Understanding the mechanisms of Interleukin-1α processing and secretion A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2020 Victor Sebastian Tapia Olivares School of Biological Sciences Division of Neuroscience and Experimental Psychology 1 Contents List of Figures ...................................................................................................... 4 Abbreviations ....................................................................................................... 5 Abstract ............................................................................................................... 7 Declaration .......................................................................................................... 8 Copyright Statement ............................................................................................ 9 Thesis Format .................................................................................................... 10 Acknowledgements ............................................................................................ 11 Chapter 1. General Introduction ......................................................................... 12 1.1. Inflammation ........................................................................................... 13 1.2. Regulation of the inflammatory response ................................................ 13 1.3. IL-1 signalling .......................................................................................... 15 1.4. IL-1 expression ....................................................................................... 17 1.5. IL-1 sequence ......................................................................................... 18 1.6. IL-1 sub-cellular localization .................................................................... 22 1.7. Mechanisms of IL-1 activation ................................................................. 23 1.7.1. Calpain ......................................................................................... 25 1.7.2. Inflammasome activation .............................................................. 25 1.7.3. Non-canonical inflammasomes: Caspases-4/5/11 ........................ 29 1.7.4. Caspase-8 .................................................................................... 30 1.7.5. Granzymes ................................................................................... 30 1.7.6. Extracellular activation of IL-1 ....................................................... 31 1.8. Mechanisms of IL-1 secretion.................................................................. 32 1.8.1. Passive IL-1α release from death cells ......................................... 32 1.8.2. GSDMD-dependent IL-1 secretion ................................................ 32 1.8.3. Alternative mechanisms of IL-1 secretion ..................................... 35 1.9. Role of IL-1α in physiology and disease .................................................. 37 1.9.1. IL-1α in acute inflammation ........................................................... 37 1.9.2. IL-1α in chronic inflammation ........................................................ 38 1.9.3. IL-1 therapy .................................................................................. 40 1.10. Summary and aims .......................................................................... 42 Chapter 2. The three cytokines IL-1β, IL-18 and IL-1α share related but distinct routes...................................................................................................................43 2.1. Paper title and authors ............................................................................ 44 2.2. Abstract ................................................................................................... 46 2.3. Introduction ............................................................................................. 47 2.4. Results .................................................................................................... 48 2.5. Discussion ............................................................................................... 61 2 2.6. Experimental procedures ........................................................................ 65 2.7. References .............................................................................................. 70 Chapter 3. Regulation of IL-1α release by nuclear localization ........................... 74 3.1. Paper title and authors ............................................................................ 75 3.2. Abstract ................................................................................................... 76 3.3. Introduction ............................................................................................. 77 3.4. Results .................................................................................................... 78 3.5. Discussion ............................................................................................... 84 3.6. Experimental procedures ........................................................................ 85 3.7. References .............................................................................................. 89 3.8. Supplementary Figures ........................................................................... 91 Chapter 4. A TurboID-based proximity labelling approach to study the IL-1α interactome .........................................................................................................92 4.1. Paper title and authors ............................................................................ 93 4.2. Abstract ................................................................................................... 94 4.3. Introduction ............................................................................................. 95 4.4. Results .................................................................................................... 96 4.5. Discussion ............................................................................................. 102 4.6. Experimental procedures ...................................................................... 103 4.7. References ............................................................................................ 107 4.8. Supplementary figures .......................................................................... 110 Chapter 5. General Discussion ........................................................................ 111 5.1. Summary............................................................................................... 112 5.2. General discussion ................................................................................ 113 5.2.1. Degrees of IL-1α signalling ......................................................... 113 5.2.2. Mechanisms of IL-1α processing and secretion .......................... 115 5.3. Experimental considerations and future prospects ................................ 116 5.3.1. Insights in IL-1 secretion ............................................................. 116 5.3.2. Nuclear localization .................................................................... 118 5.3.3. Pro-domain selectivity................................................................. 118 5.3.4. IL-1α interactors ......................................................................... 119 5.4. Concluding remarks .............................................................................. 120 Chapter 6. General Bibliography ...................................................................... 121 Word count: 42701 3 List of Figures Figure 1.1 - IL-1 signalling pathway and negative regulators. ............................ 16 Figure 1.2 - Protein interactions, post-translational modifications and cleavage sites for IL-1. ...................................................................................................... 21 Figure 1.3 - Calpain and inflammasome-dependent IL-1 activation. ................... 24 Figure 1.4 - Canonical inflammasomes. ............................................................. 28 Figure 1.5 - Unconventional secretory pathways of IL-1. .................................... 33 Figure 2.1 - IL-1β release after inflammasome activation depends on plasma membrane permeability. .................................................................................... 49 Figure 2.2 - ASC speck release depends on cell lysis. ....................................... 53 Figure 2.3 - IL-18 release during inflammasome activation depends on plasma membrane permeability. .................................................................................... 55 Figure 2.4 - IL-1α processing and release depends on calpains 1 and 2. ........... 57 Figure 2.5 - IL-1α release can occur independently of cell lysis. ........................ 60 Figure 2.6 - IL-1α is released from viable MEFs. ................................................ 62 Figure 3.1 - Expression constructs of pro-IL-1α. ................................................ 79 Figure 3.2 - Nuclear localization regulates processing and secretion of pro-IL-1α. .......................................................................................................................... 81 Figure 3.3 - Effects of NLS mutation on IL-1α secretion. .................................... 83 Figure 4.1 - Characterization