The UBE2L3 ubiquitin conjugating enzyme: interplay with inflammasome signalling and bacterial ubiquitin ligases Matthew James George Eldridge 2018 Imperial College London Department of Medicine Submitted to Imperial College London for the degree of Doctor of Philosophy 1 Abstract Inflammasome-controlled immune responses such as IL-1β release and pyroptosis play key roles in antimicrobial immunity and are heavily implicated in multiple hereditary autoimmune diseases. Despite extensive knowledge of the mechanisms regulating inflammasome activation, many downstream responses remain poorly understood or uncharacterised. The cysteine protease caspase-1 is the executor of inflammasome responses, therefore identifying and characterising its substrates is vital for better understanding of inflammasome-mediated effector mechanisms. Using unbiased proteomics, the Shenoy grouped identified the ubiquitin conjugating enzyme UBE2L3 as a target of caspase-1. In this work, I have confirmed UBE2L3 as an indirect target of caspase-1 and characterised its role in inflammasomes-mediated immune responses. I show that UBE2L3 functions in the negative regulation of cellular pro-IL-1 via the ubiquitin- proteasome system. Following inflammatory stimuli, UBE2L3 assists in the ubiquitylation and degradation of newly produced pro-IL-1. However, in response to caspase-1 activation, UBE2L3 is itself targeted for degradation by the proteasome in a caspase-1-dependent manner, thereby liberating an additional pool of IL-1 which may be processed and released. UBE2L3 therefore acts a molecular rheostat, conferring caspase-1 an additional level of control over this potent cytokine, ensuring that it is efficiently secreted only in appropriate circumstances. These findings on UBE2L3 have implications for IL-1- driven pathology in hereditary fever syndromes, and autoinflammatory conditions associated with UBE2L3 polymorphisms. 2 Acknowledgments First and foremost, I would like to extend my most sincere thanks to my supervisor Avinash. Having the opportunity to join a brand-new lab has taught me so many things at and away from the bench and being able to learn from you first hand has been invaluable. Not every student gets the opportunity to work so closely with their supervisor and I will be forever grateful for your mentorship. I would like to thank my lab mates Julia and Pippa, you two have been the best lab mates ever. Thank you for always being around, not only have you been great colleagues, you have been fantastic and supportive friends. Without the two of you, the 4th floor would have been a very different place, it was a pleasure working with you both. Julia, thank you for all your contributions to the paper, it would have taken three times as long without you. Thank you to the other inhabitants of the 4th floor, old and new Dan, Amy, Sophie, and Nancy, you couldn’t ask for better people to share a lab space with. Thank you to my supervisees who helped me with my bacteria side projects Anja, Youssef, and Razia. I would also like to thank all the members of the CMBI who I have crossed paths with over the years in particular Vani, Miles, Robbie, Sina, Agnes, Angie thank you for making it such a great place to work. Thank you Sanika, who entered the fray a little later but made the last year and half of my PhD that little bit easier. Finally, thank you to my parents, my sister and the rest of my family whose continued support has made my academic studies possible. Mum and Dad thank you for giving me the opportunity to pursue what I love to do. 3 Declaration of originality I hereby declare that the work presented in this thesis is my own, any work carried out by anyone other than myself be it published and unpublished has been acknowledged and clearly credited in the relevant figure legends. Any resources or ideas that are from the work of others has been reference and a full bibliography can be found at the end of this thesis. Copyright declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 4 Table of contents Abstract ...................................................................................................................................... 2 Acknowledgments...................................................................................................................... 3 Declaration of originality ........................................................................................................... 4 Copyright declaration ................................................................................................................ 4 Tables and figures .................................................................................................................... 10 Abbreviations ........................................................................................................................... 15 Chapter 1 – Introduction ......................................................................................................... 18 1.1 Introduction to pattern recognition .............................................................................. 19 1.2 Toll-like receptor signalling ............................................................................................ 20 1.2.1 MyD88 dependent activation of NF-κB .................................................................. 22 1.2.2 TRIF dependent signalling ....................................................................................... 24 1.3 Inflammasomes .............................................................................................................. 25 1.3.1 Principles of inflammasome activation................................................................... 25 1.3.3 NLRP3 inflammasome ............................................................................................. 28 1.3.4 NLRC4 inflammasome ............................................................................................. 31 1.3.5 AIM2 inflammasome ............................................................................................... 33 1.3.6 Non-canonical inflammasome activation ............................................................... 34 1.3.7 PYRIN inflammasome .............................................................................................. 35 1.3.8 NLRP1 inflammasome ............................................................................................. 37 1.3.9 Other inflammasomes ............................................................................................ 38 1.4 Caspase-1 Substrates and Targets ................................................................................. 39 1.4.1 pro-IL-1β .................................................................................................................. 40 1.4.2 pro-IL-18 .................................................................................................................. 42 1.4.3 IL-1α ........................................................................................................................ 43 1.4.4 Gasdermin D ........................................................................................................... 44 1.5 Inflammasome signalling during bacterial infection ......................................................... 46 1.5.1 Pathogen sensing by inflammasomes .................................................................... 46 1.5.2 Bacterial circumvention of inflammasome sensing and mechanisms ................... 48 1.5.3 Host-protective properties of inflammasomes during bacterial infection ............. 49 1.6 Role of inflammasomes in inheritable illnesses ................................................................ 51 1.7 Novel downstream targets of inflammasomes ................................................................. 53 1.8 Enzymes of the ubiquitin cascade ..................................................................................... 55 5 1.9 UBE2L3: an atypical E2 conjugating enzyme ..................................................................... 58 1.10 Aims and Objectives ......................................................................................................... 63 Chapter 2 – Materials and Methods ....................................................................................... 64 2.1 Eukaryotic and bacterial cell growth conditions............................................................ 65 2.1.1 Eukaryotic cell culture ............................................................................................. 65 2.1.2 Bacterial growth conditions for infection ............................................................... 66 2.2 Bacterial strains, plasmids, and primers ........................................................................ 69 2.2.1 Bacterial strains ....................................................................................................... 69 2.2.2 Bacterial and eukaryotic expression plasmids ........................................................ 70 2.2.4 Primers used during this study ..............................................................................