Defining how TCR complex mediated signaling is regulated by neddylation

描述（由申请人提供）：类泛素化修饰是一种重要的翻译后修饰，我的研究计划的目标是定义如何操纵翻译后修饰来调节T细胞受体（TCR）复合物信号，T细胞激活和效应功能，最终治疗免疫介导的疾病。一个重要的未解之谜是类化修饰是如何调节t细胞功能的。R21提案中的研究将在这个知之甚少的领域探索并提供基础数据。在我们的初步研究之前，尚不清楚类化修饰如何调节T细胞受体（TCR）复合物信号传导和T细胞功能。我们使用了一种高度选择性的nedd8激活酶（NAE1）抑制剂，它可以阻断所有蛋白质类化修饰，作为确定类化修饰抑制是否影响t细胞功能的工具。我们发现抑制类黄酮化导致IL-2产生增加，IL-2作为TCR信号强度的生物学读数，以及增殖增强，激活标记物表达增加，体外Tregs发育增强。此外，我们用类化化抑制剂治疗SKG小鼠，这些小鼠由于已知的TCR复合物信号缺陷而发展为炎性关节炎，并观察到体内关节炎进展减少和产生IL-2的T细胞数量增加。总的来说，这些研究表明，类化化抑制剂（一种已经用于恶性肿瘤患者的i期临床试验的药物）可以增强t细胞功能，并可能成为自身免疫性疾病的治疗选择。因此，我们假设，定义在T细胞中被类化并调节TCR复合物信号的蛋白质将使我们更深入地了解操纵T细胞功能以治疗免疫介导疾病的方法。为了开始填补我们对类化修饰如何调节TCR复合物信号传导的理解的空白，我们将采取一种无偏倚的方法，并通过以下具体目标来检验我们的假设：为了鉴定受TCR复合物信号修饰的蛋白，我们将进行凝胶内电泳（DIGE）和质谱分析。目标2。为了确定在TCR复合物信号上被诱导类化的蛋白是否抑制TCR信号，我们将敲低该蛋白的表达，并产生表达该蛋白不可类化突变体的T细胞系。我们将量化IL-2的产生和激活标记物的诱导，作为TCR信号强度的生物学读数。为了实现这些目标，我们已经生成了必要的试剂，并组建了一个具有蛋白质组学专业知识的合作团队来进行拟议的研究。在这些研究的结论中，我们将通过识别可诱导类化的蛋白质并验证其在调节TCR复合物信号和T细胞效应功能中的重要性，扩展我们对TCR复合物信号传导调节的知识。定义类化修饰如何调节TCR复合物信号传导，将为在各种免疫介导的疾病中特异性操纵t细胞功能带来新的机会。

DESCRIPTION (provided by applicant): Neddylation is an essential posttranslational modification, and a goal of my research program is to define how posttranslational modifications can be manipulated to regulate T cell receptor (TCR) complex signaling, T-cell activation, and effector functions to ultimately treat immune-mediated disease. An important unanswered question is how neddylation regulates T-cell function. The studies in this R21 proposal will explore and provide foundational data in this poorly understood area. Prior to our preliminary studies, it was unknown how neddylation regulates T-cell receptor (TCR) complex signaling and T cell function. We used, a highly selective NEDD8-activating enzyme (NAE1) inhibitor that blocks all protein neddylation, as a tool to determine if inhibition of neddylation impacts T-cell function. We found that inhibition of neddylation leads to increased IL-2 production, which serves as a biological readout for TCR signal strength, as well as enhanced proliferation, increased activation marker expression, and enhanced Tregs development in vitro. In addition, we treated SKG mice, which develop inflammatory arthritis due to a known defect in TCR complex signaling, with the neddylation inhibitor and observed reduced arthritis progression and increased numbers of IL-2 producing T cells in vivo. Collectively, these studies suggest that the neddylation inhibitor, a drug already being used in phase-I clinical trials for patients with malignancies, enhances T-cell function and may be a treatment option for autoimmune disease. Thus we hypothesize that defining proteins that are neddylated in T cells and regulate TCR complex signaling will lead to greater insights into ways to manipulate T cell-function to treat immune mediated disease. To begin to fill the gap in our understanding of how neddylation regulates TCR complex signaling, we will take an unbiased approach and test our hypothesis through the following specific aims: Aim 1. To identify proteins that are neddylated upon TCR complex signaling, we will perform difference in-gel electrophoresis (DIGE) and mass spectrometry. Aim 2. To determine if the proteins that are inducibly neddylated upon TCR complex signaling inhibit TCR signaling, we will knockdown expression of the protein and generate T cell lines that express a non-neddylatable mutant of the protein. We will quantitate IL-2 production and activation marker induction as a biological readout for TCR signal strength. To achieve these goals, we have generated the necessary reagents and have assembled a collaborative team with proteomic expertise to perform the proposed studies. At the conclusion of these studies, we will have expanded our knowledge of the regulation of TCR complex signaling by identifying proteins that are inducibly neddylated and validating their importance in regulating TCR complex signaling and T- cell effector functions. Defining how neddylation regulates TCR complex signaling should lead to new opportunities to specifically manipulate T-cell function in a variety of immune mediated diseases.