RelA依赖于甲基化的泛素化对NF-κB功能及固有免疫反应的负调控作用

NF-κB是一类重要转录因子，调节炎症、免疫反应、细胞存活和肿瘤发生。NF-κB信号通路失调会造成炎症和肿瘤等多种疾病。目前对NF-κB调控机制，尤其是负调控机制认识尚不全面。申请人最近的工作首次发现NF-κB受甲基化负调控，甲基转移酶Set9介导的RelA亚基的甲基化会引发RelA泛素化和降解，揭示了“甲基化-泛素化-蛋白降解”这一新的NF-κB负调控路径，但甲基化引起泛素化的机制还不清楚。我们推测一个甲基化依赖的泛素连接酶E3介导这一过程。本研究通过体外筛选初步发现了一个多亚基E3（ECS-WSB1）能特异性识别并泛素化甲基化的RelA。在此基础上，我们拟在分子和细胞水平上研究该E3识别甲基化RelA并介导其泛素化的机制及它对NF-κB功能的调节作用；还将用CRISPR技术在巨噬细胞中敲除该E3，分析它对巨噬细胞固有免疫应答的调节作用，期望为炎症和肿瘤等疾病的防治提供新策略和新靶点。

NF-κB is a family of transcription factors that play critical roles in regulating inflammatory and immune responses, cell survival, and tumorigenesis. Deregulation of NF-κB signaling pathway has been implicated in many diseases such as inflammatory diseases and cancer. Mechanisms regulating NF-κB activation, particularly negative mechanisms, are incompletely understood at the present time. Recently, we have demonstrated for the first time that NF-κB is down-regulated by methylation, and that methylatransferase Set9-mediated methylation of the RelA subunit causes ubiquitination and degradation of RelA, establishing methylation-ubiquitination-degradation as a previously uncharacterized cascade for negative regulation of NF-κB. However the molecular mechanism underlying the methylation-dependent ubiquitination remains elusive. We hypothesize that a methylation-dependent ubiquitin ligase E3 mediates this process. In a biochemical screen we have discovered a multi-subunit E3 (ECS-WSB1) that can specifically recognize methylated RelA and promote its ubiquitination. Based on these preliminary findings, we propose to study how this E3 ubiqitinates RelA in a methylation-dependent manner and how it plays a role in regulating NF-κB action at the molecular and cellular level. Furthermore, we will generate knockout of this E3 in THP1 macrophage cells by using the newly developed CRISPR/CAS9 technique and explore the regulatory role of this E3 in the innate immune response of macrophage cells. This study may provide new therapeutic strategies and targets for the prevention and treatment of inflammatory diseases and cancer.