p38IP对T细胞受体信号通路及Treg细胞发育的调节和机制

The negative regulators in TCR signaling have a central role in appropriate activation of T cells, maintenance of T cell homeostasis, tolerance and prevention of autoimmune disease. Our preliminary data showed that p38IP associated with TAK1 in resting T cells, TCR stimulation induced a quick dissociation and then a stronger association between p38IP and TAK1. And p38IP inhibited TCR-induced TAK1 ubiquitiation and IL-2 production. Therefore, we propose that p38IP negatively regulates TCR signaling via targeting TAK1. We will study the detail mechanism underlying TCR-induced dynamical association between p38IP and TAK1. By using MS/MS and western blot analysis, we will analyze the proteins associate with p38IP and TAK1 before and after TCR stimulation, and try to find if there is a potential deubiquitinase involved in p38IP-mediated inhibition of TAK1 ubiquitiation. Then, we will check how p38IP regulates the association between TAK1 and its binding proteins TABs and the potential deubiquitinase. Next, we will map the domains on p38IP that are responsible for binding with TAK1 or inhibition of TAK1 ubiquitination before and after TCR stimulation, and analyze their role in regulation of TCR-induced TAK1 dynamical activation and T cell activation. Furthermore, we will make the T-cell-conditional p38IP-knockout mice and check T cell development. By the retroviral transduction of bone marrow and bone marrow transplantation and reconstitution, we will demonstrate the function and mechanism of p38IP in Treg development and maturation. Through this study, we hope to find a novel mechanism in regulation of TCR signaling and Treg cell development.

TCR信号通路中负调控因子确保T细胞被恰当激活,维护T细胞自稳耐受及防止自身免疫性疾病发生.预研究表明静息T细胞中p38IP与激酶TAK1结合,TCR刺激下先解离再更强结合且抑制TAK1泛素化,推测p38IP经TAK1负调节TCR信号通路.将研究p38IP动态调节TAK1的具体机制,经质谱及western分析静息及刺激时p38IP及TAK1蛋白复合物组成,探究p38IP对TAK1与其结合蛋白TABs等结合的调节,寻找p38IP可能利用的去泛素化酶;靶定p38IP在静息及刺激时起抑制作用的功能域,比较p38IP及其抑制功能域对TCR诱导TAK1活性动态和T细胞活化的调节.制作T细胞条件性p38IP敲除小鼠,分析p38IP对T细胞发育包括Treg发育的调节;籍逆转录病毒载体及骨髓移植重建,比较p38IP及其抑制功能域对Treg发育的调节.诠释调节TCR信号通路及Treg发育的新机制.

免疫受体信号的负调控是防止免疫系统过度活化和维持免疫稳态的重要机制，发现新的免疫受体信号的负调控因子将为干预治疗相关疾病提供新思路。 在本研究中，我们采用的主要方法有高通量RNA测序分析受p38IP调节的细胞因子、质谱分析蛋白质复合物、免疫共沉淀及western blot分析p38IP与TAK1及TABs蛋白相互作用及动态结合、体外激酶反应鉴定p38IP对TAK1活性的影响、流式及Elisa鉴定细胞因子的生成、体外泛素化反应鉴定p38IP结合的去泛素化酶，以及风湿病关节炎患者PBMC中分析p38IP的表达及其与相关信号蛋白的关联等。我们发现p38IP蛋白抑制多种免疫受体信号通路，其中包括TCR受体信号通路和LPS信号通路，且在自身免疫疾病类风湿关节炎患者外周血单个核细胞中p38IP蛋白水平显著下调。研究进一步揭示，p38IP采用双重调控方式抑制免疫受体信号通路中的关键蛋白激酶TAK1活性：（1）通过竞争性结合TAK1从而解离TAK1-TAB2激酶复合物。 TAK1的活化主要依赖于其结合蛋白TAB2介导的非锚定多聚泛素链与TAK1的结合。由于p38IP的竞争性结合，阻断了TAK1与TAB2及其结合的多聚泛素链的接触，从而抑制TAK1活化。TAK1-p38IP复合物与TAK1-TAB2复合物在静息细胞中达到一定的平衡。有趣的是，免疫信号刺激会诱导p38IP与TAK1发生瞬时解离，利于促进TAK1活化，之后再结合，从而抑制TAK1过度活化。究其动态结合原因，发现非锚定多聚泛素链可以像接力棒般从TAB2向TAK1传递；还发现TAB2和TAK1结合泛素链之后分别对TAK1和p38IP有更强的结合亲和力。在这两种因素的交织作用下，刺激诱导p38IP与TAK1发生动态结合，精确调控TAK1活化。（2）免疫信号刺激后，p38IP还作为接头蛋白特异性地将去泛素化酶USP4招募到活化的TAK1，去除TAK1上共价和非共价结合的泛素链。因此，p38IP可通过感应TAK1活性，精准调控TAK1活化，从而防止免疫信号的过活化。研究还进一步制备并利用p38IP条件敲除小鼠，发现p38IP在T细胞发育特别是Treg发育中具有重要作用。本研究不仅发现了新的免疫受体信号通路负性调控因子，也为认识p38IP生物学功能提供了新视角。