title: Reversible thiol oxidation in the H2O2-dependent activation of the transcription factor Pap1.
abstract: Reversible thiol oxidation is both a mark of hydrogen peroxide (H2O2) toxicity and an initiator of signalling events. H2O2 sensors contain exposed and reactive cysteine residues, which become transiently oxidized as an activation mechanism. In fission yeast, the Pap1 (pombe AP-1) transcription factor is normally cytosolic, and upon H2O2 stress it undergoes post-translational modifications impairing its nuclear export; genetic evidences suggested the formation of a disulphide bond in Pap1 as a triggering activation event. Nuclear Pap1 is then recruited to about 50-80 promoters and induces an adaptation response. We have now dissected the role of all seven cysteine residues in Pap1 using genetic and proteomic techniques, and we show that four of them are required for Pap1 to be activated by H2O2 stress. Thus, mutants lacking each one of these cysteine residues display sensitivity to peroxides. Furthermore, these mutant proteins do not become oxidized by H2O2 and cannot bind to promoters or trigger the Pap1-dependent gene expression program. We also demonstrate, by proteomic analysis of reduced and oxidized Pap1, that these four cysteine residues are reversibly oxidized upon H2O2 stress. Our study suggests that not just one but probably two disulphide bonds are required to promote the important conformational changes that trigger Pap1 activation and nuclear accumulation.
authors.affiliation: Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, C/Dr. Aiguader 88, E-08003 Barcelona, Spain.
authors.forename: Isabel A
authors.initials: IA
authors.lastname: Calvo
authors.forename: José
authors.initials: J
authors.lastname: Ayté
authors.forename: Elena
authors.initials: E
authors.lastname: Hidalgo
mesh_terms: D000494:Allosteric Regulation; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D006861:Hydrogen Peroxide / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D000073718:Pancreatitis-Associated Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport / Q000235:genetics; D040901:Proteomics; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D000073718:Pancreatitis-Associated Proteins; C105096:Pap1 protein, S pombe; C000612141:REG3A protein, human; D029702:Schizosaccharomyces pombe Proteins; D006861:Hydrogen Peroxide; D003545:Cysteine
keywords: Disulphide bond; Fission yeast; H2O2 sensor; Pap1; Redox cascade