Thioltransferase (glutaredoxin) reactivates the DNA-binding activity of oxidation-inactivated nuclear factor I.
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.
Bandyopadhyay
S
S
Lerner Research Institute, Department of Cancer Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Starke
D W
DW
Mieyal
J J
JJ
Gronostajski
R M
RM
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
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