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: 8385513
• 负责人: AMNON ALTMAN
• 金额: $ 41.92万
• 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/8385513
• 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）：蛋白质棕榈酰化是对众多蛋白质的运输，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导中起着关键作用。迈向该领域的两个主要突破包括发现了介导蛋白质棕榈酰化的棕榈酰酰基转移酶（PATS）的大型家族（DHHC家族），以及开发基于高度敏感和定量蛋白质组学的方法，用于全球质量分析棕榈酰蛋白质组。 TCR诱导的T细胞激活取决于高度富集棕榈酰蛋白的多蛋白脂质筏相关复合物。在2006年，我们发现，抗抗T细胞在适配器LAT的棕榈酰化中显示出选择性缺陷，从而导致LAT的细胞内保留，并且未能促进TCR诱导的T细胞激活。在NIH R21赠款支持的初步研究中，我们确定了几个候选人：i）增强lat棕榈酰化； ii）与之缔合； iii）在厌食的T细胞中下调；和/或IV）是TCR诱导的IL-2产生所必需的。此外，我们与B. Cravatt博士的实验室进行了合作，以对厌氧和对照T细胞进行基于蛋白质组学的蛋白质棕榈酰化分析。这些研究为以下研究提供了合理的基础：目标1。我们将确认并确定lat-palmitoylating Pats并分析其底物特异性；研究PAT敲低对LAT及其棕榈酸盐的稳定性，周转和分类的影响；并分析这些拍打对T细胞激活和体外厌食的各个方面的敲击的影响，包括T细胞分化和细胞因子产生。 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。我们将通过使用稳定的同位素在细胞培养物中使用稳定的同位素标记（SILAC）在PAT敲低T细胞中识别DHHC15的棕榈酰蛋白质组，并鉴定DHHC15的生理PAT底物，并鉴定出与氨基酸（SILAC）中的氨基酸标记（SILAC），并用酸性脂肪酸类似酸类似酸17- octadecyciccycycycycycycycycy coupplotic（SILAC）。碱性脂肪酸掺入内源性棕榈酰化位点后，将使用点击化学混合并将重型膜蛋白质组混合并耦合到生物素 - 氮杂，并在链霉亲蛋白 - 琼脂糖珠中富集，使用多维蛋白质识别技术（Mudpit Technology（Mudpit）（Mudpit）（Mudpits）上的大量spececeprosectorssspectorsexsection spececeportor spececeporne spececeportor spececeporne spececretrone。这些研究将首次建立T细胞中选定PAT的功能和底物特异性，揭示蛋白质棕榈酰化改变在T细胞活化和厌食中的作用，并为对T细胞生物学中蛋白质棕榈酰化的机械方面的理解铺平了道路，并验证了DHHC蛋白AS AS Prundices sease sease sease seases中的蛋白质机械方面。