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: 8238986
• 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 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8238986
• 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 Delivery Systems; 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. PUBLIC HEALTH RELEVANCE: Many cellular proteins can be modified by addition of a lipid moiety in a process termed protein palmitoylation, which plays an important role in regulating the localization, trafficking and function of these proteins. Based on our preliminary findings that documented a defect in this process in T lymphocytes that can poorly respond to antigen stimulus (i.e., are anergic), we will study the enzymes that mediate protein palmitoylation and their importance in T cell activation. Results of the proposed study may validate these enzymes as potential drug targets, and open the way to rational design of enzyme inhibitors.

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，对于在细胞生长和信号转导中发挥关键作用的众多蛋白质的运输、定位和功能是必需的。最近推动该领域发展的两项重大突破包括发现介导蛋白质棕榈酰化的棕榈酰酰基转移酶 (PAT) 大家族（DHHC 家族），以及开发用于棕榈酰蛋白质组全局分析的高灵敏度和定量蛋白质组学方法。 TCR 诱导的 T 细胞激活依赖于富含棕榈酰蛋白的多蛋白脂筏相关复合物。 2006年，我们发现无反应性T细胞在接头LAT的棕榈酰化中表现出选择性缺陷，导致LAT在细胞内滞留并且无法促进TCR诱导的T细胞激活。在 NIH R21 资助支持的初步研究中，我们确定了几种候选 PAT：i) 增强 LAT 棕榈酰化； ii) 与其进行身体接触； iii) 在无反应性 T 细胞中下调；和/或iv)是TCR诱导的IL-2产生所必需的。此外，我们还与 B. Cravatt 博士的实验室合作，对无能 T 细胞与对照 T 细胞中的蛋白质棕榈酰化进行基于蛋白质组学的全局分析。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 LAT 棕榈酰化 PAT 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；并分析敲低这些 PAT 对体外和体内 T 细胞活化和无反应性的各个方面的影响，包括 T 细胞分化和细胞因子产生。目标 2. 我们将通过进行成像/共定位研究，绘制底物关联和棕榈酰化所需的 LAT 及其 PAT 中的相应关键区域，并分析废除 DHHC 蛋白-LAT 相互作用的 LAT 突变对 Lat 无效背景中 LAT 功能的影响，来表征和研究 LAT 和 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物中氨基酸的稳定同位素标记 (SILAC) 与炔基脂肪酸类似物 17-十八氰酸的代谢标记相结合，鉴定 PAT 敲低 T 细胞中 DHHC15 的生理 PAT 底物。炔基脂肪酸掺入内源性棕榈酰化位点后，将使用点击化学将重膜和轻膜蛋白质组混合并与生物素叠氮化物偶联，在链霉亲和素-琼脂糖珠上富集，进行胰蛋白酶处理，并在高分辨率质谱仪上使用多维蛋白质鉴定技术 (MudPIT) 进行分析。这些研究将首次确定 T 细胞中选定 PAT 的功能和底物特异性，揭示改变的蛋白质棕榈酰化在 T 细胞活化和无反应中的作用，并为大大提高对 T 细胞生物学中蛋白质棕榈酰化机制的理解以及验证 DHHC 蛋白作为免疫疾病药物靶点铺平道路。 公共卫生相关性：许多细胞蛋白质可以通过在称为蛋白质棕榈酰化的过程中添加脂质部分来进行修饰，该过程在调节这些蛋白质的定位、运输和功能方面发挥着重要作用。我们的初步发现记录了 T 淋巴细胞在这一过程中存在缺陷，对抗原刺激反应不佳（即无反应性），根据我们的初步发现，我们将研究介导蛋白质棕榈酰化的酶及其在 T 细胞激活中的重要性。拟议研究的结果可能会验证这些酶作为潜在的药物靶点，并为酶抑制剂的合理设计开辟道路。