RIP1蛋白的氧化和磷酸化调控的体内功能研究

Necroptosis is a type of programmed cell death involving in many pathological processes, including bacterial or viral infection, and sterile inflammation. RIP1 (Receptor interacting serine/threonine kinase 1) is an important intracellular signaling mediator and one of its role is regulation of necroptosis. We have demonstrated that the upregulated mitochondrial ROS by RIP3 in tumor necrosis factor-α (TNF) treated cells directly oxidize three specific cysteines in RIP1, which leads to RIP1 autophosphorylation on S161. The S161 phosphorylated RIP1 promotes formation of RIP1-RIP3 necrosome and subsequent necroptosis. The RIP3-ROS-RIP1-RIP3 signaling circle in the pathway of necroptosis is a positive feedback loop, and the proposed research here is to understand its in vivo function. This project will generate RIP1 S161N, RIP1 S161E and RIP1 3CS mutant mice and address the following questions: 1) we will focus on the physio-pathological function of RIP1 S161 phospho-defect/mimic mutation or ROS insensitive mutation in mice and the underlying mechanisms; 2) we will compare the lethality and pathogenicity phenotype of RIP1 knockout (KO) mice with these three mutant strains and thus distinguish different in vivo functions of RIP1; 3) we will cross these mutant stains with “death-related” KO mice to further elucidate the function of RIP1 oxidation and S161 phosphorylation. This project will bring new insight into the function of necroptosis and provide new ideas for development of new treatment of necroptosis-related diseases.

程序性细胞坏死（Necroptosis）高度受调控且参与到了机体的多种病理过程中。RIP1是调控Necroptosis的一个重要分子。我们的研究表明，RIP3在被TNF激活后所上调的线粒体ROS可以直接特异地氧化RIP1，促进RIP1在S161位点自磷酸化，进而促进RIP1-RIP3坏死小体形成和Necroptosis发生。这是一个由RIP3-ROS-RIP1-RIP3组成的正反馈环，其体内功能是本项目研究的内容。本项目将构建出三株突变型小鼠：1）探索RIP1 S161位点自磷酸化的缺失/获得，或者其对ROS感应的缺失对小鼠生理病理功能的影响及其分子机制；2）通过与RIP1 KO小鼠的对比，分解RIP1的不同功能；3）通过与死亡相关基因的KO小鼠杂交，以期定位RIP1氧化/S161自磷酸化介导的Necroptosis的体内功能。本项目的开展，对全面解析程序性坏死并协助疾病治疗具有重要意义。

本项目是项目负责人前期工作的延续（2017发表于Nature Communications）。它以小鼠作为研究对象，探究了RIP1蛋白的三种突变形式（RIP1 3CS，RIP1 S161E，RIP1 S161N）对小鼠在生理病理上的影响和潜在的作用机制。本项目发现，1）将RIP1的三个与介导程序性细胞坏死相关的半胱氨酸突变为丝氨酸后（RIP1 3CS），小鼠出现了围产期死亡的表型，进一步研究发现，3CS纯合突变小鼠在各组织均出现了病变，且这种致死性可以被Casp8和RIP3的双敲除或者Casp8和Mlkl的双敲除所挽救。2）RIP1 S161E的纯合突变小鼠对TNF引起的SIRS更加敏感，此敏感性主要来源于体细胞，且是由Caspase-8和RIP3同时介导的。3）RIP1 S161N的纯合突变小鼠能抵抗TNF引起的SIRS，且此抗性是由体细胞介导的。本项目通过对RIP1的三个突变小鼠品系的研究，揭示了RIP1蛋白在小鼠正常发育过程中和在系统性炎症反应过程中的重要作用，为治疗由RIP1介导的各种疾病提供了理论依据。