Hyperthermia protects mice against the lethal effects of endotoxin.

Recently, investigators have reported that heat shock proteins (HSPs) can protect isolated cells from cytotoxicity induced by two important mediators of sepsis: interleukin-1 and tumor necrosis factor. The present study was undertaken to examine the hypothesis that transient whole body hyperthermia could decrease mortality from subsequent challenge with gram-negative endotoxin. We demonstrate that heat pretreatments improved long-term survival fivefold in a mouse endotoxin model and this was correlated with the production of HSPs. There was a marked difference in individual organ expression of the inducible 72-kDa heat shock protein (HSP72). Heat treatments caused significant HSP72 formation in lung, liver, kidney, and small intestine, but much lesser formation in heart, brain, and abdominal wall muscle. Additional experiments demonstrated that the protective effect of hyperthermic treatments against an endotoxin challenge occurred early, i.e., 1 and 2 h after heating, was maximal at 12 h, and had significantly diminished by 48 h. The formation and decay of HSP72 demonstrated a time course that paralleled the survival curve from endotoxin challenge, thus suggesting a possible role for HSP72 in the protective effect. Surprisingly, and in contrast to studies reported in incubated cells, endotoxin alone did not cause significant formation of HSP72 in vivo.

Genetic evidence for a functional relationship between Hsp104 and Hsp70.

The phenotypes of single Hsp104 and Hsp70 mutants of the budding yeast Saccharomyces cerevisiae provide no clue that these proteins are functionally related. Mutation of the HSP104 gene severely reduces the ability of cells to survive short exposures to extreme temperatures (thermotolerance) but has no effect on growth rates. On the other hand, mutations in the genes that encode Hsp70 proteins have significant effects on growth rates but do not reduce thermotolerance. The absence of a thermotolerance defect in S. cerevisiae Hsp70 mutants is puzzling, since the protein clearly plays an important role in thermotolerance in a variety of other organisms. In this report, examination of the phenotypes of combined Hsp104 and Hsp70 mutants uncovers similarities in the functions of Hsp104 and Hsp70 not previously apparent. In the absence of the Hsp104 protein, Hsp70 is very important for thermotolerance in S. cerevisiae, particularly at very early times after a temperature upshift. Similarly, Hsp104 plays a substantial role in vegetative growth under conditions of decreased Hsp70 protein levels. These results suggest a close functional relationship between Hsp104 and Hsp70.

The role of heat-shock proteins in thermotolerance.

The role of heat-shock proteins (hsps) in thermotolerance was examined in the budding yeast Saccharomyces cerevisiae and in the fruit fly Drosophila melanogaster. In yeast cells, the major protein responsible for thermotolerance is hsp 100. In cells carrying mutations in the hsp 100 gene, HSP 104, growth is normal at both high and low temperatures, but the ability of cells to survive extreme temperatures is severely impaired. The loss of thermotolerance is apparently due to the absence of the hsp 104 protein itself because, with the exception of the hsp 104 protein, no differences in protein profiles were observed between mutant and wild-type cells. Aggregates found in mutant cells at high temperatures suggest that the cause of death may be the accumulation of denatured proteins. No differences in the rates of protein degradation were observed between mutant and wild-type cells. This, and genetic analysis of cells carrying multiple hsp 70 and hsp 104 mutations, suggests that the primary function of hsp 104 is to rescue proteins from denaturation rather than to degrade them once they have been denatured. Drosophila cells do not produce a protein in the hsp 100 class in response to high temperatures. In this organism, hsp 70 appears to be the primary protein involved in thermotolerance. Thus, the relative importance of different hsps in thermotolerance changes from organism to organism.

Hsp104 is required for tolerance to many forms of stress.

Heat-shock proteins (hsps) are induced by many types of stress. In Saccharomyces cerevisiae, a mutation in the HSP104 gene, a member of the highly conserved hsp100 gene family, reduces the ability of log-phase fermenting cells to withstand high temperatures after mild, conditioning pretreatments. Here, we examine the expression of hsp104 and its importance for survival under many different conditions. Hsp104 is expressed at a higher level in respiring cells than in fermenting cells and is required for the unusually high basal thermotolerance of respiring cells. Its expression in stationary phase cells and spores is crucial for the naturally high thermotolerance of these cell types and for their long-term viability at low temperatures. The protein is of critical importance in tolerance to ethanol and of moderate importance in tolerance to sodium arsenite. Thus, the hsp104 mutation establishes the validity of a long-standing hypothesis in the heat-shock field, namely, that hsps have broadly protective functions. Further, that a single protein is responsible for tolerance to heat, ethanol, arsenite and long-term storage in the cold indicates that the underlying causes of lethality are similar in an extraordinary variety of circumstances. Finally, the protein is of little or no importance in tolerance to copper and cadmium, suggesting that the lethal lesions produced by these agents are fundamentally different from those produced by heat.

hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.

hsp82 is one of the most highly conserved and abundantly synthesized heat shock proteins of eucaryotic cells. The yeast Saccharomyces cerevisiae contains two closely related genes in the HSP82 gene family. HSC82 was expressed constitutively at a very high level and was moderately induced by high temperatures. HSP82 was expressed constitutively at a much lower level and was more strongly induced by heat. Site-directed disruption mutations were produced in both genes. Cells homozygous for both mutations did not grow at any temperature. Cells carrying other combinations of the HSP82 and HSC82 mutations grew well at 25 degrees C, but their ability to grow at higher temperatures varied with gene copy number. Thus, HSP82 and HSC82 constitute an essential gene family in yeast cells. Although the two proteins had different patterns of expression, they appeared to have equivalent functions; growth at higher temperatures required higher concentrations of either protein. Biochemical analysis of hsp82 from vertebrate cells suggests that the protein binds to a variety of other cellular proteins, keeping them inactive until they have reached their proper intracellular location or have received the proper activation signal. We speculate that the reason cells require higher concentrations of hsp82 or hsc82 for growth at higher temperatures is to maintain proper levels of complex formation with these other proteins.