Global protein palmitoylation and DHHC proteins in T cell activation and anergy

T 细胞活化和无能中的全局蛋白棕榈酰化和 DHHC 蛋白

• 批准号: 8766542
• 负责人: AMNON ALTMAN
• 金额: $ 44.6万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8766542
• 关键词: Acids; Amino Acids; Antigens; Azides; Biochemical; Biological; Biotin; CD28 gene; Cell Culture Techniques; Cellular biology; Chemistry; Collaborations; Complex; Coupled; Data; Defect; Deletion Mutation; Development; Disease; Drug Targeting; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Failure; Family; Family member; Fatty Acids; Future; Grant; Human Pathology; Image; Immune System Diseases; In Vitro; Interleukin-2; Label; Laboratories; Light; Lipids; Malignant Neoplasms; Maps; Mediating; Membrane; Membrane Microdomains; Metabolic; Methods; Mutation; Palmitates; Palmitic Acylation Site; Physiological; Play; Post-Translational Protein Processing; Process; Production; Protein Analysis; Proteins; Proteome; Proteomics; Publishing; RNA Interference; Reporter; Research Institute; Resolution; Role; Signal Transduction; Sorting - Cell Movement; Specificity; Stable Isotope Labeling; Stimulus; Substrate Specificity; T cell differentiation; T-Cell Activation; T-Lymphocyte; Technology; Time; Transferase; United States National Institutes of Health; Validation; anergy; base; cell growth; cytokine; design; fatty acid analog; improved; in vivo; inhibitor/antagonist; knock-down; mass spectrometer; member; nervous system disorder; palmitoylation; protein function; response; streptavidin-agarose; trafficking

DESCRIPTION (provided by applicant): Protein palmitoylation is an essential post-translational modification necessary for trafficking, localization and function of numerous proteins that play key roles in cell growth and signaling. Two major recent breakthroughs that have advanced this field include the discovery of a large family (DHHC family) of palmitoyl acyl transferases (PATs) that mediate protein palmitoylation, and development of highly sensitive and quantitative proteomics-based methods for global analysis of the palmitoyl proteome. TCR-induced T cell activation depends on multi-protein lipid raft-associated complexes highly enriched in palmitoyl proteins. In 2006, we discovered that anergic T cells display a selective defect in the palmitoylation of the adaptor LAT, resulting in intracellular retention of LAT and failure to promote TCR-induced T cell activation. In preliminary studies supported by an NIH R21 grant, we identified several candidate PATs that: i) enhance LAT palmitoylation ; ii) physically associate with it; iii) are down-regulated in anergic T cells; and/or iv) are required for TCR-induced IL-2 production. In addition, we initiated a collaboration with Dr. B. Cravatt's laboratory to conduct a proteomics-based global analysis of protein palmitoylation in anergic vs. control T cells. These studies provide a rational basis for the following studies: Aim 1. We will confirm and identify LAT-palmitoylating PATs and analyze their substrate specificity; study the effects of PAT knockdown on the stability, turnover and sorting of LAT and its palmitate; and analyze the effects of knocking-down these PATs on various aspects of T cell activation and anergy in vitro and in vivo, including T cell differentiation and cytokine production. Aim 2. We will characterize and study the biological relevance of physical associations between LAT and LAT-palmitoylating DHHC proteins by conducting imaging/colocalization studies, mapping respective critical regions in LAT and its PATs that are required for substrate association and palmitoylation, and analyzing the effect of LAT mutations that abolish DHHC protein-LAT interactions on the functionality of LAT in a Lat-null background. Aim 3. We will compare the palmitoyl proteome in anergic vs. control T cells, and identify physiological PAT substrates of DHHC15 in PAT knockdown T cells by using stable isotope labeling with amino acids in cell culture (SILAC) coupled to metabolic labeling with the alkynyl fatty acid analog 17-octadecynoic acid. After alkynyl fatty acid incorporation into endogenous palmitoylation sites, heavy and light membrane proteomes will be mixed and coupled to biotin-azide using click chemistry, enriched on streptavidin-agarose beads, trypsinized, and analyzed using multidimensional protein identification technology (MudPIT) on a high-resolution mass spectrometer. These studies will establish for the first time the function and substrate specificity of selected PATs in T cells, reveal the role of altered protein palmitoylation in T cell activation and anergy, and pave the way to a greatly improved understanding of the mechanistic aspects of protein palmitoylation in T cell biology, and to the validation of DHHC proteins as drug targets in immunological diseases.

描述（由申请人提供）：蛋白质棕榈酰化是一种重要的翻译后修饰，是许多在细胞生长和信号传导中起关键作用的蛋白质的运输、定位和功能所必需的。最近在这一领域取得的两项重大突破包括：介导蛋白棕榈酰化的棕榈酰转移酶（PATs）大家族（DHHC家族）的发现，以及基于高灵敏度和定量的基于蛋白质组学的棕榈酰蛋白质组全局分析方法的开发。tcr诱导的T细胞活化依赖于高度富集棕榈酰蛋白的多蛋白脂筏相关复合物。在2006年，我们发现无能T细胞在适配器LAT的棕榈酰化中表现出选择性缺陷，导致LAT在细胞内保留，无法促进tcr诱导的T细胞活化。在NIH R21资助的初步研究中，我们确定了几种候选pat: 1)增强LAT棕榈酰化；Ii)物理关联；iii)在无能T细胞中下调；和/或iv)是tcr诱导的IL-2产生所必需的。此外，我们还启动了与B. Cravatt博士实验室的合作，对无能T细胞与对照T细胞中的蛋白棕榈酰化进行基于蛋白质组学的全球分析。这些研究为以下研究提供了合理的基础：目的1。我们将确认和鉴定at -棕榈酰化PATs并分析其底物特异性；研究PAT敲除对LAT及其棕榈酸酯的稳定性、周转和分选的影响；并分析敲除这些PATs对体外和体内T细胞活化和能量的各个方面的影响，包括T细胞分化和细胞因子的产生。目标2。我们将通过成像/共定位研究，绘制LAT及其pat中底物结合和棕榈酰化所需的各自关键区域，并分析在LAT零背景下，消除DHHC蛋白-LAT相互作用的LAT突变对LAT功能的影响，来表征和研究LAT与LAT-棕榈酰化DHHC蛋白之间物理关联的生物学相关性。目标3。我们将比较无能T细胞与对照T细胞中的棕榈酰蛋白质组，并通过使用细胞培养氨基酸（SILAC）的稳定同位素标记以及烷基脂肪酸类似物17-十八烯酸的代谢标记，鉴定PAT敲除T细胞中DHHC15的生理PAT底物。烷基脂肪酸掺入内源性棕榈酰化位点后，重膜和轻膜蛋白质组将通过click化学混合并偶联到生物素叠氮化物上，在链亲和素琼脂糖珠上富集，胰蛋白酶化，并在高分辨率质谱仪上使用多维蛋白鉴定技术（MudPIT）进行分析。这些研究将首次确定选定的PATs在T细胞中的功能和底物特异性，揭示改变的蛋白棕榈酰化在T细胞活化和能量中的作用，并为大大提高对T细胞生物学中蛋白棕榈酰化的机制方面的理解铺平道路，并为验证DHHC蛋白作为免疫疾病的药物靶点铺平道路。