Molecular chaperones, including the heat-shock proteins (Hsps), are a ubiquitous feature of cells in which these proteins cope with stress-induced denaturation of other proteins. Hsps have received the most attention in model organisms undergoing experimental stress in the laboratory, and the function of Hsps at the molecular and cellular level is becoming well understood in this context. A complementary focus is now emerging on the Hsps of both model and nonmodel organisms undergoing stress in nature, on the roles of Hsps in the stress physiology of whole multicellular eukaryotes and the tissues and organs they comprise, and on the ecological and evolutionary correlates of variation in Hsps and the genes that encode them. This focus discloses that (a) expression of Hsps can occur in nature, (b) all species have hsp genes but they vary in the patterns of their expression, (c) Hsp expression can be correlated with resistance to stress, and (d) species' thresholds for Hsp expression are correlated with levels of stress that they naturally undergo. These conclusions are now well established and may require little additional confirmation; many significant questions remain unanswered concerning both the mechanisms of Hsp-mediated stress tolerance at the organismal level and the evolutionary mechanisms that have diversified the hsp genes.

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Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology

The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.

Molecular chaperones in cellular protein folding.

T URNOVER OF AB ERR ANT PROT EINS IN E. COLI . . . . . . . . . . . . . . . . . 445 The Lon Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 DnaK. DnaJ. GrpE. GroEL and GroES . . . . . . . . . . . . . . . . . . . . . . . . . . 447 The Clp Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 The DegP Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452

The Function of Heat-Shock Proteins in Stress Tolerance: Degradation and Reactivation of Damaged Proteins

The Heat Shock Response: Life on the Verge of Death

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.

The heat-inducible members of the Hsp100 (or Clp) family of proteins share a common function in helping organisms to survive extreme stress, but the basic mechanism through which these proteins function is not understood. Hsp104 protects cells against a variety of stresses, under many physiological conditions, and its function has been evolutionarily conserved, at least from Saccharomyces cerevisiae to Arabidopsis thaliana. Homology with the Escherichia coli ClpA protein suggests that Hsp104 may provide stress tolerance by helping to rid the cell of heat-denatured proteins through proteolysis. But genetic analysis indicates that Hsp104 may function like Hsp70 as a molecular chaperone. Here we investigate the role of Hsp104 in vivo using a temperature-sensitive Vibrio harveyi luciferase-fusion protein as a test substrate. We find that Hsp104 does not protect luciferase from thermal denaturation, nor does it promote proteolysis of luciferase. Rather, Hsp104 functions in a manner not previously described for other heat-shock proteins: it mediates the resolubilization of heat-inactivated luciferase from insoluble aggregates.

Protein disaggregation mediated by heat-shock protein Hsp104.

I. INTRODUCTION The capacity of different individuals of the same species to survive short exposures to extreme temperatures (thermotolerance) varies over a remarkable range, commonly over several orders of magnitude. Both differences in growth conditions and differences in genetic background contribute. Although the contributions of genetic background are just beginning to be deciphered, the effects of growth conditions have been the subject of detailed and intense scrutiny. Nutrient availability, oxygen tension, diurnal rhythms, and a host of other variables exert highly reproducible effects on thermotolerance. By far the most closely studied phenomenon, however, is the tolerance afforded by short pretreatments at moderately elevated temperature. When whole organisms or cultured cells are given such pretreatments, their resistance to killing by extreme heat increases dramatically. This increased resistance, known as induced thermotolerance (Fig. 1), is observed in virtually every organism studied. Remarkably, mild heat pretreatments elicit resistance not just to high temperatures, but to an extraordinary variety of other stresses. In addition, exposure to other mild stresses elicits protection not just against higher doses of those particular stresses, but against high temperatures as well. Such tolerance-inducing treatments generally also induce the synthesis of a small number of proteins known as the heat shock proteins (hsps; Fig. 2) (Li and Laszlo 1985; Lindquist 1986; Nagao et al. 1986; Subjeck and Shyy 1986; Sanchez and Lindquist 1990; Nover 1991; Sanchez et al. 1992). Historically, many observations have suggested that hsps play a vital part in induced tolerance. For example, it is striking that hsps...

18 Heat Shock Proteins and Stress Tolerance

MOST eukaryotic cells produce proteins with relative molecular masses in the range of 100,000 to 110,000 after exposure to high temperatures1. These proteins have been studied only in yeast and mammalian cells. In Saccharomyces cerevisiae, heat-shock protein hsp104 is vital for tolerance to heat, ethanol and other stresses (ref. 2, and Y.S. et al., manuscript submitted). The mammalian hsp110 protein is nucleolar and redistributes with growth state, nutritional conditions and heat shock3,5. The relationships between hsp110, hsp104 and the high molecular mass heat-shock proteins of other organisms were unknown. We report here that hsp104 is a member of the highly conserved ClpA/ClpB protein family first identified in Escherlchla coli6 and that additional heat-inducible members of this family are present in Schizosaccharomyces pombe and in mammals. Mutagenesis of two putative nucleotide-binding sites in hsp104 indicates that both are essential for function in thermotolerance.

Hsp104 is a highly conserved protein with two essential nucleotide-binding sites.

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.

The role of heat-shock proteins in thermotolerance

http://lindquistlab.wi.mit.edu/wp-content/uploads/2013/06/Schirmer1998JBiolChem.pdf

Dawn A. Parsell

Papers

Protein disaggregation mediated by heat-shock protein Hsp104.

18 Heat Shock Proteins and Stress Tolerance

Hsp104 is a highly conserved protein with two essential nucleotide-binding sites.

The role of heat-shock proteins in thermotolerance

The ATPase Activity of Hsp104, Effects of Environmental Conditions and Mutations