TNF信号诱导的细胞坏死参与自身免疫性疾病、阿尔茨海默氏症等多种疾病过程。我们前期工作发现细胞坏死小体(RIP1/RIP3)形成功能性淀粉样纤维复合物(Cell 2012; Biochemistry 2014)。但坏死小体的精细结构以及在神经退行性疾病中的具体作用机理仍不清楚。申请人近期已获得RIP1、RIP3核心区域的高分辨率晶体结构，并且发现坏死小体在体外及细胞内与神经退行性疾病相关蛋白aβ（aβ-40及aβ-42）发生相互作用。本项目拟进一步通过X-光晶体衍射及冷冻电子显微镜来解析坏死小体蛋白复合物的三维结构；通过荧光极化、定点突变、ITC等方法研究坏死小体组装和精细调控的分子机制；运用免疫组织化学、免疫共沉淀等手段，在PC-12细胞及APP/PS1转基因小鼠中，研究坏死小体与aβ蛋白相互作用的分子机理；最终阐释细胞坏死发生的分子机制及与神经退行性疾病交互对话的作用机理。

The tumor necrosis factor (TNF) induced cell necrosis plays key roles in human diseases such as autoimmunity diseases, Alzheimer’s disease and so on. Our previous work showed that the necrosome RIP1/RIP3 forms a functional amyloid complex (Cell 2012; Biochemistry 2014). However, the structural basis of necrosome and its relationship with neurodegenerative diseases are not clear. We recently solved the high-resolution crystal structures of the key regions of RIP1/RIP3 complex, and found that RIP1/RIP3 interacted with Alzheimer’s disease related proteins aβ-40 and aβ-42 in vitro..In this proposal, we will study the structural basis of RIP1/RIP3 complex by using the combination of X-ray crystallography and cryo-EM, unveiling the molecular mechanism of RIP1/RIP3 self-assembly and regulation by using fluorescence polymerization, site specific mutagenesis and isothermal titration calorimetry methods. In addition, we will carry out the functional studies for the interaction between RIP1/RIP3 and aβ proteins in PC-12 cell line and APP/PS1 transgenic mouse using immunochemistry and co-immunoprecipitation methods, eventually benefiting our understanding for the molecular mechanism of cell necrosis, and cross-talk relationship between necrosis and neurodegenerative diseases.