The role of oxidative stress in the induction of Drosophila heat-shock proteins

The role of oxidative stress in the induction of heat-shock proteins (HSPs) was studied in Drosophila Kc cells by comparing the effects of two different inducers, temperature stress and reoxygenation following a period of anoxia, on cellular respiration, thiol status, and the accumulation of HSPs. A heat shock from 25 to 37 degrees C caused a 60% increase in the rate of O2 uptake but caused little oxidative stress as indicated by a constant level of reduced glutathione, a slight increase in oxidized glutathione, and no change in protein sulfhydryls. Heat shock resulted in a pronounced accumulation of HSPs which was not inhibited by anoxic conditions. A different HSP inducer, reoxygenation following anoxia, resulted in an overall inhibition of respiration, the appearance of CN -insensitive O2 uptake, a 50% decrease in the level of reduced glutathione and a fourfold increase in the ratio of oxidized to reduced glutathione. Despite these indicators of oxidative stress, HSP synthesis was less pronounced than observed during heat shock and was not affected by antioxidants. Oxidative stress may induce HSP synthesis in some cases but is not responsible for HSP synthesis during a heat shock.     

The effect of heat shock on primary cultures of brain capillary endothelium: inhibition of assembly of zonulae occludentes and the synthesis of heat-shock proteins

Subjecting primary cultures of bovine brain microvessel endothelial cells to thermal stress (heat shock) results in: (1) an inhibition of further tight junction assembly, (2) the disappearance and/or disassembly of tight junctions, (3) a 30-fold increase in the number of plasmic fracture (PF)-face intramembrane particles, and (4) the new and/or enhanced synthesis of at least three heat-shock polypeptides (HSPs) with molecular masses of approximately 100,000, 90,000 and 70,000. Endothelial cells which are heat-shocked and allowed to recover at 37 degrees C exhibit, within the first 2 h, a marked depression in the synthesis of HSPs and the new and/or enhanced synthesis of a 47,000 dalton "recovery" polypeptide. In later periods of recovery (2-4 h), the synthesis of this polypeptide is even more pronounced and is accompanied by the new and/or enhanced synthesis of a polypeptide(s) with a molecular mass of 35 to 37,000. The appearance of these "recovery protein(s)" in the endothelial cells is concomitant with a decrease in the number of PF-face intramembrane particles and the resumption of tight junction assembly. Results of this study suggest that some of the HSPs synthesized by thermally-stressed cultures of brain endothelial cells may activate or be directly involved in a mechanism(s) to ensure survival of these cells by decreasing membrane fluidity and stabilizing the plasma membrane of these cells. Moreover, our results also suggest that the recovery of these cells from the stress of heat shock is accompanied by the synthesis of "recovery" proteins which, in some manner, may be directly involved in, or necessary for, rapidly reversing the membrane-stabilizing effect of heat shock by promoting membrane fluidity and the apparent amplified synthesis and assembly and/or reassembly of tight junctions.     

Detailed characterization of heat shock protein synthesis and induced thermotolerance in seedlings of Sorghum bicolor L.

Tolerance of both protein synthesis and seedling growth to apreviously lethal high temperature can be induced by prior exposureto a sub-lethal temperature during which the synthesis of heatshock proteins (HSPs) occurs. In this study, a thermal gradientbar was used to measure the physiological effects of temperatureon seedlings of sorghum (Sorghum bicolor L.) in conjunctionwith studies of gene expression. The duration of HSP synthesis,both during continued high temperature treatment or on returnto normal temperatures, was found to be very finely modulatedand was dependent on the severity of the initial heat shock.The synthesis of heat shock proteins and the induction of thermotolerancewere rapid, reversible and reinducible phenomena. Maximal thermotolerancewas obtained after treatments that induced the full complementof HSPs. Subsequent treatments that repressed HSP synthesis,also abolished thermotolerance. The presence of HSPs, however,was not sufficient for the tissue to be in a thermotolerantstate and the results suggest that either their de novo synthesis,or some other factor, is required for the induction of thermotolerance.Pre-existing HSPs did not inhibit the synthesis of new HSPs.Although the kinetics of the synthesis of HSPs and the developmentof thermotolerance show a tight correlation, the kinetics ofthe decay of thermotolerance and the degradation of HSPs werenot linked. The functional state or distribution of HSPs maywell change during the recovery process. Key words: Heat shock, thermotolerance, Sorghum bicolor, growth, protein synthesis     

