Regulation of T cell anergy by palmitoyl acyl transferases

棕榈酰酰基转移酶对 T 细胞无反应性的调节

• 批准号: 7753678
• 负责人: AMNON ALTMAN
• 金额: $ 27.18万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7753678
• 关键词: Accounting; Acylation; Adaptor Signaling Protein; Address; Antibodies; Antigens; Area; Autoimmunity; Biological; Cell membrane; Cell physiology; Cysteine; Defect; Development; Drug Delivery Systems; Ectopic Expression; Enzymes; Event; Family; Family member; Future; Immune System Diseases; Immune Tolerance; Immune response; Immune system; Interleukin-2; Label; Mediating; Membrane Microdomains; Metabolic; Methods; Mitogen-Activated Protein Kinases; Molecular Profiling; Mus; Palmitates; Pathway interactions; Peripheral; Play; Process; Proteins; Receptor Signaling; Regulation; Relative (related person); Rest; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Small Interfering RNA; Substrate Specificity; System; T cell anergy; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Testing; Time; Transcription Factor AP-1; Transferase; Transplantation; Tumor Immunity; anergy; base; clinically relevant; clinically significant; driving force; expression vector; functional status; knock-down; mRNA Expression; novel; palmitoylation; prevent; public health relevance; receptor

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.

描述（由申请人提供）：T细胞无反应性在自身免疫、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反应性 T 细胞中发现了一种新的 TCR 信号传导缺陷，即棕榈酰化受损，从而导致 T 细胞激活连接子 (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严重依赖于它们的棕榈酰化。尽管蛋白质棕榈酰化已知已有超过 30 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。最近发现的哺乳动物蛋白棕榈酰酰基转移酶 (PAT) 大型（23 个成员）家族代表了该领域的重大突破。这一进展和我们最近的初步证据表明，有缺陷的 LAT 棕榈酰化实际上可能会导致无反应，成为该项目的驱动力。我们建议进行探索性/开发性研究，以表征 T 淋巴细胞中 PAT 的表达，识别 LAT 反应性 PAT，并分析它们在 T 细胞反应性和无反应性中的潜在作用。在目标 1 中，我们将使用定量实时 PCR 测定 23 种已知小鼠 PAT 在静息、激活和无能 T 细胞中的 mRNA 表达谱，并在蛋白质水平确认 T 细胞表达的 PAT mRNA 的表达。在目标2中，我们将使用所有T细胞表达的PAT的表达载体，并采用已建立的异位表达系统来筛选这些PAT增强LAT棕榈酰化的能力。将进一步测试鉴定出的 LAT 反应性 PAT 棕榈酰化一组有限的其他棕榈酰化底物的能力，以测试其相对底物特异性。在目标 3 中，我们将选择之前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 递送方法来敲低选定的 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 LAT 沉默是否对 T 细胞功能状态产生影响。反应性 PAT 会诱导类似于 T 细胞无反应性的功能状态。了解 LAT 棕榈酰化缺陷的机制及其与 T 细胞无反应性的关系，可能会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，并可能暗示底物选择性 PAT 作为自身免疫和其他免疫学的未来药物靶点疾病。公共卫生相关性：无反应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，但无法对其产生有效的免疫反应。旨在诱导或预防 T 细胞无反应性的策略对自身免疫、移植和肿瘤免疫具有重要意义。在这里，我们将研究我们发现的一种新的无反应性诱导途径，该途径由关键分子 LAT 的加工和功能缺陷组成，LAT 对于 T 淋巴细胞的发育和正常功能至关重要。