Stress-induced activation of the heat-shock response: cell and molecular biology of heat-shock factors.

Exposure of cells to environmental and physiological stress leads to an imbalance in protein metabolism, which challenges the cell to respond rapidly and precisely to the deleterious effects of stress on protein homoeostasis. The heat-shock response, through activation of heat-shock transcription factors (HSFs) and the elevated expression of heat-shock proteins and molecular chaperones, protects the cell against the accumulation of non-native proteins. Activation of HSF1 involves a complex multi-step pathway in which the inert monomer oligomerizes to a DNA-binding, transcriptionally active, trimer which relocalizes within the the nucleus to form stress-induced HSF1 granules. Attenuation of the heat-shock response involves molecular chaperones which repress the HSF1 transactivation domain and HSF-binding protein 1 (HSBP1), which interacts with the HSF1 oligomerization domain of HSF1 to negatively regulate its activity, thus insuring that the expression of chaperones is precisely determined.

In vivo and in vitro function of the Escherichia coli periplasmic cysteine oxidoreductase DsbG.

We have characterized in vivo and in vitro the recently identified DsbG from Escherichia coli. In addition to sharing sequence homology with the thiol disulfide exchange protein DsbC, DsbG likewise was shown to form a stable periplasmic dimer, and it displays an equilibrium constant with glutathione comparable with DsbA and DsbC. DsbG was found to be expressed at approximately 25% the level of DsbC. In contrast to earlier results (Andersen, C. L., Matthey-Dupraz, A., Missiakas, D., and Raina, S. (1997) Mol. Microbiol. 26, 121-132), we showed that dsbG is not essential for growth and that dsbG null mutants display no defect in folding of multiple disulfide-containing heterologous proteins. Overexpression of DsbG, however, was able to restore the ability of dsbC mutants to express heterologous multidisulfide proteins, namely bovine pancreatic trypsin inhibitor, a protein with three disulfides, and to a lesser extent, mouse urokinase (12 disulfides). As in DsbC, the putative active site thiols in DsbG are completely reduced in vivo in a dsbD-dependent fashion, as would be expected if DsbG is acting as a disulfide isomerase or reductase. However, the latter is not likely because DsbG could not catalyze insulin reduction in vitro. Overall, our results indicate that DsbG functions primarily as a periplasmic disulfide isomerase with a narrower substrate specificity than DsbC.

The heat-shock response: regulation and function of heat-shock proteins and molecular chaperones.

Exposure of cells to stresses such as heat shock, oxidant injury and heavy metals causes an imbalance in protein metabolism which challenges the cell to respond rapidly, yet precisely, to minimize the deleterious effects of environmental and physiological stress. The heat-shock response, through the activation of HSFs, results in the elevated expression of heat-shock genes and the concomitant synthesis of HSPs and molecular chaperones. Molecular chaperones function in a variety of protein biosynthetic events and protect proteins from the deleterious effects of acute or chronic stress by stabilizing and refolding protein-folding intermediates or facilitating protein degradation. The accumulation of misfolded proteins has also become a central issue to diseases of protein folding, including sickle cell haemoglobin, cystic fibrosis and prion diseases, in addition to complex multifactorial diseases such as bacterial and viral infections, myocardial ischaemia, neurodegenerative diseases and cancer.

Activation of heat shock factor 1 DNA binding precedes stress-induced serine phosphorylation. Evidence for a multistep pathway of regulation.

Exposure of mammalian cells in culture to the anti-inflammatory drugs sodium salicylate or indomethacin results in activation of heat shock factor 1 (HSF1) DNA binding activity. We have previously shown that the drug-induced HSF1 becomes associated with the heat shock elements of the hsp70 promoter, yet transcription of the hsp70 gene is not induced (Jurivich, D. A., Sistonen, L., Kroes, R. A., and Morimoto, R. I. (1992) Science 255, 1243-1245). In this study, we have examined the basis for uncoupling the heat shock transcriptional response. Comparison of heat shock and drug-induced forms of HSF1 has revealed that the transcriptionally inert drug-induced HSF1 is constitutively but not inducibly serine-phosphorylated, whereas heat shock-induced HSF1 is both constitutively and inducibly serine-phosphorylated. The transcriptionally inert intermediate represented by drug-induced HSF1 can be converted to the transcriptionally active state by a subsequent exposure to heat shock. The only detectable change in HSF1 is the acquisition of inducible serine phosphorylation. These data reveal that acquisition of the trimeric DNA binding state of HSF1 is independent of and precedes inducible phosphorylation and furthermore that inducible phosphorylation correlates with transcriptional activation.

The transcriptional regulation of heat shock genes: a plethora of heat shock factors and regulatory conditions.

The inducible regulation of heat shock gene transcription is mediated by a family of heat shock factors (HSF) that respond to diverse forms of physiological and environmental stress including elevated temperature, amino acid analogs, heavy metals, oxidative stress, anti-inflammatory drugs, arachidonic acid, and a number of pathophysiological disease states. The vertebrate genome encodes a family of HSFs which are expressed ubiquitously, yet the DNA binding properties of each factor are negatively regulated and activated in response to specific conditions. This chapter will discuss the regulation of the HSF multi-gene family and the role of these transcriptional activators in the inducible expression of genes encoding heat shock proteins and molecular chaperones.