Molecular Mechanisms of Programmed Necrosis Execution

项目摘要 坏死性凋亡是一种不依赖半胱天冬酶的程序性坏死。的激活 坏死性凋亡信号级联与多种人类疾病的发病机制有关， 包括癌症、炎症性肠病、肝损伤、胰腺炎、神经退行性疾病 疾病，以及各种病毒，细菌和真菌感染，包括SARS-CoV-2。 坏死性凋亡信号级联是由RIPK 1和RIPK 3的顺序激活介导的 促炎配体下游的激酶，如TNF α或微生物相关的 分子。MLKL是一种假激酶，其在被RIPK 3磷酸化后四聚化， 可渗透水的小孔，促使细胞膜破裂。该孔隙形成阶段导致 坏死性凋亡的坏死表型。它也是决定细胞命运的关键点， 可以在MLKL阶段停止和逆转坏死性凋亡的执行。的机制 调节MLKL激活和执行这种类型的程序性坏死是很差的 明白在这里，我们将填补我们对分子机制的理解的空白， 调节MLKL活化、四聚化和坏死性凋亡细胞死亡， 磷酸化和泛素化。我们的目标是确定MLKL的机械作用 促进或抑制MLKL四聚化的翻译后修饰事件， 鉴定通过这些事件调节MLKL驱动的坏死细胞死亡的酶。我们还旨在 确定MLKL下游需要哪些结构因素来执行 坏死性细胞死亡最后，为了验证这些酶和因子在介导 在体内坏死性凋亡，我们将测试他们的基因敲除如何影响对牛痘病毒的敏感性 感染，有助于未来发展的战略，提高宿主抗病毒 反应总的来说，这个项目将大大扩展我们对细胞的理解。 在坏死性凋亡执行阶段MLKL上游和下游的信号传导机制 并为未来的抗微生物疗法以及治疗 包括坏死性下垂的执行。

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.