Malonylation of GAPDH in the inflammatory response in macrophages Thesis submitted to the University of Dublin for the Degree of Doctor of Philosophy by Silvia Galván-Peña 13335025 School of Biochemistry and Immunology Trinity College Dublin March 2018 Declaration This thesis is submitted by the undersigned. The work herein is entirely my own, with the exception of: • Figure 3.7: this work was analysed by Steve DeHaro • Figure 3.13: this experiment was performed by PTM Biolabs • Figure 3.15A: generated by Alan Nadin • Figure 4.16 and Figre 4.17: this work was sequenced by George Royal and analysed by Steve DeHaro Silvia Galván-Peña Aknowledgements First and foremost, I would like to thank my supervisor Luke O’Neill for giving me the opportunity to do my PhD in his lab and being a great mentor over the last four years. Never in a million years would I have chosen to work on malonylation, but I will be forever grateful for the rewarding challenge that this project has been. ‘It’ll be fine Silvia, just keep plugging away’ he’d say, and he was right. I’d also like to thank my two favorite lab buddies, Sarah and Richie. Sarah’s couch has been my second home for the last two years and she always has the right words of wisdom when I need them. I’m thankful for Richie too, who has a way of returning me to sanity when I start going crazy. Not sure I would have been able to get through this PhD without them. I’d also like to thank Moritz for his RNA-IP wisdom, even if he doesn’t share my hatred for them. I’d probably hate them even more if I he hadn’t had the patience to help me through them. The rest of the O’Neill lab as well, all the many that have left as well as the ones who are still there. They are all a wonderful bunch of people I have missed terribly during my time in GSK. To Emma Koppe for teaching me the GSK ways. I don’t think I would have survived my time in Stevenage without her, she has been invaluable. To Steve DeHaro and George Royal for sticking with me and letting me traumatise them for life with the RIPSeq. I have learnt a great deal from them. To Mike Rees, for his endless patience in teaching me mass spectrometry and to Ken Fantom for letting my ignorant self get her hands on his precious LC/MS machine. To Alan Nadin for his selfless generation of the MalAMyne compound. To this day, it has been the easiest, most painless and efficient collaboration I have ever been a part of. To Paul Stout, Brian Young and Lesley King for their readily supply of bone marrow, I would not be able to do my job in GSK without them. And of course, to all the members of the Immunology Catalyst for their continuous support. I’d also like to thank my parents. I have never been lucky with anything in life, except in getting the family that I did. I would not be who I am and be where I am if it wasn’t for them, and I will never be able to thank them for all the sacrifices they have had to make to get me here, even if they don’t fully understand what ‘here’ means. I don’t think they have managed to even say ‘malonylation’ successfully once, but you can’t blame them for trying. And finally, my biggest thank you of them all, to my best friend and wonderful better half. For putting up with my bad moods, accepting all the ‘sorry, I don’t have time today’, and for listening to me talk science garbage for endless hours. Airam, you have been the best support of them all. ‘I won’t give up, no I won’t give in, until I’ve reached the end, and then I’ll start again’ - Zootopia Table of contents List of figures Abbreviations Abstract Chapter 1: Introduction 7 1.1 The innate immune system and inflammation 8 1.1.1 Macrophages as key players in inflammation 9 1.1.2 Activation of macrophages through TLRs 10 1.2 Metabolic changes in macrophages 10 1.2.1 Citrate metabolism 12 1.2.2 Succinate metabolism 16 1.3 Metabolic regulation of post-translational modifications 17 1.3.1 Lysine succinylation and malonylation and their regulation by sirt5 21 1.3.1.1 Succinylation 23 1.3.1.2 Malonylation 24 1.3.1.3 Sirt5 26 1.4 Moonlighting properties of glycolytic enzymes 27 1.4.1 The multifunctional role of GAPDH 28 1.4.1.1 GAPDH and cell death 28 1.4.1.2 GAPDH and RNA-binding 30 1.4.1.3 GAPDH as a virulence factor 33 1.5 Aims 35 Chapter 2: Materials and Methods 36 2.1 Materials 37 2.1.1 Cell culture 37 2.1.1.1 Cell culture reagents 37 2.1.1.2 Cell lines 37 2.1.1.3 Primary cells 37 2.1.1.4 Stimuli 37 2.1.1.5 Inhibitors 37 2.1.2 Western blotting 37 2.1.2.1 Western blotting reagents 37 2.1.2.2 Western blotting antibodies 38 2.1.3 Immunoprecipitation reagents 38 2.1.4 Cu(I)-catalysed click chemistry reagents 38 2.1.5 RNA extraction and PCR reagents 38 2.1.6 Luminex reagents 38 2.1.7 Transfection and plasmid reagents 39 2.1.8 Miscellaneous reagents 39 2.2 Methods 40 2.2.1 