Regulation of T cell anergy by palmitoyl acyl transferases

描述（由申请人提供）：T细胞能量在自身免疫、移植和肿瘤免疫中具有重要意义。最近，我们在抗原特异性无能T细胞中发现了一种新的TCR信号缺陷，即棕榈酰化受损，从而导致T细胞活化连接体（LAT）的脂质筏定位和功能受损。其他T细胞信号蛋白的正确定位和功能也严重依赖于它们的棕榈酰化。尽管已知蛋白棕榈酰化已有30多年的历史，但直到最近，人们对其机制基础和调控仍知之甚少。最近发现的一个新的大型（23个成员）哺乳动物蛋白棕榈酰酰基转移酶（PATs）家族代表了这一领域的重大突破。这一进展和我们最近的初步证据表明，缺陷的LAT棕榈酰化实际上可能导致能量，这是该项目的推动力。我们建议开展探索性/发展性研究，以表征PAT在T淋巴细胞中的表达，鉴定lata反应性PAT，并分析其在T细胞反应性和能量中的潜在作用。在Aim 1中，我们将使用实时荧光定量PCR测定23种已知小鼠PAT在静息、活化和无能T细胞中的mRNA表达谱，并在蛋白水平上确认T细胞表达的PAT mRNA的表达。在Aim 2中，我们将使用所有T细胞表达的PATs的表达载体，并采用已建立的异位表达系统来筛选这些PATs增强LAT棕榈酰化的能力。鉴定出的lata反应性pat将进一步测试其棕榈酰化有限的其他棕榈酰化底物的能力，以测试其相对底物的特异性。在目标3中，我们将选择在前面目标中确定的最佳候选pat进行进一步的功能分析。具体来说，我们将使用一种新颖、高效的siRNA递送方法来敲除选定的PAT的表达，并分析PAT沉默对T细胞功能状态的影响，重点是确定沉默latt反应性PAT是否会诱导类似T细胞能量的功能状态。了解有缺陷的LAT棕榈酰化的机制及其与T细胞能量的关联可能为可逆蛋白棕榈酰化如何调节T细胞命运和功能的关键方面提供重要线索，并可能暗示底物选择性pat作为自身免疫和其他免疫疾病的未来药物靶点。公共卫生相关性：能量是免疫耐受的一种重要形式，其中免疫系统的T淋巴细胞尽管能够识别抗原，但不能对其产生有效的免疫反应。旨在诱导或阻止T细胞能量的策略在自身免疫、移植和肿瘤免疫中具有重要意义。在这里，我们将研究我们发现的一种新的能量诱导途径，它由一个关键分子LAT的加工和功能缺陷组成，LAT是T淋巴细胞发育和正常功能所必需的。

DESCRIPTION (provided by applicant): T cell anergy has important implications in autoimmunity, transplantation and tumor immunity. Recently, we identified a novel TCR signaling defect in antigen-specific anergic T cells, namely, impaired palmitoylation and, consequently, lipid raft localization and function, of linker for activation of T cells (LAT). Proper localization and function of other T cell signaling proteins is also critically dependent on their palmitoylation. Although protein palmitoylation has been known for >30 years, its mechanistic basis and regulation has, until very recently, been poorly understood. The recent discovery of a novel, large (23 member) family of mammalian protein palmitoyl acyl transferases (PATs) represents a major breakthrough in this area. This advance and our more recent preliminary evidence that defective LAT palmitoylation may actually cause anergy serve as a driving force for this project. We propose to conduct exploratory/developmental studies in order to characterize the expression of PATs in T lymphocytes, identify LAT-reactive PAT(s), and analyze their potential role in T cell responsiveness and anergy. In Aim 1, we will use quantitative real-time PCR to determine the mRNA expression profile of 23 known mouse PATs in resting, activated and anergic T cells, and confirm expression of T cell-expressed PAT mRNAs at the protein level. In Aim 2, we will use expression vectors of all T cell-expressed PATs, and employ an established ectopic expression system in order to screen the ability of these PATs to enhance the palmitoylation of LAT. Identified LAT-reactive PATs will be further tested for their ability to palmitoylate a limited set of other palmitoylation substrates as a test of their relative substrate specificity. In Aim 3, we will select the best candidates PATs identified in previous aims for further functional analysis. Specifically, we will use a novel, highly efficient method for siRNA delivery in order to knock-down the expression of selected PATs, and analyze the effects of PAT silencing on the functional status of T cells, with emphasis on determining whether silencing of LAT-reactive PATs will induce a functional state resembling T cell anergy. Understanding the mechanism that underlies defective LAT palmitoylation and its association with T cell anergy may provide critical clues on how reversible protein palmitoylation regulates key aspects of T cell fate and function, and potentially implicate substrate-selective PATs as future drug targets in autoimmunity and other immunological diseases. PUBLIC HEALTH RELEVANCE: Anergy is an important form of immune tolerance, in which T lymphocytes of the immune system, despite being able to recognize an antigen, cannot generate an effective immune response to it. Strategies aimed at inducing or preventing T cell anergy have significant implications in autoimmunity, transplantation, and tumor immunity. Here we will investigate a novel pathway for anergy induction discovered by us, which consists of defective processing and function of a key molecule, LAT, which is essential for the development and proper function of T lymphocytes.