Defining how TCR complex mediated signaling is regulated by neddylation

描述(申请人提供)：去甲肾上腺素是一种基本的翻译后修饰，我的研究计划的一个目标是确定如何操纵翻译后修饰来调节T细胞受体(TCR)复合体信号、T细胞激活和效应器功能，从而最终治疗免疫介导性疾病。一个重要的悬而未决的问题是，内分泌如何调节T细胞的功能。R21提案中的研究将探索并提供这一鲜为人知的领域的基础数据。在我们的初步研究之前，还不知道neddyling如何调节T细胞受体(TCR)复合体信号和T细胞功能。我们使用了一种高选择性的NEDD8激活酶(NAE1)抑制剂，它可以阻断所有蛋白质的脱氢酶，作为确定抑制脱氢酶是否影响T细胞功能的工具。我们发现，抑制去甲基化导致IL-2的产生增加，这是TCR信号强度的生物读数，并在体外促进增殖，增加激活标志物的表达，促进Tregs的发育。此外，我们对因TCR复合体信号缺陷而发生炎性关节炎的SKG小鼠进行了治疗，观察到体内关节炎进展缓慢，产生IL-2的T细胞数量增加。总而言之，这些研究表明，这种已经用于恶性肿瘤患者I期临床试验的药物可以增强T细胞功能，并可能成为治疗自身免疫性疾病的一种选择。因此，我们假设，定义T细胞中需要的蛋白质并调节TCR复合体信号将导致对如何操纵T细胞功能以治疗免疫介导性疾病的更深入的了解。为了填补我们对neddyling如何调控TCR复合体信号的理解上的空白，我们将采取公正的方法，通过以下具体目标来检验我们的假设：目的1.为了识别TCR复合体信号转导所需的蛋白质，我们将进行差异凝胶内电泳(DGE)和质谱分析。目的2.为了确定被诱导依赖于TCR复合体信号转导的蛋白质是否抑制TCR信号转导，我们将敲除该蛋白的表达，并产生表达该蛋白的不可突变的T细胞系。我们将量化IL-2的产生和激活标志物的诱导，作为TCR信号强度的生物读数。为了实现这些目标，我们已经产生了必要的试剂，并组建了一个具有蛋白质组专业知识的合作团队来执行拟议的研究。在这些研究的结论中，我们将通过识别可诱导缺失的蛋白质并验证它们在调节TCR复合体信号和T细胞效应器功能中的重要性，来扩大我们对TCR复合体信号调节的知识。明确neddyling如何调节TCR复合体信号应该会带来新的机会，特别是在各种免疫介导性疾病中操纵T细胞功能。 与公共卫生相关：我们已经发现，翻译后蛋白质的一种基本修饰--脱氧核糖核酸能抑制T细胞受体(TCR)复合体的信号传递。这个R21建议的目标是确定缺失的蛋白质和调控TCR复合体信号的缺失(S)机制。发现调节TCR复合体信号的蛋白质的新的翻译后修饰将增强我们操纵T细胞激活和功能的能力，最终目标是治疗免疫介导性疾病。

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. PUBLIC HEALTH RELEVANCE: We have discovered that neddylation, an essential posttranslational protein modification, inhibits signaling through the T cell receptor (TCR) complex. The goal of this R21 proposal is to identify neddylated proteins and mechanism(s) by which neddylation regulates TCR complex signaling. Discovering new posttranslational modifications of proteins that regulate TCR complex signaling will enhance our ability to manipulate T-cell activation and function, with the ultimate goal of treating immune mediated disease.