Mechanisms of H₂O₂-induced signal transduction

PhD ThesisReactive oxygen species (ROS), including H2O2, are produced as unavoidable by-products of aerobic respiration, leading to oxidative stress and the initiation and development of many diseases, particularly age-associated diseases. Cells have evolved antioxidant and repair enzymes to protect against ROS, including the 2-Cys peroxiredoxins (Prx) family, a group of extremely abundant, highly conserved peroxidases. H2O2 initiates protective cell responses that include increasing expression of these enzymes. Counterintuitively, typical 2-Cys Prx have also been shown to have important roles in promoting stress-induced signal transduction, and as molecular chaperones, independent of their thioredoxin peroxidase activity. Although H2O2’s function as a signalling molecule is now well-established, the targets of H2O2 signals and the mechanisms by which these targets are regulated are poorly characterised. Studies in yeast models have provided some of the best evidence to date for the positive signalling roles of low levels of H2O2 to drive adaptive responses to limit damage. For example, in the fission yeast Schizosaccharomyces pombe, different levels of H2O2 activate distinct signal transduction pathways and transcriptional responses that protect cells against the toxic effects of increased ROS. Previous work has established that the single typical 2-Cys Prx, Tpx1, is essential for H2O2-induced gene expression. In S. pombe, Tpx1 is needed to promote the H2O2-induced activation of the AP-1-like transcription factor Pap1 and the p38/JNK related mitogen activated protein kinase (MAPK), Sty1, by mechanisms involving oxidation of cysteine residues. The overall aim of this project was to use biochemical and genetic approaches to investigate the molecular mechanisms underlying H2O2- sensing in the genetically amenable model organism, S. pombe. In this study we have identified that Tpx1 stimulates inactivation of the protein tyrosine phosphatase (PTP) Pyp1, to promote activation of the Sty1 pathway in response to low concentrations of H2O2. We have identified that Pyp1 undergoes multiple post-translational modifications, including oxidation and 4 phosphorylation in response to H2O2. We have examined the role of these modifications in regulating H2O2-induced activation of Sty1, identifying that H2O2-induced formation of a disulphide with thioredoxin, and stress-induced phosphorylation by the MAPKK Wis1, are important for maintaining Pyp1 function in cells exposed to increasingly stressful conditions. Moreover, we have also investigated the relationship between related PTP in the control of H2O2-induced Sty1 activation, identifying an unexpected role for Pyp1 in promoting the expression of the stress-induced PTP, Pyp2. Oxidation of the catalytic cysteine in the glycolytic GAPDH enzyme, Tdh1, has previously been shown to be important for H2O2-induced activation of Sty1. Indeed, GAPDH’s susceptibility to H2O2-induced oxidation of its catalytic cysteine has been proposed to have evolved as an important protective response to H2O2. Here we show that although a second cysteine in the active site of Tdh1 is important for the H2O2-induced oxidation of Tdh1 and oxidative stress resistance, this oxidation is not important for the H2O2-induced activation of Sty1. Having found that Tpx1 is required for the oxidation of Pap1, Sty1, Pyp1 and Tdh1, we have also investigated whether Tpx1 act as direct H2O2 transducers to promote the oxidation of these and other proteins. This has provided further insight into the mechanisms involved in activation of Pap1, and also identified many potential new candidates for Tpx1-dependent regulation by H2O2. These include a conserved MAPK activated kinase, Srk1, that we show inhibits cell cycle progression by a mechanism that is partially dependent on Tpx1. In summary, this study has identified new mechanisms involved in protective responses to increases in H2O2. In particular, it has provided new insight into how cells regulate the activity of stress-activated MAPK and GAPDH to coordinate appropriate responses to rises in ROS.Biotechnology and Biological Sciences Research Council (BBSRC