Resolution, detection, and characterization of redox conformers of human HSF1.
We describe here an experimental protocol for the resolution, detection, and quantitation of the reduced and oxidized conformers of human heat shock factor 1 (hHSF1) and report on the effects in vitro and in vivo of redox-active agents on the redox status, structure, and function of hHSF1. We showed that diamide, a reagent that promotes disulfide bond formation, caused a loss of immunorecognition of the monomeric hHSF1 protein in a standard Western blot detection procedure. Modification of the Western blot procedure to include dithiothreitol in the equilibration and transfer buffers after gel electrophoresis allowed for the detection of a compact, intramolecularly disulfide cross-linked oxidized hHSF1 (ox-hHSF1) in the diamide-treated sample. The effect of diamide was blocked by pretreatment with N-ethylmaleimide and was reversed by dithiothreitol added to the sample prior to gel electrophoresis. Incubation with nitrosoglutathione at 42 degrees C also promoted the conversion of HSF1 to ox-HSF1; at 25 degrees C, however, nitrosoglutathione was by itself without effect but blocked the formation of ox-hHSF1 in the presence of diamide. The disulfide cross-linked ox-hHSF1 was monomeric and resistant to the in vitro heat-induced trimerization and activation. The possibility that ox-HSF1 may occur in oxidatively stressed cells was evaluated. Treatment of HeLa cells with 2 mm l-buthionine sulfoximine promoted the formation of ox-HSF1 and blocked the heat-induced activation of HSF DNA binding activity. Our result suggests that hHSF1 may have integrated redox chemistry of cysteine sulfhydryl into its functional responses.
Manalo
D J
DJ
Graduate Program in Cell and Developmental Biology and Department of Cell Biology and Neuroscience, Rutgers State University, Piscataway, New Jersey 08854-8082, USA.
Liu
A Y
AY
D015153:Blotting, Western / Q000379:methods; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006367:HeLa Cells; D000076249:Heat Shock Transcription Factors; D006358:Hot Temperature; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D026422:S-Nitrosoglutathione; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D004220:Disulfides; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; D009603:Nitroso Compounds; D013439:Sulfhydryl Reagents; D014157:Transcription Factors; D003958:Diamide; D009569:Nitric Oxide; D019328:Buthionine Sulfoximine; D026422:S-Nitrosoglutathione; D005978:Glutathione; D005033:Ethylmaleimide; D004229:Dithiothreitol