinases were analysed after polyacrylamide gel electrophoresis using renaturing gelatine gels. Proteinases of 24, 29, and 130 kDa were shown to be inhibited by heat shock and were further characterized as to their properties. A major part of the heat shock-induced inhibition is probably due to suppression of de novo synthesis of proteinases as deduced from experiments with cycloheximide. During several hours of recovery from heat shock, the inhibition of overall protein degradation and ATP-independent proteinases is reversed. Azocasein assays as well as pulse-chase experiments further showed that ATP-dependent protein degradation is only slightly affected by heat shock. Two ATP-binding proteinases of about 60 and 160 kDa even show an increased activity after heat shock. The degradation rate of heat shock proteins is inhibited by heat shock treatment, indicating that they are degraded by ATP-independent proteinases. Western blot analysis of a ∼40-kDa degradation product of HSP70 containing its amino terminal portion revealed a reduction in the amount of this peptide after heat shock.     

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.     

Analysis of the expression of the two major proteins of the 70 kilodalton mammalian heat shock family

This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.     

Changes in Protein Composition and the H+-ATPase Activity of the Plasma Membrane during Induction of Callus from Tuber Tissues of Jerusalem Artichoke

Changes in composition and synthesis of the proteins in plasmamembranes during early periods of induction of callus from tubertissues of Jerusalem artichoke were examined in relation toanalogous changes in H⁺-ATPase activity. By the 12th h of culture,vanadate-sensitive ATPase activity had increased more than 3.5-fold.The level of a polypeptide with a molecular mass of 97 kDa,which putatively corresponded to a subunit of the plasma membraneH⁺-ATPase, also showed a similar increase. Increases in ATPaseactivity and in the level of the 97-kDa polypeptide occurredindependently of the presence of 2,4-D in the culture mediumbut the rate of increase in both cases was slightly higher fortissue disks cultured with 2,4-D than for the control disksin medium without 2,4-D for the first 12 h of culture. The increasein the level of the 97-kDa polypeptide may be ascribed predominantlyto synthesis de novo during the early period of culture. Enhancedsynthesis of the 97-kDa polypeptide in the cultured tissuesmay have resulted in the increases in ATPase activity. Sinceauxin itself may stimulate H⁺-ATPase activity, the activatedH⁺-ATPase may be further stimulated in tissue disks culturedwith 2,4-D. The H⁺-ATPase activated in this way may produceconditions that facilitate the induction of callus from tubertissues of Jerusalem artichoke during the early period of culture. (Received July 13, 1992; Accepted October 19, 1992)     

Effect of heat shock on the intracellular distribution of proteins

Exposure of cells to heat induces thermotolerance, a transient resistance to subsequent heat challenges. It has been shown that thermotolerance is correlated in time with the enhanced synthesis of heat shock proteins. In this study, the association of induced heat shock proteins with various cellular fractions was investigated and the heat-induced changes in skeletal protein composition in thermotolerant and control cells was compared. All three major heat shock proteins induced in Chinese hamster fibroblasts after a 46 degrees C, 4-min heat treatment (70, 87, and 110 kDa) were purified with the cytoplasmic fraction, whereas only the 70-kDa protein was also found in other cell fractions, including that containing the cellular skeleton. Immediately after a second heat treatment at 45 degrees C for 45 min, the 110-kDa protein from thermotolerant cells also purified extensively with the cellular skeletal fraction. In this regard, the 110-kDa protein behaved similarly to many other cellular proteins, since we observed an overall temperature-dependent increase in the total labeled protein content of the high-salt-resistant cellular skeletal fraction after heat shock. Pulse-chase studies demonstrated that this increased protein content gradually returned to normal levels after a 3-hr incubation at 37 degrees C. The alteration or recovery kinetics of the total labeled protein content of the cellular skeletal fraction after heat shock did not correlate with the dramatic increase in survival observed in thermotolerant cells. The relationship between heat shock proteins and thermotolerance, therefore, does not correlate directly with changes in the heat-induced cellular alterations leading to differences in protein fractionation.     

