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: 8590200
• 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/8590200
• 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)：蛋白质棕榈酰化是许多蛋白质运输、定位和功能所必需的翻译后修饰，这些蛋白质在细胞生长和信号传递中起关键作用。最近两项重大突破推动了该领域的发展，包括发现了一个介导蛋白质棕榈酰化的棕榈酰酰基转移酶(PATs)大家族(DHHC家族)，以及开发了基于高灵敏度和定量的蛋白质组学方法来对棕榈酰基蛋白质组进行全局分析。TCR诱导的T细胞活化依赖于高度富含棕榈酰基蛋白质的多蛋白脂筏相关复合体。2006年，我们发现无能T细胞在LAT的棕榈酰化过程中表现出选择性缺陷，导致LAT滞留在细胞内，无法促进TCR诱导的T细胞活化。在NIH R21拨款支持的初步研究中，我们确定了几个候选PATs：i)增强LAT棕榈酰化；ii)物理上与其相关；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，我们开始与B·克拉瓦特博士的实验室合作，以蛋白质组学为基础，对无能T细胞和对照T细胞中的蛋白质棕榈酰化进行全球分析。这些研究为以下研究提供了合理的基础：目的1.确认和鉴定LAT-Palmitoylating PATs并分析其底物特异性；研究PAT敲除对LAT及其棕榈酸酯稳定性、周转和分选的影响；分析敲除这些PATs在体内外对T细胞活化和无能的各个方面的影响，包括T细胞分化和细胞因子的产生。目的2.我们将通过进行成像/共定位研究，定位底物结合和棕榈酰化所需的LAT及其PAT各自的关键区，以及在Lat缺失背景下分析取消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和LAT-Palmitoylating DHHC蛋白之间的物理关联的生物学相关性。目的3.比较无能T细胞和对照T细胞的棕榈酰基蛋白质组，并利用细胞培养中氨基酸的稳定同位素标记(SILAC)和炔基脂肪酸类似物17-十八烷基诺酸的代谢标记来鉴定PAT基因敲除T细胞中DHHC15的生理底物。在将炔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混合并使用点击化学与生物素叠氮偶联，在链霉亲和素-琼脂糖珠上浓缩，胰酶消化，并在高分辨率质谱仪上使用多维蛋白质识别技术(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，揭示改变的蛋白质棕榈酰化在T细胞激活和无能中的作用，并为极大地提高对蛋白质棕榈酰化在T细胞生物学中的机制的理解以及验证DHHC蛋白质作为免疫疾病的药物靶标铺平道路。