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The Regulation of Deubiquitinases by Oxidative Stress in Saccharomyces

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The Regulation of Deubiquitinases by Oxidative Stress in Saccharomyces The regulation of deubiquitinases by oxidative stress in Saccharomyces cerevisiae. Faye Elizabeth Jade Curtis Thesis submitted for the degree of Doctor of Philosophy Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Newcastle University September 2018 Declaration I certify that this thesis is my own work, except where acknowledged. Work in this thesis has not been previously submitted for another degree or qualification at this or any other university. [i] [ii] Abstract Reactive oxygen species (ROS) are produced as by-products of cellular processes. In eukaryotes high levels of ROS cause oxidative stress, leading to intracellular damage and age-related diseases, whereas low levels of ROS are vital for many cellular functions including signal transduction and cell proliferation. It is therefore essential that cells can sense the different types and levels of ROS to ensure that they respond in an appropriate manner. Ubiquitination is a post- translational modification which plays vital roles in fundamental cellular processes, including protein degradation and many signalling pathways. Ubiquitination of substrates occurs in a cycle involving the coordinated activity of conjugation and deconjugation enzymes and, interestingly, most ubiquitin pathway enzymes utilise catalytic cysteines for function. Recent work from our lab and others has begun to reveal that the relative sensitivity of these cysteines to oxidation is important for sensing the different types and levels of ROS. In the present study we hypothesised that deubiquitinases (dUbs), which remove ubiquitin from substrates through the activity of an active site catalytic cysteine, may also be regulated by ROS. Hence to test this hypothesis, the tractability and powerful genetic tools available in the model eukaryote Saccharomyces cerevisiae were utilised to investigate the potential regulation and function of all dUbs in this organism in response to different oxidising agents. Excitingly, an initial screen of available S. cerevisiae dUb gene deletion mutants and strains expressing epitope-tagged dUbs identified wide and varied responses of dUbs to different oxidising agents. For example, several dUbs were found to be important for cell survival under different oxidative stress conditions. Furthermore, specific dUbs were also found to be modified in response to specific oxidising agents. In particular, further investigations into specific dUbs observed that Ubp12 was reversibly oxidised into a HMW intramolecular disulphide complex in response to H2O2 but no other oxidising agents tested. Significantly, the catalytic cysteine of Ubp12 was shown to be essential for this complex, suggesting that oxidation regulates Ubp12 activity. Another dUb, Ubp2, was also found to be oxidised in response to H2O2. Other work has shown that Ubp2 and Ubp12 regulate mitochondrial dynamics, and therefore the present study led to a model [iii] suggesting that the regulation of Ubp12 and Ubp2 by H2O2 may regulate mitochondrial dynamics in response to different levels of H2O2. In contrast to Ubp2 and Ubp12, further investigations into another dUb, Ubp15, was found to be oxidised into HMW complexes in response to both H2O2 and diamide. Interestingly, these Ubp15 HMW complexes have different mobilities suggesting differences in the way Ubp15 responds to different oxidising agents. Significantly, similar to Ubp12, the catalytic cysteine of Ubp15 was essential for the formation of the H2O2-induced HMW complex. Ubp15 has previously been implicated in the regulation of cell cycle progression, and consistent with this link the present results suggest that Ubp15 may be important for regulating G1 phase arrest/delay, and for regulating the release into S phase, following H2O2 treatment. Collectively, the work described here has begun to provide insight into how cells sense and respond to different types and levels of ROS by regulating specific dUbs. Furthermore, the yeast dUbs are conserved in higher eukaryotes and hence these studies have potential implications for the regulation of fundamental processes such as the cell cycle and mitochondrial dynamics. [iv] Acknowledgements Firstly, I would like to thank my supervisor Brian Morgan, for his constant support, advice, enthusiasm, and total confidence in me throughout this PhD. I would also like to acknowledge the BBSRC for funding my research, and David Stead at Aberdeen University for completing the mass spectrometry and computational analysis. Secondly, I would like to thank everyone in the Morgan, Quinn, Whitehall, and Veal labs, past and present, for helping with ideas and insight in this project. But more importantly for providing cake, chocolate and for making the four years fun! I would especially like to thank Callum- for keeping me sane (ish!), and Zoe-for the much needed coffee breaks, wine nights and random chat. Thirdly, I would like to thank my family, Mam and Dad, and even you Scall, for the encouragement and support over all the years and for providing endless love (and gin) to get me to here. Finally, I would like to thank Luke. You have been there to celebrate the good days and to cheer me up after the bad days. You have made everything better, and I definitely couldn’t have done it without you. [v] [vi] Table of Contents Declaration ......................................................................................................... i Abstract ........................................................................................................ iii Acknowledgements ............................................................................................... v Table of Contents ................................................................................................. vii Abbreviations ...................................................................................................... xiii List of Figures ...................................................................................................... xv List of Tables ...................................................................................................... xix Chapter One: Introduction ..................................................................................... 1 1.1. Ubiquitin and ubiquitin-like modifications…………………….... ………......1 1.1.1. Ubiquitin and ubiquitin-like conjugation .................................... …...……..3 1.1.1.1. Ubiquitin conjugation ....................................................................... 3 1.1.1.2. Types of ubiquitin conjugation ....................................................... 10 1.1.1.2.1. Mono- and multi-ubiquitination ....................................................... 10 1.1.1.2.2. Poly-ubiquitination ......................................................................... 13 1.1.1.3. Targets and functions of ubiquitination .......................................... 15 1.1.1.3.1. Ubiquitination and cell cycle regulation .......................................... 15 1.1.1.3.2. Ubiquitination and DNA repair ....................................................... 16 1.1.1.3.3. Ubiquitination and protein localisation ........................................... 17 1.1.1.3.4. Ubiquitination and mitochondrial regulation ................................... 17 1.1.1.4. Ubiquitin-like modifications ............................................................ 18 1.1.2. Deubiquitination ..................................................................................... 20 1.1.2.1. JAMM family .................................................................................. 23 1.1.2.2. USP family ..................................................................................... 24 1.1.2.3. UCH family ..................................................................................... 27 1.1.2.4. OTU family ..................................................................................... 27 1.1.2.5. DUb specificity ............................................................................... 28 1.1.2.5.1. Ubiquitin vs ubiquitin-like specificity ............................................... 28 1.1.2.5.2. Substrate specificity ....................................................................... 29 1.1.2.5.3. Type of chain linkage ..................................................................... 29 1.1.2.5.4. Protein partners ............................................................................. 31 1.1.2.5.5. Outstanding questions about dUb specificity ................................. 32 1.1.2.6. DUbs in disease ............................................................................. 32 1.1.3. Regulation of ubiquitin/ubiquitin-like pathway enzymes ......................... 33 1.2. Reactive oxygen species ....................................................................... 35 [vii] 1.2.1. Types and sources of ROS..................................................................... 35 1.2.1.1. The mitochondrial electron transport chain .................................... 36 1.2.1.2. Transition metals ............................................................................ 37 1.2.1.3. The immune response ................................................................... 37 1.2.1.4. Xenobiotics..................................................................................... 39 1.2.1.5. UV and ionising radiation ............................................................... 40 1.2.2. Effects of ROS .......................................................................................
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