Examination of cadmium-induced expression of the small heat shock protein gene, hsp30, in Xenopus laevis A6 kidney epithelial cells

Cadmium is a highly toxic environmental pollutant that has been classified as a human carcinogen. Toxicological responses to cadmium exposure include respiratory diseases, neurological disorders and kidney damage. In the present study, we have characterized the effect of cadmium on the accumulation of the small heat shock protein (HSP), HSP30, in Xenopus laevis A6 kidney epithelial cells. Incubation of A6 cells with cadmium chloride induced the accumulation of HSP30 protein and hsp30 mRNA. While HSP70 protein and hsp70 mRNA accumulation were also induced, the relative levels of actin remained relatively unaffected. Elevated levels of HSP30 were detected in cells undergoing prolonged exposure of cells to cadmium chloride or in cells recovering from cadmium chloride treatment. Immunocytochemical analysis of cadmium chloride-treated A6 cells revealed HSP30 accumulation primarily in the cytoplasm in a punctate pattern supplemented with larger HSP30 staining structures. Also, HSP30 co-localized with the F-actin cytoskeleton at higher cadmium chloride concentrations. The combination of mild heat shock temperatures plus cadmium chloride concentrations employed in this study resulted in a synergistic accumulation of HSP30 protein and hsp30 mRNA. Finally, in contrast to heat shock, prior exposure of Xenopus A6 cells to cadmium chloride treatment, sufficient to induce the accumulation of HSPs, did not protect the cells against a subsequent thermal challenge.     

Heat shock proteins and chilling sensitivity of mung bean hypocotyls

Excised mung bean (Vigna radiata L.) hypocotyl sections wereexposed to 40 C for up to 4 h in the presence or absence of50 µM cycloheximide (CHX) before being held at a non-chilling(20 C) or chilling (2.5 C) temperature. Mung bean hypocotyltissue is chilling sensitive, and the rate of solute leakageis highly correlated with the extent of chilling injury. A 3h heat shock at 40 C reduced chilling-induced solute leakageby up to 40%, but leakage was similar to non-heat-shocked hypocotylswhen CHX was present. Specific proteins were labelled when hypocotylswere exposed to [³⁵S] methionine during the last hour of heatshock. The nine most intense bands on the autoradiographs ofSDS-PAGE gels of extracted protein corresponded to molecularweights of 114, 79, 73, 70, 60, 56, 51, 46, and 18 kDa. The18 kDa band reached a maximum after 1 h at 40 C and then rapidlydecreased in intensity as the heat shock continued, becomingundetectable at 4 h. The four most intense bands after 3 h at40 C corresponded to molecular weights of 79, 70, 51, and 46kDa. The synthesis of these four hsps was markedly reduced whenthe hypocotyl sections were exposed to CHX during heat shock.During chilling for 6 d, the levels of hsps 79 and 70 remainedsignificantly higher in tissue that was heat shocked prior tochilling than in tissue that was not heat shocked. In contrast,the levels of hsps 51 and 46 were similar in bothheat-shockedand control tissues. Heat-shock-induced chilling tolerance waslost between 6 and 9 d ofstorage at 2.5 C; this loss coincidedwith the decay of hsps 79 and 70 to control levels. These resultssuggest that heat shock induces an increase in both chillingtolerance and the de novo synthesis of specific heat shock proteins;namely hsps 79 and 70. This is the first report showing a relationshipbetween heat-shock-induced chilling tolerance and specific heat-shock-inducedproteins. Key words: Ion leakage, protein synthesis, Vigna radiata     

Expression and regulation of casein kinase 2 during heat shock in Verticillium dahliae.

A homogeneous preparation of casein kinase 2 has been isolated from the phytopathogenic fungus Verticillium dahliae (the parasite of cotton). The enzyme consists of three subunits with molecular masses of 53, 41, and 38 kDa. Highly specific immune serum against casein kinase 2 has been obtained. By means of immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunochemical isolation on protein A-Sepharose, it is shown that the amount of casein kinase 2 increases under heat shock conditions (at least in part due to the synthesis de novo), while the synthesis of the majority of other proteins falls. The activity of casein kinase 2 is supressed during heat shock and so does not correlate with its content. The results give an evidence for the two-step model of casein kinase 2 regulation during heat shock.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Heat and sodium arsenite act synergistically on the induction of heat shock gene expression in Xenopus laevis A6 cells

