Redox-dependent regulation of the conformation and function of human heat shock factor 1.
We present here evidence that redox-dependent thiol-disulfide exchange can provide a mechanism regulating the conformation and activity of the human heat shock transcription factor 1 (HSF1). Diamide and dithiothreitol were reagents used respectively to promote and reverse disulfide cross-link, and the resolution and detection of redox conformers of HSF1 were done according to recently published methods [Manalo, D. J., and Liu, A. Y.-C. (2001) J. Biol. Chem. 276, 23554-23561]. We showed that preincubation of the latent HSF1 monomer with diamide inhibited the in vitro heat-induced activation and trimerization of HSF1 and caused the formation of ox-hHSF1, a compact, disulfide cross-linked HSF1 conformer detectable in immuno-Western blot assay. These effects of diamide were dose-dependent and were rapidly and quantitatively reversed by dithiothreitol. Cysteine site-specific mutants of HSF1 were constructed and used to determine which of the five cysteine residues may be engaged in disulfide cross-link. Analysis of the in vitro transcribed and translated HSF1 proteins showed that while mutation of C1 (amino acid 36) and C2 (amino acid 103) had no effect on the redox sensitivity of HSF1, the mutation of C3 (amino acid 153) or double mutation of C4 and C5 (amino acids 373 and 378, respectively) to serine rendered HSF1 insensitive to diamide and prevented its conversion to ox-HSF1. HSF1 with a single cysteine to serine mutation at either the C4 or C5 position gave different ox-HSF1 conformers in the presence of diamide, suggesting that C3 could be disulfide cross-linked to either C4 or C5. The possibility that HSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional response in higher mammalian cells is discussed.
Manalo
Dominador J
DJ
Graduate Program in Cell and Developmental Biology, Rutgers State University of New Jersey, Piscataway, New Jersey 08854-8082, USA.
Lin
Zheng
Z
Liu
Alice Y-C
AY
D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D000076249:Heat Shock Transcription Factors; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D014157:Transcription Factors
D017931:DNA Primers; D004268:DNA-Binding Proteins; D004220:Disulfides; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; D014157:Transcription Factors; D003545:Cysteine