Cell culture 40 2.2.1.1 Growth and maintenance of cell lines 40 2.2.1.2 Cryo-preservation of cells 40 2.2.1.3 Generation of mouse bone marrow-derived macrophages 40 2.2.1.4 Generation of human blood monocyte-derived macrophages 41 2.2.2 Western blotting 42 2.2.2.1 Preparation of cell lysates 42 2.2.2.2 Sodium Dodecyl Sulphate – Polyacrylamide gel electrophoresis (SDS-PAGE) 42 2.2.2.3 Electrophoretic transfer of proteins 43 2.2.2.4 Blocking of non-specific binding sites 43 2.2.2.5 Antibody probing 43 2.2.2.6 Testing of antibody specificity 44 2.2.2.7 Stripping and reprobing of PVDF membrane 44 2.2.2.8 Invitrogen western blot system 44 2.2.3 Protein immunoprecipitation 45 2.2.3.1 GAPDH and succinyl-lysine immunoprecipitation 45 2.2.3.2 Malonyl-lysine immunoprecipitation 46 2.2.3.3 Myc immunoprecipitation 46 2.2.4 BCA assay 46 2.2.5 Identification of malonylated proteins by liquid chromatography – mass spectrometry (LC/MS) 47 2.2.5.1 Trypsin in-gel digestion 47 2.2.5.2 LC/MS analysis 47 2.2.6 Identification of post-translational modification by LC/MS 48 2.2.6.1 Trypsin in-gel digestion 48 2.2.6.2 LC/MS analysis 48 2.2.7 MalAMyne Cu(I)-catalysed click chemistry 48 2.2.7.1 Labelling of cells with MalAMyne 48 2.2.7.2 Cu(I)-catalysed biotin click chemistry 49 2.2.7.3 Streptavidin affinity enrichment of biotinylated proteins 49 2.2.8 RNA analysis 49 2.2.8.1 RNA extraction using RNeasy kit 49 2.2.8.2 RNA extraction using QIAzol 49 2.2.8.3 Reverse Transcription PCR (RT-PCR) 50 2.2.8.4 Real Time quantitative PCR using Taqman® gene expression assays 50 2.2.8.5 Design and validation of SYBR primers 51 2.2.8.6 Real Time quantitative PCR using SYBR green 51 2.2.8.7 Data analysis 52 2.2.9 Enzyme Linked Immunosorbent Assay (ELISA) 52 2.2.10 Meso Scale Discovery (MSD) assay 52 2.2.11 Luminex cytokine assay 53 2.2.11.1 Antibody-bead pre-coupling 53 2.2.11.2 Luminex assay 53 2.2.12 Malonyl-CoA ELISA 54 2.2.13 GAPDH enzymatic assay 54 2.2.13.1 Enzymatic assay as absorbance versus time 54 2.2.13.2 Enzymatic assay as absorbance versus substrate concentration 55 2.2.13.3 Colorimetric GAPDH enzymatic assay 55 2.2.14 Cytotoxicity assay 55 2.2.15 RNA-Immunoprecipitation (RIP) 56 2.2.15.1 Immunoprecipitation 56 2.2.15.2 RNA isolation 56 2.2.16 RIP-Seq 57 2.2.16.1 Immunoprecipitation and RNA isolation 57 2.2.16.2 Library generation 57 2.2.16.2.1 Ribo-Zero deplete and fragment RNA 57 2.2.16.2.2 cDNA synthesis 58 2.2.16.2.3 3’ ends adenylation and adapter ligation 59 2.2.16.2.4 DNA fragments enrichment 59 2.2.16.3 Library quality check and quantification 60 2.2.16.4 Sequencing and analysis 60 2.2.17 Bacterial work 60 2.2.17.1 Bacterial transformation 60 2.2.17.2 Bacterial culture for minipreps and maxipreps 61 2.2.18 Site-directed mutagenesis 61 2.2.18.1 Mutagenesis reaction 61 2.2.18.2 Digestion of amplification products and transformation 62 2.2.18.3 Sequencing 62 2.2.19 Transfection 63 2.2.19.1 Plasmid transfection 63 2.2.19.2 siRNA transfection 63 2.2.20 Agarose gel electrophoresis 63 2.2.21 Cloning 63 2.2.21.1 Genomic amplification 64 2.2.21.2 DNA purification and digestion 64 2.2.21.3 Ligation reaction 64 2.2.22 Luciferase assay 65 2.2.23 Seahorse analysis of oxygen consumption 66 2.2.24 FLAG affinity chromatography 67 2.2.24.1 Resin preparation 67 2.2.24.2 Column chromatography and elution 67 2.2.25 Statistical analysis 67 Chapter 3: Results – Investigating the role of succinylation and malonylation in macrophages 68 3.1 Introduction 69 3.2 Characterizing the succinylation and malonylation profiles of activated macrophages 71 3.2.1 LPS increases protein succinylation 71 3.2.2 Other TLR ligands can also increase protein succinylation 71 3.2.3 LPS and R848 increase protein malonylation 76 3.2.4 Identification of LPS-induced malonylated proteins 76 3.3 Characterizing the succinylation and malonylation of GAPDH 84 3.3.1 LPS induces GAPDH succinylation 84 3.3.2 R848 is unable to induce succinylation of GAPDH 84 3.3.3 LPS induces GAPDH malonylation at lysine 213 89 3.4 Investigating the regulation of LPS-induced malonylation 95 3.4.1 LPS increases malonyl-CoA levels 95 3.4.2 Malonyl-CoA boosts IL10 production, while reducing TNFα and pro-IL1β in response to LPS 95 3.4.3 LPS modulates sirt5 expression levels 98 3.4.4 Sirt5 KO macrophages have impaired cytokine production in response to LPS 98 3.4.5 Blocking of transcription prevents the induction of malonylation by LPS 98 3.5 Discussion 103 Chapter 4: Results – Investigating the role of GAPDH in macrophages 108 4.1 Introduction 109 4.2 Investigating the role of GAPDH enzymatic activity 111 4.2.1 LPS increases GAPDH enzymatic activity 111 4.2.2 Inhibition of GAPDH enzymatic activity blocks cytokine production 111 4.2.3 Inhibiting glycolysis through 2DG has different effects to GAPDH inhibition alone.