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。

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.