Molecular Mechanisms of Programmed Necrosis Execution

PROJECT SUMMARY Necroptosis is a caspase-independent type of programmed necrosis. The activation of the necroptosis signaling cascade is implicated in the pathogenesis of various human diseases, including cancer, inflammatory bowel disease, liver injury, pancreatitis, neurodegenerative disorders, and a diverse range of viral, bacterial, and fungal infections, including SARS-CoV-2. The necroptosis signaling cascade is mediated by the sequential activation of RIPK1 and RIPK3 kinases downstream of pro-inflammatory ligands such as TNF or microbe-associated molecules. MLKL is a pseudokinase that tetramerizes upon phosphorylation by RIPK3 to form water-permeable pores that drive cell membrane rupture. This pore formation stage leads to the necrotic phenotype of necroptosis. It is also a critical point of cell fate determination, as necroptosis execution can be halted and reversed at the MLKL stage. The mechanisms regulating MLKL activation and execution of this type of programmed necrosis are poorly understood. Here, we will fill in the gaps of our understanding of the molecular mechanisms that regulate MLKL activation, tetramerization, and execution of necroptotic cell death via phosphorylation and ubiquitination. We aim to determine the mechanistic roles of the MLKL post-translational modification events in promoting or suppressing MLKL tetramerization and identify the enzymes regulating MLKL-driven necrotic cell death via these events. We also aim to determine which structural factors are required downstream of MLKL to execute the necroptotic cell death. Finally, to validate the roles of these enzymes and factors in mediating necroptosis in vivo, we will test how their genetic knockouts affect sensitivity to Vaccinia virus infection, contributing to the future development of strategies for enhancing host anti-viral response. Overall, this project will significantly expand our understanding of the cellular signaling mechanisms upstream and downstream of MLKL at the necroptosis execution stage and pave the way for future anti-microbial therapies, as well as treatments for diseases that involve necroptosis execution.