RESUMO
Redox control of the transcription factor c-Jun maps to a single cysteine in its DNA binding domain. However, the nature of the oxidized state of this cysteine and, thus, the potential molecular mechanisms accounting for the redox regulation of c-Jun DNA binding remain unclear. To address this issue, we have analyzed the purified recombinant c-Jun DNA binding domain for redox-dependent thiol modifications and concomitant changes in DNA binding activity. We show that changes in the ratio of reduced to oxidized glutathione provide the potential to oxidize c-Jun sulfhydryls by mechanisms that include both protein disulfide formation and S-glutathiolation. We provide evidence that S-glutathiolation, which is specifically targeted to the cysteine residue located in the DNA binding site of the protein, may account for the reversible redox regulation of c-Jun DNA binding. Furthermore, based on a molecular model of the S-glutathiolated protein, we discuss the structural elements facilitating S-glutathiolation and how this modification interferes with DNA binding. Given the structural similarities between the positively charged cysteine-containing DNA binding motif of c-Jun and the DNA binding site of related oxidant-sensitive transcriptional activators, the unprecedented phenomenon of redox-triggered S-thiolation of a transcription factor described in this report suggests a novel role for protein thiolation in the redox control of transcription.
Assuntos
DNA/química , DNA/metabolismo , Glutationa/metabolismo , Proteínas Proto-Oncogênicas c-jun/química , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação , Gráficos por Computador , Homeostase , Humanos , Cinética , Zíper de Leucina , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
This study addresses potential molecular mechanisms underlying the inhibition of the transcription factor c-Jun by nitric oxide. We show that in the presence of the physiological sulfhydryl glutathione nitric oxide modifies the two cysteine residues contained in the DNA binding module of c-Jun in a selective and distinct way. Although nitric oxide induced the formation of an intermolecular disulfide bridge between cysteine residues in the leucine zipper site of c-Jun monomers, this same radical directed the covalent incorporation of stoichiometric amounts of glutathione to a single conserved cysteine residue in the DNA-binding site of the protein. We found that covalent dimerization of c-Jun apparently did not affect its DNA binding activity, whereas the formation of a mixed disulfide with glutathione correlated well with the inhibition of transcription factor binding to DNA. Furthermore, we provide experimental evidence that nitric oxide-induced S-glutathionylation and inhibition of c-Jun involves the formation of S-nitrosoglutathione. In conclusion, our results support the reversible formation of a mixed disulfide between glutathione and c-Jun as a potential mechanism by which nitrosative stress may be transduced into a functional response at the level of transcription.