title: Thioltransferase (glutaredoxin) reactivates the DNA-binding activity of oxidation-inactivated nuclear factor I.
abstract: The reversible oxidative inactivation of transcription factors has been proposed to be important in cellular responses to oxidant stress and in several signal transduction pathways. The nuclear factor I (NFI) family of transcription factors is sensitive to oxidative inactivation due to the presence of a conserved, oxidation-sensitive cysteine residue within the NFI DNA-binding domain. Here we show that restoration of the DNA-binding activity of oxidized NFI-C can be catalyzed in vitro by the cellular enzyme thioltransferase (glutaredoxin) coupled to GSH and GSSG reductase. To test whether GSH-dependent pathways play a role in the maintenance of NFI activity in vivo, we used buthionine sulfoximine, an agent that inhibits GSH synthesis, and N-acetylcysteine, an agent that can replenish intracellular GSH. Pretreatment of HeLa cells with buthionine sulfoximine greatly potentiated the inactivation of NFI by the oxidizing agent diamide. Inclusion of N-acetylcysteine in the culture medium during the recovery period following diamide treatment increased the extent of restoration of NFI activity. These results suggest that maintenance of the DNA-binding activity of NFI proteins during oxidant stress in vivo requires a GSH-dependent pathway, likely involving thioltransferase-catalyzed reduction of the oxidation-sensitive cysteine residue on NFI.
authors.affiliation: Lerner Research Institute, Department of Cancer Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: S
authors.initials: S
authors.lastname: Bandyopadhyay
authors.forename: D W
authors.initials: DW
authors.lastname: Starke
authors.forename: J J
authors.initials: JJ
authors.lastname: Mieyal
authors.forename: R M
authors.initials: RM
authors.lastname: Gronostajski
mesh_terms: D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D011506:Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051840:Y-Box-Binding Protein 1
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D054477:Glutaredoxins; D051839:NFI Transcription Factors; C497647:NFIC protein, human; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D004247:DNA; D010088:Oxidoreductases; D005978:Glutathione