RIPK1泛素化调控细胞死亡分子机理的研究

The interaction of TNFa with TNF receptor-1 activates three downstream signaling pathways, including NF-κB, apoptosis and necroptosis. Necroptosis is a form of regulated necrosis, which depends on the kinase activity of the receptor interacting protein kinase 1 (RIPK1). Necroptosis is closely involved in human diseases such as ischemia reperfusion injury, inflammatory bowl diseases and neurodegenerative diseases. Previous studies have shown that RIPK1 is subject to extensive ubiquitination modification in TNFa signaling pathway which plays a very important role in regulating its activation and functions. However, it is still now unclear how ubiquitination of RIPK1 regulates its activation and function in two established TNF signaling complexes, TNFR1 complex I and complex II (necrosome), to medicate signaling switch from cell survival to cell death, and what ubiquitin ligases regulate ubiquitination of RIPK1 in these complexes. Based on our extensive experience in studying the signaling mechanism of necroptosis and my expertise in mass spectrometry, we plan to perform a systemic study on the function of RIPK1 ubiquitination. The aim of this study is to investigate the key ubiquitination sites and ubiquitination linkage types in TNFR1 complex and necrosome, discover the E3 ubiquitin ligases and ubiquitin binding proteins which regulates RIPK1 function using mass spectrometry. This study is supposed to uncover how ubiquitination of RIPK1 regulates cell death medicated by TNFa signaling pathway, providing insights into development of the diagnostic and therapeutic targets for Necroptosis related human diseases.

肿瘤坏死因子TNFa诱导的下游信号可分为三条通路，分别介导细胞的存活，凋亡和坏死。依赖于受体相互作用蛋白激酶RIPK1激酶活性的细胞坏死（Necroptosis）是一种可被调控的细胞死亡形式，且密切参与多种人类疾病。以往的研究表明，RIPK1的泛素化修饰参与调节TNFa下游的信号通路，决定细胞是生存还是死亡的不同命运。目前，RIPK1的泛素化修饰如何调控其在TNFR1复合体和坏死复合体中的功能，如何介导TNFa下游信号通路由生存转向死亡，以及其泛素化修饰被哪些泛素酶调节都尚未研究清楚。本项目拟对RIPK1泛素化位点的功能进行系统的研究，探索RIPK1在TNFR1复合体和坏死复合体中关键的泛素化位点和泛素化类型，寻找调控RIPK1泛素化的关键酶和泛素结合蛋白，揭示RIPK1泛素化对TNFa信号通路介导的细胞死亡的调控，为寻找治疗相关疾病的药物靶标提供有力的科学支持。

RIPK1是TNFR1、TLRs通路中调控细胞死亡和炎症的重要蛋白。RIPK1的激酶活性在TNFR1通路中发挥着促进死亡和炎症的作用，而它的支架功能发挥着促进存活的作用。是什么机制使RIPK1具有两种相反的功能目前尚不清楚。RIPK1蛋白本身具有多个泛素化位点，这些泛素化位点对于调控RIPK1激酶活性的激活以及是否影响RIPK1与其他蛋白相互作用并不清楚。本工作通过系统地突变RIPK1泛素化赖氨酸位点，筛选出人源RIPK1死亡结构域的627位赖氨酸以及其同源的鼠RIPK1612位赖氨酸对于介导RIPK1泛素化，RIPK1同源二聚及异源结合TNFR1，TRADD和FADD非常重要。本工作通过系统地突变RIPK1泛素化赖氨酸位点，筛选出人源RIPK1死亡结构域的627位赖氨酸以及其同源的鼠RIPK1612位赖氨酸对于介导RIPK1泛素化，RIPK1同源二聚及异源结合TNFR1，TRADD和FADD非常重要。通过质谱分析RIPK1肽段的泛素化修饰，我们发现人RIPK1 K627及鼠RIPK1 K612的泛素化调控RIPK1的整体泛素化模式，促进RIPK1 N端蛋白激酶结构域和中间结构域泛素化水平，抑制死亡结构域的泛素化。RIPK1 K612R突变抑制了RIPK1-DD与RIPK1-DD的同源二聚，以及RIPK1-DD与TNFR1-DD,TRADD-DD 和FADD-DD的异源二聚。我们发现K612R突变抑制TNF刺激引起的RIPK1的激活，从而抑制RIPK1依赖的细胞凋亡和坏死。然而，RIPK1K612R/K612R骨髓来源的巨噬细胞在TLR3/4通路中促进RIPK3的激活及RIPK3/RIPK1/MLKL复合物的形成，促进Caspase-1的剪切，IL-1beta的释放以及炎症反应。人源RIPK1 K627及鼠源RIPK1 K612的泛素化在TNFR1和TLRs信号通路中起到了不同的调控功能。本工作突显了RIPK1 DD和hRIPK1 K627/mRIPK1 K612泛素化对于调控RIPK1激活的重要作用，揭示了细胞的不同死亡形式包括凋亡，坏死和caspase-1激活之间进行交互作用的分子机制。