转录因子硝基化修饰对Th17细胞可塑性分化的调控作用研究

T cells can differentiate into many subsets of different functions under different physiological and pathological conditions. The abnormalities of Th17 cell subsets are related to many major diseases such as tumor and autoimmune diseases. The post-translational modification of transcription factor plays an important role in the differentiation of Th17 cells. Among them, nitration modification, active in the tumor microenvironment, inflammatory local tissue, is an important factor of T cell differentiation under pathological condition. However, nitration modification is different from enzymatic phosphorylation or acetylation. It is NO-mediated nonspecific modification. Iit is difficult to obtain a specific structure of the nitro-modified protein by using common chemical methods or biological methods. As a result, it is difficult to study on function of nitro-modified protein;In this project, the protein biosynthetic element of nitrotyrosine can be transformed into T cells, which is pre-established in our lab, to establish a kind of protein expression system which can be regulated in T cells. To investigate the effects of nitrification on the differentiation of Th17 cells, such as RORγt, STATA3 and FOXP3, and to investigate the effect of nitrification on the differentiation of Th17 cells at the molecular level.This project will provide a basis for in-depth understanding of the pathogenesis of autoimmune diseases.

T细胞可以在不同生理病理条件下分化为许多不同功能的亚群，其中Th17细胞亚群的分化异常与肿瘤、自身免疫等许多重大疾病相关；转录因子翻译后修饰对Th17细胞分化有重要影响。硝基化修饰在肿瘤微环境、炎症局部组织中异常活跃，是病理状态下影响T细胞分化的一种重要因素。但硝基化修饰不同于酶催化的磷酸化、乙酰化等修饰，是NO介导的非特异性修饰，普通化学方法或生物学方法都很难获得特定结构的硝基化修饰蛋白，难以开展相关机理研究；本项目拟利用课题组前期建立的蛋白质定点硝基化修饰系统，将能识别硝基酪氨酸的蛋白质生物合成元件转化入T细胞中，建立一种可调控的，能在T细胞内对特定转录因子进行定点硝基化修饰的翻译系统，探讨RORγt、STATA3和FOXP3等关键转录因子的硝基化修饰对Th17细胞分化的影响，在分子水平阐明转录因子的硝基化修饰对Th17细胞分化的调控机制。为深入理解自身免疫性疾病的发病机理提供基础。

T细胞可以在不同生理病理条件下分化为许多不同功能的亚群，其中Th17细胞亚群的分化异常与肿瘤、自身免疫等许多重大疾病相关；转录因子翻译后修饰对Th17细胞分化有重要影响。硝基化修饰在肿瘤微环境、炎症局部组织中异常活跃，是病理状态下影响T细胞分化的一种重要因素。但硝基化修饰不同于酶催化的磷酸化、乙酰化等修饰，是NO介导的非特异性修饰，常规分子生物学方法难以对其动态修饰过程进行调控。本课题组以前期建立的非天然氨基酸引入平台为基础，设计并进化获得了能在大肠杆菌和哺乳动物细胞中定点引入3-硝基酪氨酸的表达系统；利用iNOS-/-基因敲除小鼠，验证了NO对Th17细胞的分化具有调节作用，并通过免疫共沉淀等方法确定了转录因子RORγt的硝基化对Th17细胞分化的影响；进一步结构分析结果显示，在转录因子RORγt中存在346位Tyr残基的硝基化修饰；利用腺病毒作为载体，将前期筛选的3-硝基酪氨酸引入系统和346位突变为TAG的RORγt转化至iNOS-/-鼠的naïve CD4+T细胞中，进一步验证了RORγt第346位酪氨酸硝基化对Th17细胞分化的抑制作用。本项目研究成果为细胞内动态硝基化修饰的研究提供新的工具和技术基础。