Necrosome Activation and Degradation in Macrophages

Abstract

Necroptosis is a recently discovered form of cell death that is distinct from apoptosis that promotes inflammation. Necroptosis is induced upon engagement of various classical death receptors such as tumor necrotic factor receptor (TNFR) and Fas ligand, and also by engagement of toll like receptors involved in recognition of various pathogen associated molecular patterns such as lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (poly I:C). Necroptosis is induced by the activation of receptor-interacting protein kinase-1 (RipK1) and receptor-interacting protein kinase-3 (RipK3) that leads to phosphorylation-driven trimerization and consequent translocation of the mixed lineage kinase domain-like (MLKL) protein to the cell membrane, resulting in cell rupture. The formation of this necrosome complex is highly regulated by phosphorylation and ubiquitination. The precise mechanisms through which members of the necrosome assemble sequentially in macrophages are not clear. Herein I have evaluated the mechanisms that promote the assembly and degradation of the necrosome in macrophages. In the first aim of my thesis I revealed that during early necrosome formation RipK3 destabilizes the complex by promoting degradation of interacting proteins. RipK3 promoted K48-ubiquitin dependent proteasomal degradation of various necrosome interacting proteins such as RipK1, CASPASE-8, and FADD. This degradation event occurred independently of other ubiquitin editing enzymes such as cIAP1/2, A20 and CYLD that are known to edit RipK1. I further show that this degradation of early necrosomal proteins is mediated by the E3-ubiquiting ligase Triad3a (RNF216). Knocking-down Triad3a prevented degradation of necrosome proteins, enhanced necroptosis and production of inflammatory cytokines. In the second section of my thesis, I evaluated the mechanisms responsible for necrosome signalling following engagement of type-I interferon receptor. I show that the downstream Janus kinase (JAKs) kinases (TYK2 and JAK1) are required for necroptosis in macrophages. My results indicate that IFNAR1 signalling promotes phosphorylation of RipK3 and MLKL, the two events that are required for necroptosis. Furthermore, I observed that type-I interferon signalling promotes the transcription of MLKL and the phosphorylation driven oligomerization of MLKL which is necessary for pore formation in cells. Finally, inhibition of the p38MAPK pathway abrogated the resistance of Ifnar1-/- macrophages. The results presented in this thesis reveal mechanisms that regulate necrosome signalling and will help identify potential therapeutic targets that could be exploited in chronic inflammatory conditions.