去泛素化酶USP1调控NLRP3炎症小体活化及其分子机制

NLRP3 inflammasome is a multi-protein platform which comprises NLRP3, ASC and caspase-1 and plays crucial roles in multiple diseases. Investigation of the regulatory mechanisms of NLRP3 inflammasome activation has important scientific significance and application value in elucidating the mechanism of the immune responses and developing novel ways of immunomodulatory therapy. We identified a candidate protein, deubiquitination enzyme USP1, which might interact with NLRP3 by co-immunoprecipitation (Co-IP) and liquid chromatography coupled with tandem mass spectrometry. An association between USP1 and NLRP3/ASC could be detected using Co-IP. USP1 siRNA knockdown and specific inhibitor treatment both greatly inhibited NLRP3/ASC protein expression and IL-1β secretion. These data suggested that USP1 played crucial roles in the activation of NLRP3 inflammasome and NLRP3/ASC may be the targets of USP1. However, the exact mechanisms of USP1 in regulating NLRP3 inflammasome activation and the potential roles of USP1 in NLRP3 inflammasome related diseases remain unknown. In the present proposal, we plan to use varieties of molecular and immunology techniques and several disease models (such as alum or monosodium urate induced acute peritonitis, folic acid-induced acute tubular necrosis, etc.), to explore the functions of USP1 in the regulation of NLRP3 inflammasome activity, reveal the possible molecular mechanisms (whether USP1 regulates NLRP3 inflammasome activation via deubiquitinating NLRP3 and ASC), clarify the roles of USP1 in NLRP3 inflammasome related diseases and supply theoretical basis for immunomodulatory therapy.

NLRP3炎症小体是由NLRP3、ASC和caspase-1组成的分子平台，其活化强度与多种疾病的发生密切相关；其调控机制研究对阐明免疫反应产生及调控的机理，寻找免疫调节疗法的新途径具有重要意义。我们筛选出新的NLRP3结合蛋白去泛素化酶USP1；免疫共沉淀证实USP1可特异性结合NLRP3和ASC；USP1干扰及抑制剂均显著抑制NLRP3、ASC蛋白表达及IL-1β分泌；提示USP1在NLRP3炎症小体活化中发挥作用，且靶点可能是NLRP3和ASC。但具体调控机制及其在炎症小体相关疾病中的作用尚不清楚。本研究拟利用分子生物学、免疫学技术及急性腹膜炎、急性肾小管坏死等疾病模型，探讨USP1在NLRP3炎症小体活化调控中的作用并阐明其分子机制（即其调节作用是否通过去除NLRP3和ASC的泛素化实现）；探讨USP1在NLRP3炎症小体相关疾病中的作用，为探寻相关疾病的免疫调节疗法提供理论依据。

NLRP3炎症小体活化失衡将导致多种疾病的发生，如感染性疾病、痛风、动脉粥样硬化、阿尔茨海默氏病及癌症等。因此，深入揭示NLRP3炎症小体活化的调控机制，对于阐明相关疾病发生、发展的机理，寻找免疫调节治疗的新途径具有重要意义。NLRP3表达是NLRP3炎症小体活化的限速步骤，也是调控的重要方式。在本项目资助下，我们以巨噬细胞为研究模型，对NLRP3的调控机制进行研究，并取得了阶段性成果。负责人作为通讯作者在The Journal of Experimental Medicine、Nature Communications、PNAS、Cell Reports、The Journal of Immunology等杂志发表论文7篇；2016年获得国家自然科学基金优秀青年基金资助；2016年入选山东大学首批齐鲁(仲英)青年学者；2018年获得国家自然科学基金委和英国医学科学院牛顿高级学者项目资助。.我们发现UAF1通过募集USP1抑制NLRP3泛素化修饰，抑制其降解；通过募集USP12/46抑制p65泛素化降解，促进NLRP3表达；进而促进NLRP3炎症小体活化(图1)。UAF1-USP1特异性抑制剂ML323抑制NLRP3炎症小体活化，并缓解NLRP3相关炎症性疾病。E3泛素连接酶TRIM31可介导NLRP3发生K48偶联的泛素化修饰，促进其通过蛋白酶体途径降解，进而抑制NLRP3炎症小体活化。这一系列研究成果对于阐明NLRP3相关疾病发生、发展的机理，寻找免疫调节治疗的新途径具有重要意义。