NLRP3磷酸化调控炎症小体激活的功能和机制研究

Inflammasomes are key signaling platforms that play a critical role in innate immunity. NLRP3 inflammasome, which is comprised of the NOD-like intracellular receptor, NLRP3, the adaptor protein, ASC, and the inflammatory caspase, caspase-1, is quickly assembled in response to both the infection- and damage-derived signals. NLRP3 inflammasome thus acts as a key signaling pathway and protects host from invading pathogens and sterile tissue damages by mediating the immune recognition and inflammatory responses. Although NLRP3 has been intensively studied, the detailed mechanisms underlying the activation of NLRP3 inflammasome is still elusive. Reveal the precise regulation of NLRP3 activation provide more insight to understand the innate immune responses and the inflammatory diseases. To do so, we have performed a MS-based identification of the PTM of NLRP3, and found that NLRP3 is phosphorylated during the activation of NLRP3 inflammasome at a highly conserved serine site. Our preliminary study suggested that the phosphorylation of this serine site is critical for NLRP3 activation. Importantly, with the knock-in mice bearing the NLRP3 phosphorylation site mutatant, we demonstrated that this phosphorylation of NLRP3 is essential for the activation of NLRP3 inflammasome. Based on this, we propose to study the role of NLRP3 phosphorylation in regulation of inflammasome activation: i) to observe the phosphorylation kinetics with site-specific antibody; ii) to analysis the kinase for NLRP3 phosphorylation and to investigate the function of the kinase in regulating NLRP3 inflammasome; iii) to explore the mechanism that how this phosphorylation regulate NLRP3 inflammasome, including the complex assembly, the release of pro-inflammatory cytokines and pyroptosis, etc.; iv) use the in vitro reconstitution of NLRP3 assay to evaluate the function of phosphorylation-deficient mutation of NLRP3; finally, with the phosphorylation-site-mutant knock-in mice and the NLRP3-mutation-derived autoimmune disease mice, we are planning on evaluating the physiological function of NLRP3 phosphorylation in vivo. Thus, our study will reveal a novel mechanistic evidence that this phosphorylation on NLRP3 is critical for inflammasome activation and is important for the further study of NLRP3 inflammasome-related human diseases.

NLRP3炎症小体在介导机体免疫识别和炎症反应中发挥核心作用，其调控异常导致自身免疫和肿瘤等重要疾病的发生。因此，NLRP3炎症小体激活调控是免疫学前沿领域的关键科学问题。我们利用质谱发现炎症小体激活过程中NLRP3蛋白质的一个磷酸化修饰并通过构建该修饰位点突变的基因敲入小鼠证明该磷酸化为NLRP3炎症小体激活所必需。据此重要发现，本项目拟深入阐明磷酸化调控NLRP3炎症小体激活的机制及其生物学意义：通过位点特异抗体分析NLRP3磷酸化的发生规律；鉴定相应激酶并研究其对炎症小体的调控作用；通过研究炎症小体组装、炎症因子释放、细胞焦亡等过程解析该磷酸化如何调控炎症小体激活；利用上述基因敲入小鼠及NLRP3突变所致免疫疾病小鼠，在整体水平评价NLRP3磷酸化的生理作用。综上，本研究将揭示NLRP3磷酸化调控炎症小体激活的新机制，回答免疫学领域的关键问题并为相关疾病的治疗研究提供重要科学依据。

机体对病原微生物的快速识别是固有免疫系统产生抗感染免疫反应的关键环节之一。研究发现，模式识别受体是天然免疫系统的关键感受器，能够识别来自不同病原体的特定分子模式，从而迅速启动机体的免疫反应抵抗病原体感染。.NLRP3是NLR模式识别受体家族中研究最为广泛的受体之一，能够识别病原体及组织损伤相关的分子模式。我们的研究发现磷酸化是NLRP3炎症小体预激活阶段（Priming）的关键分子事件，证明了该修饰在NLRP3炎症小体激活中的重要调控作用，拓展了人们对固有免疫应答机制的理解与认识，为NLRP3介导自身免疫综合征等疾病提供了潜在治疗靶点。.蛋白质cGAS近年被揭示是胞质DNA的关键感受器，能够感知胞质DNA并激活免疫反应，是宿主免疫系统识别病毒感染的重要模式识别受体。研究表明，cGAS不仅在抗病毒免疫反应中发挥了关键作用，其功能的异常激活也是一类自身免疫疾病发生的主要原因。因此，寻找有效控制cGAS活性的手段并探究其调控机理，是针对这类疾病发展治疗手段的关键。我们的研究从cGAS的调控机理解析入手，发现了乙酰化修饰对cGAS活性的调控作用并揭示了其调控规律。研究鉴定到，当cGAS的K384、K394和K414三个赖氨酸位点中任何一个位点发生乙酰化修饰时，均会使cGAS失去活性。进而，我们发现阿司匹林可以使cGAS在上述关键位点上发生乙酰化从而抑制其活性。此外，我们还鉴定到了cGAS的关键调控蛋白质——G3BP1。机制研究表明G3BP1与cGAS相互作用，能够促进cGAS发生寡聚化进而更高效地结合DNA。缺失G3BP1显著抑制细胞中cGAS的活性。重要的是，我们发现天然化合物EGCG（来源于绿茶）能够通过破坏cGAS-G3BP1寡聚物，抑制cGAS激活。综上所述，我们的发现不但揭示了DNA感受器cGAS的关键调控机制，还发现了针对该靶点的有效抑制剂，为艾卡迪综合征等无药可治的自身免疫疾病的治疗提供了潜在治疗策略。