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The Role of PGAM5 in Regulating Viral Infection and the Pathogenesis of Intestinal Inflammation

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The Role of PGAM5 in Regulating Viral Infection and the Pathogenesis of Intestinal Inflammation The role of PGAM5 in regulating viral infection and the pathogenesis of intestinal inflammation Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Yuqiang Yu 1 Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 1st July 2021 Vorsitzender des Promotionsorgans: Prof. Dr. Wolfgang Achtziger Gutachter/in: Prof. Dr. Falk Nimmerjahn Prof. Dr. Christoph Becker 2 Table of contents Table of contents 1 Abstract .................................................................................................................. 6 2 Zusammenfassung................................................................................................. 7 3 Introduction ............................................................................................................ 9 3.1 PGAM5 ............................................................................................................. 9 Structure and expression ........................................................................... 9 PGAM5 acts as a regulator in cell death pathways................................... 10 The role of PGAM5 in Wnt/β-catenin signaling pathway ........................... 14 PGAM5 in the NLRP3 inflammasome ...................................................... 14 PGAM5 activity is linked to multimerization .............................................. 15 3.2 Cellular defense against viruses ...................................................................... 16 Toll-like receptors ..................................................................................... 16 RIG-I-like receptors .................................................................................. 19 MAVS is a key factor in the RLR pathway ................................................ 20 IFNs and antiviral responses .................................................................... 23 Modeling RNA viral infection .................................................................... 24 3.3 IBD .................................................................................................................. 25 3.4 Aims of the project .......................................................................................... 26 4 Material and Methods ........................................................................................... 27 4.1 Materials ......................................................................................................... 27 4.2 Animals .......................................................................................................... 31 4.3 Cell line .......................................................................................................... 31 4.4 Methods ......................................................................................................... 32 MEF isolation ........................................................................................... 32 Generation of HeLa knock-out cells using CRISPR/Cas9 technology ...... 32 PGAM5 overexpression ........................................................................... 33 Cell stimulation and infection .................................................................... 33 Genotyping .............................................................................................. 33 Dextran sulfate sodium (DSS)-induced colitis ........................................... 35 3 Table of contents Gene expression analysis ........................................................................ 35 Protein analysis ........................................................................................ 36 Histological analysis ................................................................................. 40 Immunocytochemistry .............................................................................. 42 Statistical analysis .................................................................................... 43 5 Results .................................................................................................................. 44 5.1 PGAM5-MAVS interaction regulates TBK1/ IRF3 dependent antiviral responses 44 Intracellular poly(I:C) delivery induces the formation of PGAM5 multimers 44 PGAM5 deficiency attenuates intracellular poly(I:C)-induced IFNβ expression .............................................................................................................. 48 PGAM5 overexpression rescued IFNβ expression in PGAM5 deficient HeLa cells 52 PGAM5 functions upstream of TBK1 ........................................................ 54 The formation of PGAM5 multimers and MAVS aggregates are independent of each other .......................................................................................................... 57 PGAM5 interacts with MAVS .................................................................... 59 PGAM5 regulates VSV-induced IFNβ expression and inhibits VSV replication ............................................................................................................... 60 5.2 PGAM5 regulates IFN-stimulated genes (ISG) expression .............................. 63 PGAM5 regulates extracellular poly(I:C)-induced STAT1 phosphorylation. 63 PGAM5 deficiency up-regulates extracellular poly(I:C)-induced SOCS3 expression. ............................................................................................................. 66 PGAM5 positively regulates IFN-stimulated STAT1 phosphorylation and downregulates SOCS3 expression. ........................................................................ 67 5.3 PGAM5 is dispensable for poly(I:C)-induced small intestinal inflammation. ..... 71 5.4 PGAM5 is dispensable for the development of experimental colitis. ................ 74 6 Discussion ............................................................................................................ 77 6.1 PGAM5 regulates IFNβ expression and antiviral response. ............................. 77 4 Table of contents 6.2 The mechanisms of PGAM5-dependent antiviral pathways. ............................ 79 6.3 PGAM5 is dispensable for multiple organ injury induced in vivo. ..................... 83 6.4 Working model ................................................................................................ 84 7 References ............................................................................................................ 85 8 List of abbreviations. ......................................................................................... 100 9 List of Tables. ..................................................................................................... 103 10 List of figures ..................................................................................................... 104 11 Curriculum vitae. ......................................................... Error! Bookmark not defined. 12 Conference Presentations ................................................................................. 106 13 Declaration ......................................................................................................... 107 5 Zusammenfassung 1 Abstract Viral infections trigger host innate immune responses, characterized by the production of type-I interferons (IFN) including IFNβ. IFNβ induces cellular antiviral defense mechanisms by expressing IFN-stimulated genes (ISG), thereby contributing to pathogen clearance. Accumulating evidence suggests that mitochondria constitute a crucial platform for the induction of antiviral immunity and cell death. The mitochondrial protein phosphoglycerate mutase family member 5 (PGAM5) has been implicated in a broad range of biological processes including certain cell death pathways and NLRP3 inflammasome activation. The hypothesis of this thesis was that PGAM5 is involved in regulating cellular immune defense and cell death in the gut. Thus, this thesis aimed to investigate functional roles of PGAM5 via in vitro and in vivo models. Initially, poly(I:C) was used to mimic RNA virus infection in HeLa cells and the presence of intracellular RNA leads to PGAM5 multimer formation and co-localization at aggregated mitochondria. Furthermore, this thesis showed that PGAM5 deficiency specifically attenuated IFNβ expression induced by intracellular poly(I:C) but not when poly(I:C) was added into the medium. Decreased phosphorylation levels of IRF3 and TBK1 in PGAM5 deficient cells further confirmed these finding. On the molecular level, a direct interaction of PGAM5 with the mitochondrial antiviral-signaling protein (MAVS) was demonstrated. Finally, this thesis verified the functional role of PGAM5 in the process of viral infection. PGAM5 deficient cells, upon infection with vesicular stomatitis virus (VSV), revealed diminished Ifnβ expression and increased VSV replication. In addition, this thesis also demonstrated that PGAM5 is important for regulating ISG responses. PGAM5 deficient cells exhibited decreased phosphorylation levels of STAT1 and expression of ISG when challenged with three different types of IFNs. Mechanistically, PGAM5 deficiency significantly up-regulated SOCS3 expression. These in vitro data supported the initial hypothesis about the functional role of PGAM5 in cellular immune defense. However, the hypothesis about cell death in gut could not be proved in vivo. PGAM5 is
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