新CYLD相互作用蛋白SPATA2对Toll样受体介导的信号通路和炎症小体激活的调控

Ubiquitination is a pivotal post-translational modification regulating immune and inflammatory responses and, when deregulated, contributes to the pathogenesis of various autoimmune and inflammatory diseases as well as cancer. The process of ubiquitination is tightly controlled by a large family of ubiquitin-specific proteases, termed deubiquitinases (DUBs). Due to their association with various human diseases, DUBs have become a promising source of therapeutic targets. However, successful development of DUB-based therapeutic drugs requires characterization of individual DUBs. CYLD is one of the critical DUBs regulating immune and inflammatory responses, but the molecular mechanisms underlying its functions remain poorly understood. This proposal is based on innovative preliminary findings from the PI’s group. Using the recently developed BioID screening approach, we identified a new CYLD-interacting protein, SPATA2. We generated SPATA2 knock-out mice and demonstrated that when challenged with the bacterial endotoxin LPS, these mutant animals displayed much higher level of serum pro-inflammatory cytokines, including TNF-α, IL-6, IL-1β and IL-18, and severer mortality, compared to their wild-type littermates. These data establish SPATA2 as a novel anti-inflammatory factor and suggest a ubiquitin-dependent mechanism involving the interplay between SPATA2 and CYLD. Based on our preliminary studies, we hypothesize that SPATA2 may regulate Toll-like receptor signaling and inflammasome activation through coordinating the DUB function of CYLD. To test this hypothesis, we will investigate how SPATA2 inhibits Toll-like receptor signaling and inflammasome activation at the molecular and cellular levels, and explore the role of SPATA2 malfunction in the onset and progression of inflammatory and autoimmune diseases using SPATA2 knock-out mice. We will also examine the role of SPATA2-CYLD interaction in the anti-inflammatory functions of SPATA2. The completion of this study would lead to identification and characterization of a new player in inflammatory and immune responses, thereby providing deeper insights into the mechanism underlying the action of CYLD. These findings will in turn be valuable for the design of new therapeutic strategies for the treatment of inflammatory and autoimmune diseases.

蛋白质泛素化受去泛素酶（deubiquitinase，DUB）严格调控。DUB功能紊乱与多种疾病相关，是极具前景的制药靶标，但药物开发依赖对DUB功能和调节机制的深入理解。CYLD是调节炎症和免疫反应的重要DUB，其活性和底物特异性调节机制尚不清楚。我们通过新方法BioID筛查发现了新的CYLD互作蛋白SPATA2，并构建了SPATA2敲除小鼠。用细菌内毒素LPS刺激小鼠时，敲除小鼠表现出比对照更高水平促炎细胞因子(TNF-α、IL-6、IL-1β和 IL-18)和更早死亡，表明SPATA2是个新抗炎症因子，可能依赖CYLD和去泛素化起作用。推测SPATA2通过CYLD调节TLR信号通路和炎症小体激活。将围绕这一假说从分子细胞水平揭示SPATA2作用机制，并用小鼠模型探讨其对炎症发生的作用。本项目开展有望鉴定一个新抗炎因子，加深对CYLD调节机制的认识，为炎症相关疾病治疗提供新策略和靶点。

NLRP3炎症小体是由多个蛋白组装而成的复合物。作为天然免疫的重要组成部分，它能激活Caspase-1，诱导细胞焦亡和白细胞介素1β（IL-1β）等促炎细胞因子的成熟与分泌，引发炎症反应。它的异常活化导致多种自身炎症性疾病，还与糖尿病和阿尔茨海默病等疾病密切相关。本项目运用BioID筛查去泛素化酶CYLD的相互作用蛋白时发现接头蛋白Spata2是新的CYLD结合蛋白。感染性休克模型造模显示敲除Spata2基因的小鼠对内细菌毒素LPS更加敏感，表现为更早死亡和更高水平的血清IL-1β和IL-18，提示Spata2抑制炎症小体活化。体外实验表明，Spata2特异性抑制多种信号诱导的NLRP3炎症小体的活化，CYLD也具有类似的抑制作用，而且Spata2与CYLD形成复合物发挥这种抑制作用。有意思的是，细胞定位实验显示Spata2主要定位于中心体，而且能够招募CYLD至中心体。进一步机制研究表明Spata2-CYLD与中心体激酶PLK4相互作用，通过去除PLK4 K63连接的泛素化促进PLK4与NLRP3的开关蛋白NEK7结合，造成NEK7的Ser204位点磷酸化；该磷酸化能够抑制NEK7与NLRP3的结合，从而负调控NLRP3炎症小体活化。该研究首次发现去泛素化酶CYLD 在接头蛋白Spata2的介导下对NLRP3炎症小体活化的调控作用，阐明了以PLK4-NEK7-NLRP3为核心的调控机制，揭示了中心体对炎症小体的重要调节作用，为NLRP3相关炎症疾病的治疗提供了潜在新靶点和策略。