Action of Lipolytic Enzymes

脂肪分解酶的作用

• 批准号: 10323245
• 负责人: EDWARD A DENNIS
• 金额: $ 40.29万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10323245
• 关键词: Address; Adopted; Allosteric Site; Anabolism; Biological Assay; Calmodulin; Catalytic Domain; Cells; Drug Targeting; Eicosanoids; Enzymes; Fatty Acids; Goals; Grant; Hispanic Populations; Human; Hydrolase; In Vitro; Inflammation; Intervention; Lipase; Lipids; Measures; Membrane; Methods; Micelles; Molecular; Molecular Conformation; Mutation; Nature; PLA2G6 gene; Phosphatidylinositol 4,5-Diphosphate; Phospholipase; Phospholipase A2; Phospholipids; Physiological; Platelet Activating Factor; Play; Proteins; Recombinants; Regulation; Role; Specificity; Triglycerides; Water; Work; clinical development; disorder control; fatty liver disease; genome wide association study; group V secretory phospholipase A2; interest; lipidomics; lipoprotein-associated phospholipase A(2); macromolecule; macrophage; member; mutant; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; oxidized lipid; polyunsaturated fat

PROJECT SUMMARY/ABSTRACT The overall goal of this grant in recent years has been to describe in molecular detail the mechanism of action of physiologically important human forms of phospholipase A2 (PLA2). During the course of these studies, we have discovered that the activity of this superfamily of enzymes depends critically on the interaction of two large macromolecules (the protein and the large lipid aggregate), where the orientation of the enzyme with respect to the plane of the lipid-water interface can have a dramatic effect on activity. The nature of this interaction has been challenging to explore, but we have now shown that association of the membrane or micelle interface with the enzyme causes an allosteric activation through a resulting conformational change. This renewal application will extend our current studies on the pure recombinant human cytosolic Group IVA cPLA2, secretory Group V sPLA2, Ca2+-independent Group VIA iPLA2, and lipoprotein-associated PLA2/PAF (platelet-activating factor) acetyl hydrolase Group VIIA LpPLA2. During the renewal period, we will focus on three new directions. First, we will explore the further role of additional allosteric sites on iPLA2 (for ATP and calmodulin) and cPLA2 (for PIP2) for enzyme regulation and as drug targets. Second, we will expand and apply what we learned with phospholipases to triglyceride lipases starting with PNPLA3, which contains a patatin-like domain and is homologous to the catalytic domain of iPLA2. PNPLA3 is of great interest because GWAS studies have shown that a natural mutation (I148M) enriched in the Hispanic population leads to an increase in nonalcoholic steatohepatitis (NASH), the advanced form of nonalcoholic fatty liver disease (NAFLD). Our lipidomics analysis shows that the pure recombinant human mutant PNPLA3 has decreased triglyceride hydrolase activity and our MD studies show that the catalytic site has adopted to a triacylglyceride substrate rather than the phospholipid substrate in iPLA2. Third, we will explore the functioning and physiological role of the various intracellular phospholipase A2s in relevant intact cells, where the actual specificity will depend on the proximity and availability of optimal phospholipid molecular species. Although we have developed a novel lipidomics assay of PLA2 specificity and function in vitro, one barrier to progress in this field is the lack of methods for determining PLA2 activity in living cells. To address this issue, we have developed a new platform for measuring PLA2 specificity and inhibition ex vivo in macrophage cells in culture. This work has and will generate important widely applicable novel information on how physiologically important phospholipases and triacylglycerol lipases interact with the lipid-water interfaces of membranes, micelles and lipid droplets to compete physiologically in selecting their substrates. This work should enable us to fully explain and integrate at a structural level the resulting specificity of multiple members of the PLA2 superfamily acting in vitro with the specific molecular species of phospholipids hydrolyzed and the specific fatty acids released as well as correlating with ex vivo specificity.

项目概要/摘要 近年来这项资助的总体目标是详细描述分子机制 生理上重要的人类磷脂酶 A2 (PLA2) 的作用。在这些过程中 研究中，我们发现这个酶超家族的活性主要取决于 两个大分子（蛋白质和大脂质聚集体）的相互作用，其中 相对于脂质-水界面平面的酶可以对活性产生巨大影响。这 这种相互作用的本质一直难以探索，但我们现在已经证明， 膜或胶束与酶的界面通过产生的结果引起变构激活 构象变化。此次更新申请将扩展我们目前对纯重组体的研究 人胞浆 IVA 组 cPLA2、分泌 V 组 sPLA2、Ca2+ 独立 VIA 组 iPLA2 和 脂蛋白相关PLA2/PAF（血小板激活因子）乙酰水解酶VIIA组LpPLA2。期间 更新期间，我们将重点关注三个新方向。首先，我们将探讨额外的作用 iPLA2（用于 ATP 和钙调蛋白）和 cPLA2（用于 PIP2）上的变构位点，用于酶调节和作为药物 目标。其次，我们将扩展并应用我们在磷脂酶方面学到的知识到甘油三酯脂肪酶 从 PNPLA3 开始，它包含 patatin 样结构域，与 的催化结构域同源 iPLA2。 PNPLA3 引起了人们极大的兴趣，因为 GWAS 研究表明，它有一个自然突变 (I148M) 西班牙裔人口的丰富导致非酒精性脂肪性肝炎（NASH）的增加 晚期非酒精性脂肪肝（NAFLD）。我们的脂质组学分析表明，纯 重组人突变体 PNPLA3 降低了甘油三酯水解酶活性，我们的 MD 研究表明 催化位点已采用三酰甘油底物而不是磷脂底物 iPLA2。第三，我们将探讨各种细胞内磷脂酶的功能和生理作用 相关完整细胞中的 A2，其中实际特异性将取决于最佳的接近度和可用性 磷脂分子种类。尽管我们已经开发出一种新的 PLA2 特异性脂质组学检测方法 和体外功能，该领域进展的一个障碍是缺乏确定 PLA2 活性的方法 活细胞。为了解决这个问题，我们开发了一个新的平台来测量 PLA2 特异性和 培养物中巨噬细胞的离体抑制。这项工作已经并将产生重要的、广泛适用的 关于生理上重要的磷脂酶和三酰甘油脂肪酶如何与 膜、胶束和脂滴的脂水界面在生理上竞争选择 他们的底物。这项工作应该使我们能够在结构层面上充分解释和整合由此产生的结果 PLA2 超家族多个成员在体外与特定分子种类作用的特异性 磷脂水解和特定脂肪酸释放以及与离体特异性相关。