Abnormal induction of heat shock proteins in an Escherichia coli mutant deficient in adenosylmethionine synthetase activity.

Most prototrophic strains of Escherichia coli become restricted for methionine at 44 degrees C. A mutant strain (RG62 metK) in which the level of S-adenosylmethionine synthetase activity is only 10 to 20% of normal shows constitutive expression of one of the heat shock proteins, the lysU gene product, lysyl-tRNA synthetase form II, at 37 degrees C. These findings suggested a possible linkage between methionine metabolism and heat shock. We examined the induction of heat shock polypeptides in strain RG62 (metK) and in its parent, RG (metK+), from which it was derived by spontaneous mutation. Exponential-phase cultures of the two strains were pulse-labeled with [3H]leucine shortly after a shift from 37 to 44 degrees C, and the total cellular polypeptides were examined by two-dimensional electrophoresis. The results confirmed the constitutive production of the lysU gene product previously reported for strain RG62, but also revealed that the induction of 2 of the 17 heat shock polypeptides, C14.7 and G13.5, was markedly depressed. Otherwise the heat shock induction pattern was similar in timing and magnitude in the two strains. Transformation of the mutant strain with a plasmid, pK8, containing the metK coding sequence and promoter region as a 1.8-kilobase insert into pBR322 restored normal induction of C14.7 and G13.5, but did not prevent constitutive expression of the lysU gene product in the medium required for growth of this strain. The three heat shock polypeptides abnormally controlled in strain RG62 are the three polypeptides which are not induced when rapid synthesis of the htpR gene product is induced by isopropyl-beta-D-thiogalactopyranoside at 28 degree C (R. A. VanBogelen, M. A. Acton, and F. C. Neidhardt, Genes Dev. 1:525-531, 1987). We postulate that induction of these three polypeptides involves metabolic signals in addition to the synthesis of the htpR gene product and that strain RG62 (metK) fails to produce the signals involved in induction of C14.7 and G13.5 on a shift-up in temperature and produces the signal related to lysU induction even at 37 degree C.     

Archaebacterial heat-shock proteins

The response to heat shock was examined in seven archaebacterial strains from the genus Halobacterium. Upon heat shock each strain preferentially synthesized a limited number of proteins which fell into three narrow mol. wt. ranges. Further examination of the heat-shock response in H. volcanii revealed that heat-shock protein (hsp) synthesis was greatest at 60°C. Synthesis of hsps at this induction temperature was both rapid and transient. Cells recovered their normal protein synthesis patterns rapidly upon returning to their normal growth temperature following heat shock. H. volcanii cells also responded with a `heat shock-like' response to salt dilution, a natural environmental stress for these organisms. These results indicate that the heat shock or stress response which is charactertistic of eukaryotic and eubacterial cells is also present among members of the archaebacterial genus Halobacterium.     

Limited stress response in Streptococcus pneumoniae.

In Streptococcus pneumoniae, heat shock induces the synthesis of 65-, 73-, and 84-kDa proteins, and ethanol shock induces a 104-kDa protein. In this study, the 65-, 84-, and 104-kDa proteins were identified as members of the GroEL, ClpL and alcohol dehydrogenase families, respectively, and the general properties of the stress response of S. pneumoniae to several other stresses were characterized. However, several stresses which are known to induce stress responses in Escherichia coli and Bacillus subtilis failed to induce any high molecular weight heat-shock proteins (HSPs) such as GroEL and DnaK homologues. A minor temperature shift from 30 to 37 C triggered induction of the homologues of DnaK and GroEL of E. coli. These features may provide a foundation for evaluating the role of heat-shock proteins relative to the physiology and pathogenesis of pneumococcus.     

