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.—Klatt, P., Molina, E. P., de Lacoba, M. G., Padilla, C. A., Martínez‐Galisteo, E., Bárcena, J. A., Lamas, S. Redox regulation of c‐Jun DNA binding by reversible S‐glutathiolation. FASEB J . 13, 1481–1490 (1999)

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis, cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adapt cell metabolism and integrate several intracellular and redox signaling to promote cell survival in an inflammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administration of tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies at advanced stages.

Glutaredoxin is generally a glutathione‐dependent hydrogen donor for ribonucleotide reductase and also catalyses general glutathione (GSH)‐disulfide‐oxidoreduction reactions in the presence of NADPH and glutathione reductase. A Glutaredoxin from human placenta was purified to homogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was monitored by the activity with hydroxyethyl disulfide as a substrate. Values of pI for glutaredoxin were obtained by IEF; the pI of the protein shifted from 7.3 in its fully reduced state to 9.0 in the oxidized state after treatment with excess hydroxyethyl disulfide. The glutaredoxin preparation showed GSH‐dependent hydrogen‐donor activity with recombinant mouse ribonucleotide reductase, it exhibited dehydroascorbate reductase activity as well as hydroxyethyl‐disulfide‐reducing activity. The amino acid sequence (residues 3–104) of glutaredoxin was determined by peptide sequencing and residues 1, 2 and 105 by cDNA sequence analysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins ‐Cys22‐Pro‐Tyr‐Cys25‐ and three additional half‐cystine residues; two of these in positions 78 and 82. The sequence was similar to other known mammalian glutaredoxins (about 80% identities), with important differences such as one additional Cys residue (Cys7) and no Met residue. The sequence of human glutaredoxin was compared to that of Escherichia coli glutaredoxin with known three‐dimensional structure in solution to indentify conserved residues and predict a structure from alignment. In particular the GSH‐binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire glutaredoxin gene (grx) and flanking sequences was isolated from a human spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) was determined, including the complete grx gene.