Genetic approaches to understand peroxiredoxin-mediated H2O2 signalling mechanisms Zoe Elizabeth Underwood Thesis submitted for the degree of Doctor of Philosophy Faculty of Medical Sciences Institute for Cell and Molecular Biosciences Newcastle University September 2018 Declaration I certify that this thesis contains my own work, except where acknowledged, and that no part of this material has been previously submitted for a degree or any other qualification at this or any other University. The work in this thesis is also supported by the publications: Veal EA, Underwood ZE, Tomalin LE, Morgan BA, Pillay CS. Hyperoxidation of Peroxiredoxins: Gain or Loss of Function? Antioxidants & Redox Signaling 2018 28(7):574-590. Tomalin LE, Day AM, Underwood ZE, Smith GR, Dalle Pezze P, Rallis C, Patel W, Dickinson BC, Bahler J, Brewer TF, Chang CJ, Shanley DP, Veal EA. Increasing extracellular H2O2 produces a bi-phasic response in intracellular H2O2, with peroxiredoxin hyperoxidation only triggered once the cellular H2O2-buffering capacity is overwhelmed. Free Radical Biology & Medicine 2016 95:333-48. i ii Acknowledgements I would firstly like to thank my supervisor, Elizabeth Veal, for her invaluable insight and guidance over the past four years. I would also like to acknowledge the Biotechnology and Biological Sciences Research Council (BBSRC) for funding this research. In addition, I would like to thank my secondary supervisor David Lydall, and my panel Alberto Sanz and Nick Watkins. I would also like to thank Peter Banks and Adrian Blackburn in the High Throughput Screening Facility at Newcastle University, for use of the robotics and the statistical analysis performed for the SGA and QFA analysis in this thesis. Additionally, I would like to thank Alex Laude in the Bio-Imaging Unit for his expertise and use of the confocal microscope. As well as, Francesco Bruni in the Wellcome Trust Centre for Mitochondrial Research for his advice and supervision in mitochondrial fractionation techniques. In addition, I would like to thank all the supervisors and members of the Quinn, Veal, Morgan and Whitehall labs, past and present, for discussion and advice throughout this project. Thanks to Alison, Lewis, Johny, Heather, Clare, Anu, Faye M, Pippa, Melanie, Alex, Slava, Callum, Yasmin, Martin and Grace for all of the cake and fun times over the last 4 years. Special thanks to Faye and Hannah for their friendship, support and encouragement throughout. Finally, thanks to my Mum, Dad, Nan, Grandad, Gran, and my brother Simon, for their continuous love and support and for always believing I could succeed. iii iv 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. v 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 C169S and H2O2-dependent activation of Sty1 in tpx1 -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. vi Contents List of Figures ................................................................................................................... xv List of Tables .................................................................................................................... xx List of Abbreviations ...................................................................................................... xxi Chapter 1. Introduction .................................................................................................... 1 1.1 Reactive Oxygen Species ........................................................................................... 1 1.1.1 Sources of ROS ................................................................................................... 1 1.1.2 Antioxidant defences ........................................................................................... 2 1.1.2.1 Superoxide dismutase ................................................................................... 2 1.1.2.2 Catalase ......................................................................................................... 2 1.1.2.3 Glutathione peroxidase ................................................................................. 2 1.1.2.4 Thioredoxin peroxidase ................................................................................ 3 1.1.2.5 Thioredoxin ................................................................................................... 3 1.1.2.6 Glutathione .................................................................................................... 3 1.1.3 Oxidative stress.................................................................................................... 6 1.1.4 Oxidative stress in ageing .................................................................................... 6 1.2 ROS and signal transduction ...................................................................................... 7 1.2.1 Mechanism of thiol oxidation/disulphide formation ........................................... 9 1.2.2 H2O2 as a signalling molecule ........................................................................... 10 1.2.3 Adaptive responses to H2O2 .............................................................................. 11 1.2.4 Positive signalling at low H2O2 concentrations ................................................. 14 1.3 Role of 2-Cys Prx in H2O2 sensing and signalling ................................................... 15 1.3.1 The 3 classes of peroxiredoxins ......................................................................... 15 1.3.2 Peroxiredoxins as antioxidants .......................................................................... 17 1.3.3 Peroxiredoxins as chaperone proteins ..............................................................