Heat shock protein (HSP) synthesis was studied in the Xenopus epithelial cell line A6 in response to heat and sodium arsenite, either singly or together. Temperatures of 33-35 degrees C consistently brought about the synthesis of HSPs at 87, 73, 70, 54, 31, and 30 kilodaltons (kDa), whereas sodium arsenite at 25-100 microM induced the synthesis of HSPs at 73 and 70 kDa. In cultures exposed to 10 microM sodium arsenite at 30 degrees C, HSP synthesis in the 68- to 73-kDa and 29- to 31-kDa regions was much greater than the HSP synthesis in response to each treatment individually. RNA dot blot analysis using homologous genomic subclones revealed that heat shock induced the accumulation of HSP 70 and 30 mRNAs. The sizes of the HSP 70 and 30 mRNAs determined by Northern hybridization were 2.7 and 1.5 kilobases, respectively. Sodium arsenite (10-100 microM) also induced the accumulation of both HSP 70 and 30 mRNAs. Finally, a mild heat shock (30 degrees C) plus a low concentration of sodium arsenite (10 microM) acted synergistically on HSP 70 and 30 mRNA accumulation in A6 cells. Thus sodium arsenite and heat act synergistically at the level of both HSP synthesis and HSP mRNA accumulation.     

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.     

Heat shock response in mycoplasmas, genome-limited organisms.

We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JA1 and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 heat shock proteins in A. laidlawii JA1, and 7 heat shock proteins in M. capricolum was observed by electrophoretic analysis of proteins from heat-shocked cells in sodium dodecyl sulfate-polyacrylamide gels. In all three strains, major heat shock proteins (66 to 68 and 26 to 29 kilodaltons [kDa]) were found. The 66- to 68-kDa protein cross-reacted with antibody to Escherichia coli DnaK protein, suggesting that this heat shock protein has been conserved in spite of major reductions in genetic complexity during mycoplasma evolution. A. laidlawii also contained a 60-kDa protein that cross-reacted with eubacterial GroEL protein and a 40-kDa protein that cross-reacted with E. coli RecA protein. Unlike with coliphages, the mycoplasma virus L2 progeny yield was not increased when virus was plated on heat-shocked A. laidlawii host cells. However, UV-irradiated L2 virus could be host cell reactivated by both A. laidlawii SOS repair and heat shock systems.     

Calcium is involved in regulation of the synthesis of HSPs in suspension-cultured sugar beet cells under hyperthermia

Exposure of plants to elevated temperatures induces a complex set of changes that enable plants to adapt following heat stress. In order to test the effect of Ca²⁺ on heat shock-induced changes in cell protein synthesis the incorporation of [ 35 S]methionine into protein was studied in cultured sugar beet ( Beta vulgaris L.) cells incubated in media containing different calcium concentrations. Heat shock inhibited the synthesis of non-heat shock proteins (non-HSPs) and promoted the synthesis of a set of HSPs, typical of plants. The synthesis of non-HSPs was greatly inhibited by external Ca²⁺ removal by treatment of the cells with ethylene glycol-bis( β -aminoethylether)- N,N,N',N'- tetraacetic acid. In contrast, extracellular Ca²⁺ appeared not to be strictly required for the de novo production of HSPs, but this cation exerted different effects on the synthesis of individual HSPs. Cell injury increased if the cells were exposed simultaneously to high temperature and Ca²⁺-deficient medium. Recovery of HSP synthesis and reduced cell injury were observed after addition of exogenous calcium to Ca²⁺-depleted cells. These findings are consistent with a Ca²⁺ requirement for the survival of the cells under heat shock, and likely for the development of cell thermotolerance.     

Secreted heat shock proteins in sunflower suspension cell cultures

Sunflower suspension cell cultures were subjected to different heat treatments and the electrophoretic patterns of heat-induced endocellular and secreted proteins were analyzed. In response to heat shock (3 h at 40°C), sunflower cells synthesized new polypeptides and secreted them into the medium, while the synthesis of other polypeptides was suppressed. Two major polypeptides of about 50 and 32 kDa were strongly induced. The two-dimensional electrophoretic analysis showed that the 32-kDa band is composed of at least four different polypeptides. Western blotting hybridizations of secreted proteins with various lectins were performed. The 32-kDa band gave a positive signal with concanavalin A. Received: 8 March 1996 / Revision received: 30 September 1996 / Accepted: 15 October 1996