Regulation Of G Protein Palmitoylation

G 蛋白棕榈酰化的调控

• 批准号: 6673578
• 负责人: TERESA L Z JONES
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6673578
• 关键词: G protein; acyltransferase; esterase; fatty acylation; human tissue; intermolecular interaction; laboratory rat; matrix assisted laser desorption ionization; molecular site; palmitates; posttranslational modifications; protein purification; protein structure function; proteomics

Heterotrimeric G proteins sit at the cytoplasmic face of membranes and transduce signals from cell surface receptors to intracellular effectors. The heterotrimer consists of the alpha subunit that binds GTP and the beta and gamma subunits that form a tight complex. Most of the G alpha subunits undergo palmitoylation, the reversible, post-translational addition of palmitate to an amino-terminal cysteine residue. Palmitoylation increases the membrane affinity of the G alpha subunit as well as modulating protein interactions with the beta/gamma subunits, receptors and effectors. Activation of G proteins by a receptor increases the palmitate turnover on the alpha subunit. The enzymes responsible for palmitoylation and depalmitoylation of proteins have been elusive. To better understand the regulation of this cycle, we are investigating the enzyme responsible for G-protein palmitoylation. We have partially purified PAT activity from rat liver and human erythrocytes with a significantly higher degree of enrichment compared to previous reports by using affinity chromatography to an inhibitor of PAT activity and to palmitoyl CoA. In collaboration with Lewis Pannell (NIDDK), we analyzed the partially purified products by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and identified ~50 proteins. To test these candidates, we developed a novel expression system and assay that allows facile expression of properly folded proteins.

异三聚体G蛋白位于膜的细胞质面，并从细胞表面受体转移到细胞内效应子。异三聚体由结合GTP和形成紧密复合物的beta和伽马亚基的α亚基组成。大多数Gα亚基都经历了棕榈酰化，可逆的，翻译后的棕榈酸酯在氨基末端半胱氨酸残基中添加。棕榈酰化增加了Gα亚基的膜亲和力，以及调节蛋白质与β/γ亚基，受体和效应子的相互作用。通过受体激活G蛋白会增加alpha亚基上的棕榈酸酯周转率。负责蛋白质棕榈酰化和去氨酰二酰化的酶是难以捉摸的。为了更好地了解该周期的调节，我们正在研究负责G蛋白棕榈酰化的酶。与以前的报道相比，通过使用亲和色谱对PAT活性抑制剂和棕榈酸COA的报道，我们具有从大鼠肝脏和人类红细胞中的部分纯化的PAT活性。与Lewis Pannell（NIDDK）合作，我们通过矩阵辅助的激光解吸/电离飞行时间（MALDI-TOF）质谱法分析了部分纯化的产物，并确定了〜50个蛋白质。为了测试这些候选者，我们开发了一种新颖的表达系统和测定，可轻松表达正确折叠的蛋白质。