title: Resolution, detection, and characterization of redox conformers of human HSF1.
abstract: 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.
authors.affiliation: Graduate Program in Cell and Developmental Biology and Department of Cell Biology and Neuroscience, Rutgers State University, Piscataway, New Jersey 08854-8082, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Manalo
authors.forename: A Y
authors.initials: AY
authors.lastname: Liu
mesh_terms: 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
chemical_list: 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