A systems biology approach to investigate the role of peroxiredoxins in responses to hydrogen peroxide

PhD ThesisMaintenance of redox balance is essential for fundamental physiological processes, including growth, differentiation, migration, and circadian rhythms. However, the mechanisms by which cells maintain this balance and sense acute, dynamic redox changes under signalling and/or stress conditions are poorly defined. 2-cys peroxiredoxins (Prxs) are abundant, ubiquitous thioredoxin peroxidases with important roles in ageing and cancer. Counter-intuitively, Prxs are sensitive to hydrogen peroxide (H2O2)-induced inhibition of this peroxidase activity by hyperoxidation to a thioredoxin-resistant form. Prx hyperoxidation has been proposed to have various functions in signalling and protein homeostasis. However, the circumstances which cause Prx to become hyperoxidized in vivo remain unclear. The fission yeast Schizosaccharomyces pombe contains a single, well-studied 2-Cys Prx with important roles in responses to H2O2. Using quantitative in vivo and in vitro kinetic data, multiple mathematical models have been developed to investigate when the S. pombe peroxiredoxin Tpx1 becomes hyperoxidised in vivo. This approach suggested that Prx hyperoxidation occurs when the peroxide-buffering capacity of a cell becomes saturated. This was confirmed experimentally using both S. pombe and human cell lines, which suggested that the thiol-proteome is responsible for this peroxide buffering capacity. Accordingly, we propose that Prx hyperoxidation signals that the cells’ antioxidant defences are overcome and that repair mechanisms need to be deployed to limit damage and restore homeostasis. We have also used an integrated modelling and experimental approach to investigate the roles of Tpx1 in promoting the H2O2-induced oxidation (activation) of the AP-1-like transcription factor Pap1. This has revealed that Tpx1 has 2 roles, participating as a direct H2O2-transducer to initiate Pap1 oxidation and also by competitively inhibiting the thioredoxin-like protein Txl1 from reducing Pap1. Finally, we use a simple computer model representing multiple cellular peroxidase processes to demonstrate that changes in gene expression in response to H2O2 limit H2O2-induced damage by improving the cells’ ability to inhibit increases in intracellular H2O2 concentration. Together this work has provided new insights into the cellular mechanisms responsible for the regulation of cellular responses to H2O2.student bursary awarded by the Biotechnology and Biological Sciences Research Council (BBSRC)