Study of the role of Pap1 as a sensor of H2O2 and as a transcriptional activator of stress responses in Schizosaccharimyces pombe

Resumen

Schizosaccharomyces pombe (S. pombe) has specific pathways to respond to extracellular hydrogen peroxide (H2O2), the main one being the Pap1 (pombe AP-1-like) pathway. Activation of Pap1 (oxidized state) occurs mainly at low H2O2 concentrations in a Tpx1-dependent manner. Previous genetic analysis seemed to indicate that an intramolecular disulfide bond is formed in Pap1 upon oxidative stress, which triggers the nuclear accumulation of the transcription factor and Pap1-dependent gene induction. In this work we have characterized Pap1 as a sensor of H2O2 and as a transcription factor. Regarding the role of Pap1 as a sensor of H2O2, we have now performed a detailed mutagenesis analysis of all cysteine residues in Pap1 and have identified new cysteine residues essential for its activation. Furthermore, we have confirmed this data regarding with a proteomic characterization of active and inactive Pap1. We have deciphered the role of different components of the thioredoxin system in the regulation of Pap1, the participation of some proteins such as Trx1 and Trr1 in the control of Pap1 redox state; and we have seen that the loss of Trx1 prolongs H2O2-induced Pap1-dependent gene expression. With regard to the study of Pap1 as a transcription factor, we have demonstrated here that the expression of some Pap1- induced genes such as ctt1, trr1, srx1, obr1, SPCC663.08c and caf5 have different requirements regarding Pap1 activity/subcellular localization/oxidation state. The drug resistance genes only require nuclear Pap1 for activation, whereas another subset of genes, those coding for the antioxidants catalase, sulfiredoxin or thioredoxin reductase, do need oxidized Pap1 to form a heterodimer with the constitutively nuclear transcription factor Prr1. The ability of Pap1 to bind and activate drug tolerance xvi promoters is independent on Prr1, whereas its ability to bind to the antioxidant promoters is significantly enhanced upon association with Prr1.