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

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传导中起关键作用的许多蛋白质的运输、定位和功能是必需的。最近的两项重大突破推动了这一领域的发展，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（PAT）大家族（DHHC家族），以及开发了高度灵敏和定量的基于蛋白质组学的棕榈酰蛋白质组全球分析方法。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质筏相关复合物。2006年，我们发现无反应性T细胞在衔接子LAT的棕榈酰化中显示出选择性缺陷，导致LAT的细胞内滞留和未能促进TCR诱导的T细胞活化。在NIH R21资助的初步研究中，我们鉴定了几种候选PAT：i）增强LAT棕榈酰化; ii）与其物理缔合; iii）在无反应性T细胞中下调;和/或iv）TCR诱导的IL-2产生所需。此外，我们还与B博士开展了合作。Cravatt的实验室进行了一项基于蛋白质组学的蛋白质棕榈酰化在无能与对照T细胞的全球分析。这些研究为以下研究提供了合理的依据：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敲低对LAT及其棕榈酸酯的稳定性，营业额和分选的影响;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，方法是进行成像/共定位研究，绘制LAT及其PAT中底物缔合和棕榈酰化所需的相应关键区域，并分析LAT突变对LAT无效背景中LAT功能的影响，该突变消除了DHHC蛋白-LAT相互作用。目标3。我们将比较无变应性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）结合炔基脂肪酸类似物17-十八炔酸的代谢标记来鉴定PAT敲低T细胞中DHHC 15的生理PAT底物。在将炔基脂肪酸掺入内源性棕榈酰化位点后，将重和轻膜蛋白质组混合并使用点击化学偶联至生物素-叠氮化物，在链霉亲和素-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，揭示改变的蛋白棕榈酰化在T细胞活化和无能中的作用，并为大大提高对T细胞生物学中蛋白棕榈酰化机制方面的理解以及DHHC蛋白作为免疫性疾病药物靶标的验证铺平道路。

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.