β-arrestin 2 泛素化修饰调控ERK1/2通路在巨噬细胞抗结核免疫中的效应与机制

TLR下游ERK1/2通路是影响MTB感染结局的关键信号通路，其活性受支架蛋白β-arrestin 2（βarr2）严密调控，但被感染Mφ中βarr2对ERK1/2通路的调控效应与机制不明。课题组前期发现MTB感染上调βarr2，沉默βarr2可增强促炎因子表达及Mφ抑菌活性，该过程依赖βarr2泛素化调节的ERK1/2核转位。在此基础上，拟：①检测MTB感染Mφ中βarr2泛素化修饰特性及其对ERK1/2通路的调控效应；②筛查调节βarr2泛素化的E3泛素连接酶、去泛素化酶DUB及其相互作用蛋白；③检测MTB感染Mφ中TLRs对E3、DUB及其相互作用蛋白活性的调控；④验证βarr2与E3、DUB及其相互作用蛋白对MTB感染免疫的调控。项目将阐明βarr2泛素化修饰调控ERK1/2通路、进而调节Mφ抗结核免疫反应的效应与机制，为以βarr2为靶点的抗结核免疫新疗法的开发提供依据。

The mitogen-activated protein kinase extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway is downstream of Toll like receptors (TLRs) and critical for the outcome of Mycobacterium tuberculosis (MTB) infection, which is closely regulated by the scaffold protein β-arrestin 2. However, in MTB-infected macrophages, the effects and regulatory mechanisms of β-arrestin 2 on the ERK1/2 pathway is not clear. Our previous work showed that the expression of β-arrestin 2 was upregulated in response to MTB infection, and silencing β-arrestin 2 expression promoted the expression of proinflammatory cytokines and the antibacterial activity of macrophages. These processes are dependent on the nuclear translocation of phospho-ERK1/2 regulated by ubiquitination of β-arrestin 2. Therefore, we seek to screen the E3 ubiquitin ligases, deubiquitinases and their interacting proteins dynamically ubiquitinating β-arrestin 2 in MTB-infected macrophages, and investigate their effects on β-arrestin 2 ubiquitination and ERK1/2 activity. For this purpose, we intend to: i) analyze the characteristics of β-arrestin 2 ubiqutination in MTB-infected macrophages and detect the regulatory effects of β-arrestin 2 ubiqutination on ERK1/2 activity; ii) screen E3 ubiquitin ligases, deubiquitinases and their interacting proteins functioning in β-arrestin 2 ubiquitination; iii) detect the regulatory effects on the activity of E3 ubiquitin ligases, deubiquitinases and their interacting proteins by TLRs in MTB-infected macrophages; iv) verify the effects of β-arrestin 2, E3 ubiquitin ligases, deubiquitinases and their interacting proteins during the anti-MTB immune responses of macrophages. This study will elucidate the regulatory mechanisms of the ERK1/2 pathway and anti-MTB immune responses of macrophages by β-arrestin 2 ubiquitination, and establish the basis for development of novel therapy for tuberculosis by targeting β-arrestin 2.