a Genetic approaches to understand peroxiredoxin-mediated H2O2 signalling mechanisms

Abstract

Hydrogen peroxide (H2O2) is a potentially toxic bi-product of aerobic metabolism. Whilst high concentrations cause the cellular damage observed in many diseases and ageing, low levels of H2O2 have been shown to be important for normal health and longevity. How organisms sense H2O2, and use it to signal positive responses is therefore of great interest. This project focused on using genetic techniques in 2 model eukaryotes, Schizosaccharomyces pombe and Caenorhabditis elegans, to address these questions and to elucidate mechanisms by which peroxiredoxins (Prx) affect H2O2 signal transduction. We have investigated whether a roGFP2-PRDX-2 fusion protein, expressed in C. elegans, was suitable to detect in vivo changes in intracellular H2O2 levels. Unfortunately, our data suggests that, although this roGFP2-PRDX-2 sensor shows some specificity for H2O2, it is insufficiently sensitive to detect small differences in endogenous H2O2. However, preliminary data using this sensor does suggest that there is an increase in H2O2 in anterior intestinal cells in response to infection with the fungal pathogen, Candida albicans. We have also investigated the role of cytosolic family 2-Cys Prx in mitochondrial function, providing further evidence for mitochondrial defects in tpx1Δ mutant S. pombe, and prdx-2(gk169) mutant C. elegans, and identifying the presence of a pool of Tpx1 in the mitochondrial intermembrane space (IMS). We present evidence suggesting that a direct role in the IMS may contribute to the function of Tpx1 in H2O2 resistance, and H2O2-induced activation of Pap1. The highly abundant 2-Cys Prx detoxify H2O2, but are also involved in initiating signal transduction. S. pombe has a single 2-Cys Prx, Tpx1, which is essential for transcriptional responses to H2O2. Tpx1 has multiple roles in promoting H2O2 signal transduction; for example, acting as a direct redox transducer to promote activation of the p38-related MAPK, Sty1, and also by promoting the oxidation of thioredoxins, to activate the transcription factor Pap1. To identify new candidate H2O2 regulated proteins, we have used high-throughput genetic screening of a library of mutants of non-essential S. pombe genes in two genetic backgrounds; tpx1C169S, in which the thioredoxin peroxidase activity of Tpx1 is disrupted, but Sty1 regulation unaffected, and the deletion mutant; tpx1Δ. vi From synthetic genetic array (SGA) analysis of the tpx1C169S mutant we identified 31 candidate genes that are important for growth in the absence of thioredoxin peroxidase activity. Notably, orthologues of 6 of these genes also exhibit a synthetic sick interaction with the Prx mutant tsa1Δ in S. cerevisiae, suggesting that these pathways may have a conserved essential function in the absence of the thioredoxin peroxidase activity. Moreover, SGA analysis of tpx1Δ also identified 21 candidate genes important for growth in a tpx1Δ. Interestingly, less overlap was observed between the SGA analysis of tpx1Δ with tsa1Δ. Our screening additionally identified 5 candidate genes where loss of function partially suppressed the growth defects associated with loss of tpx1. These included the single S. pombe cAMP-dependent protein kinase, pka1, and the COP9/signalosome complex protease subunit, csn5. Here we show that both proteins undergo oxidation, through the formation of direct protein-protein disulphide complexes with Tpx1, in response to 0.2 mM H2O2, thereby validating this approach as a means to identify new candidate H2O2- regulated proteins. Finally, this screening identified that the glutaredoxin, Grx1, was important for growth and H2O2-dependent activation of Sty1 in tpx1C169S -mutant cells. Further investigation suggested that Tpx1C169S is constitutively glutathionylated in the absence of Grx1. This supports the hypothesis that a redox-active peroxidatic cysteine is important for Tpx1’s signalling roles, as well as raising the possibility that Tpx1C169S may function as a glutathione peroxidase. In summary, this study has provided new targets and tools, as well as mechanistic insight, into the role of H2O2 and Prx in responses to peroxide