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: 7571748
• 负责人: AMNON ALTMAN
• 金额: $ 22.88万
• 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/7571748
• 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 regulation; 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，我们确定了一种新的TCR信号缺陷，在抗原特异性无能的T细胞，即受损的棕榈酰化，因此，脂筏定位和功能，连接器激活的T细胞（LAT）。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。虽然蛋白质棕榈酰化已经知道了>30年，但直到最近，对其机制基础和调控仍知之甚少。最近发现的一个新的，大的（23个成员）的哺乳动物蛋白质棕榈酰酰基转移酶（PAT）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，有缺陷的LAT棕榈酰化可能实际上导致无能作为这个项目的驱动力。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，鉴定LAT反应性PAT，并分析其在T细胞反应性和无反应性中的潜在作用。在目的1中，我们将使用定量实时PCR来确定23种已知小鼠PAT在静息、活化和无反应性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。目的2：利用T细胞表达的PAT的表达载体，并建立异位表达系统，筛选这些PAT增强LAT棕榈酰化的能力。将进一步测试鉴定的LAT反应性PAT的棕榈酰化一组有限的其他棕榈酰化底物的能力，作为其相对底物特异性的测试。在目标3中，我们将选择先前目标中确定的最佳候选PAT进行进一步的功能分析。具体而言，我们将使用一种新的，高效的siRNA递送方法，以敲低所选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反应性PAT的沉默是否会诱导类似T细胞无反应性的功能状态。了解缺陷性LAT棕榈酰化及其与T细胞无反应性相关的机制可能为可逆性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，并可能涉及底物选择性PAT作为自身免疫和其他免疫性疾病的未来药物靶点。公共卫生关系：无能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应答。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反应性诱导途径，该途径包括对T淋巴细胞的发育和正常功能至关重要的关键分子LAT的加工和功能缺陷。