Defining how TCR complex mediated signaling is regulated by neddylation

定义 TCR 复合物介导的信号传导如何通过 neddylation 进行调节

• 批准号: 8423677
• 负责人: LEONARD Louis DRAGONE
• 金额: $ 15.62万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8423677
• 关键词: Address; Adopted; Area; Arthritis; Autoimmune Diseases; Binding; Biological; CD4 Positive T Lymphocytes; Cell Line; Cell physiology; Cellular biology; Collaborations; Complex; Data; Defect; Development; Disease; Disease Progression; Enzymes; Event; Goals; Immune; Immunoblot Analysis; In Vitro; Interleukin-2; Knowledge; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methodology; Modeling; Molecular; Mus; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase I Clinical Trials; Phenotype; Pilot Projects; Plant Resins; Post-Translational Protein Processing; Process; Production; Proteins; Proteomics; Reagent; Receptor Signaling; Regulation; Regulatory T-Lymphocyte; Research; Rheumatoid Arthritis; Role; Signal Transduction; T-Cell Activation; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Testing; Time; Treatment Efficacy; comparative; gel electrophoresis; improved; in vivo; infancy; inhibitor/antagonist; innovation; insight; mouse model; mutant; programs; protein expression; response; tool

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.

描述（由申请人提供）：Neddylation是一种重要的翻译后修饰，我的研究项目的目标是确定如何操纵翻译后修饰来调节T细胞受体（TCR）复合物信号传导、T细胞活化和效应器功能，以最终治疗免疫介导的疾病。一个重要的未回答的问题是neddylation如何调节T细胞功能。R21建议中的研究将探索并提供这一知之甚少的领域的基础数据。在我们的初步研究之前，不知道neddylation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NEDD 8激活酶（NAE 1）抑制剂，可以阻止所有蛋白质neddylation，作为确定neddylation抑制是否影响T细胞功能的工具。我们发现，neddylation的抑制导致IL-2产生增加，其用作TCR信号强度的生物学读数，以及增殖增强，活化标志物表达增加和体外TcR发育增强。此外，我们用neddylation抑制剂治疗了SKG小鼠，这些小鼠由于TCR复合物信号传导中的已知缺陷而发生炎性关节炎，并观察到关节炎进展减少和体内产生IL-2的T细胞数量增加。总的来说，这些研究表明，neddylation抑制剂，一种已经用于恶性肿瘤患者I期临床试验的药物，增强了T细胞功能，可能是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neddylated并调节TCR复合物信号传导的蛋白质将导致对操纵T细胞功能以治疗免疫介导的疾病的方法的更深入的了解。为了开始填补我们对neddylation如何调节TCR复合物信号传导的理解中的差距，我们将采取无偏的方法并通过以下具体目标来测试我们的假设：目标1。为了鉴定在TCR复合物信号传导后被neddylated的蛋白质，我们将进行差异凝胶电泳（DIGE）和质谱分析。目标2.为了确定在TCR复合物信号传导时可诱导neddylated的蛋白质是否抑制TCR信号传导，我们将敲低蛋白质的表达并产生表达蛋白质的不可neddylatable突变体的T细胞系。我们将定量IL-2的产生和活化标志物的诱导，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，我们将通过鉴定可诱导neddylated的蛋白质并验证它们在调节TCR复合物信号传导和T细胞效应子功能中的重要性来扩展我们对TCR复合物信号传导调节的知识。确定neddylation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。