Regulation of T cell anergy by palmitoyl acyl transferases

描述(申请人提供)：T细胞无能在自身免疫、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细胞激活连接物(LAT)的脂筏定位和功能受损。其他T细胞信号蛋白的正确定位和功能也依赖于它们的棕榈酰化。尽管蛋白质棕榈酰化已有30年的历史，但直到最近，人们对它的机制基础和调控还知之甚少。最近发现了一个新的哺乳动物蛋白棕榈酰转移酶(PATS)大家族(23个成员)，这是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无能，这是该项目的推动力。我们建议进行探索性/发育性研究，以表征T淋巴细胞中PATS的表达，鉴定LAT反应性PAT(S)，并分析它们在T细胞反应性和无能中的潜在作用。在目标1中，我们将使用实时定量聚合酶链式反应来检测23个已知的小鼠PATs在静息、激活和无能T细胞中的mRNA表达谱，并在蛋白水平上证实T细胞表达的PAT mRNAs。在目标2中，我们将使用所有T细胞表达的PATs的表达载体，并利用建立的异位表达系统来筛选这些PATs促进LAT棕榈酰化的能力。已确定的LAT反应性PATs将进一步测试它们对有限的一组其他棕榈酰化底物的棕榈酰化能力，以测试它们的相对底物特异性。在目标3中，我们将选择在之前的目标中确定的最佳候选PAT进行进一步的功能分析。具体地说，我们将使用一种新的、高效的siRNA传递方法来下调选定的PATs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LAT反应性PATS是否会导致类似T细胞无能的功能状态。了解LAT棕榈酰化缺陷的机制及其与T细胞无能的关系，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关键线索，并潜在地暗示底物选择性PATS可能成为自身免疫和其他免疫性疾病的未来药物靶点。公共卫生相关性：无能是免疫耐受的一种重要形式，在这种情况下，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。旨在诱导或预防T细胞无能的策略在自身免疫、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无能诱导途径，它包括关键分子LAT的加工和功能缺陷，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.