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.     

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.     

Concurrent collapse of keratin filaments, aggregation of organelles, and inhibition of protein synthesis during the heat shock response in mammary epithelial cells

The sequence of heat shock-induced perturbations in protein synthesis and cytoskeletal organization was investigated in primary cultures of mouse mammary epithelial cells (MMEC). Exposure of the cells to 45 degrees C for 15 min caused a marked inhibition of protein synthesis through 2 h after heart. Resumption of protein synthesis began by 4 h, was complete by 8 h, and was accompanied by induction of four major heat shock proteins (HSPs) of 68, 70, 89, and 110 kD. Fluorescent cytochemistry studies indicated that heat shock elicited a reversible change in the organization of keratin filaments (KFs) and actin filaments but had a negligible effect on microtubules. Changes in the organization of KFs progressed gradually with maximal retraction and collapse into the perinuclear zone occurring at 1-2 h after heat followed by restoration to the fully extended state at 8 h. In contrast, actin filaments disappeared immediately after heat treatment and then rapidly returned within 30-60 min to their original appearance. The translocation of many organelles first into and then away from the juxtanuclear area along with the disruption and reformation of polyribosomes were concurrent with the sequential changes in distribution of KFs. The recovery of the arrangement of KFs coincided with but was independent of the resumption of protein synthesis and induction of HSPs. Thermotolerance could be induced in protein synthesis and KFs, but not in actin filaments, by a conditioning heat treatment. Neither protein synthesis nor induction of HSPs was necessary for the acquisition of thermotolerance in the KFs. The results are compatible with the possibility that protein synthesis may depend on the integrity of the KF network in MMEC. Heat shock thus can efficiently disarrange the KF system in a large population of epithelial cells, thereby facilitating studies on the functions of this cytoskeletal component.     

Heat shock response in photosynthetic organisms: membrane and lipid connections

The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membrane's physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membrane's physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38°C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54°C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36°C. Temperatures below 36°C or above 40°C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34°C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36°C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38°C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.     

Heat shock protects cultured neurons from glutamate toxicity.

Expression of heat shock proteins (HSPs) occurs in brain after ischemia and status epilepticus. We report that induction of the heat shock response in cortical cultures protects neurons from glutamate-induced excitotoxicity. Cultures heated to 42.2 degrees C for 20 min showed an overall decrease in protein synthesis but an increase in the synthesis of approximately 72 and approximately 85 kd proteins and in the levels of HSP70 mRNA. Heat shock inhibited excitotoxicity in cells exposed to glutamate at 3 or 24 hr following heat exposure, but not when the interval between heat and glutamate exposure was shortened to 15 min or lengthened to 48 hr. Protection due to heat shock required new protein synthesis, since it did not occur when protein or RNA synthesis inhibitors were added. By ameliorating excitotoxic processes, HSPs may attenuate brain injury in certain pathologic conditions.     

Heat-shock proteins 70 kDa and 19 kDa are not required for induction of embryogenesis of Brassica napus L. cv. topas microspores

It is currently accepted that 'stress' triggers induction of microspore embryogenesis, and for Brassica napus L. cv. Topas it is heat-shock. It has been postulated that the heat-shock proteins (HSPs) generated during heat stress have a central role in the induction mechanism. To test this hypothesis we developed a microspore induction procedure, using colchicine instead of heat treatment. The level of HSP70 increased significantly during and following the microspore heat treatment while sHSP19 expression was induced at the onset of heat-shock and declined after 8 h. In contrast, induction of embryogenesis with colchicine was not accompanied by elevation of HSP70 nor by induction of sHSP19, indicating that these HSPs are not required for induction of microspore embryogensis in this model system. These data refute the current hypothesis that HSPs have an essential role in triggering microspore embryogenesis.     

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.