Action of Lipolytic Enzymes

脂肪分解酶的作用

• 批准号: 10544745
• 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/10544745
• 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; Learning; Lipase; Lipids; Macrophage; Measures; Membrane; Methods; Micelles; Molecular; Molecular Conformation; Mutation; Nature; 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; member; mutant; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; oxidized lipid; polyunsaturated fat; 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; Learning; Lipase; Lipids; Macrophage; Measures; Membrane; Methods; Micelles; Molecular; Molecular Conformation; Mutation; Nature; 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; 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.

项目摘要/摘要 近年来，这笔赠款的总体目标是用分子详细描述 生理上重要的人类形式的磷脂酶A2（PLA2）的作用。在这些过程中 研究，我们发现该酶超家族的活性取决于 两种大型大分子（蛋白质和大脂质骨料）的相互作用，其中的方向 相对于脂质 - 水界面平面的酶可能会对活性产生巨大影响。这 这种互动的性质探索一直具有挑战性，但是我们现在表明 与酶的膜或胶束界面通过结果引起变构激活 构象变化。这种更新应用将扩展我们对纯重组的当前研究 人类胞质组IVA CPLA2，分泌组V SPLA2，Ca2+非依赖性组通过IPLA2和 脂蛋白相关的PLA2/PAF（血小板激活因子）乙酰水解酶组VIIA LPPLA2。在 续签时期，我们将重点关注三个新方向。首先，我们将探讨其他额外的作用 IPLA2（用于ATP和钙调蛋白）和CPLA2（用于PIP2）的变构位点用于酶调节，并作为药物 目标。其次，我们将扩展并应用我们所学的磷脂酶到甘油三酸酯脂肪酶 从pnpla3开始，该pnpla3包含一个类似patatin的结构域，与催化域同源 ipla2。 PNPLA3引起了极大的兴趣，因为GWAS研究表明自然突变（I148M） 富集西班牙裔人口会导致非酒精性脂肪性肝炎（NASH）的增加 非酒精性脂肪肝病（NAFLD）的晚期形式。我们的脂质分析分析表明纯 重组人突变体PNPLA3的甘油三酸酯水解酶活性降低，我们的MD研究表明 催化位点已用于三酰基甘油酸酯底物，而不是磷脂底物 ipla2。第三，我们将探索各种细胞内磷脂酶的功能和生理作用 相关完整单元格中的A2，其中实际特异性取决于最佳的接近性和可用性 磷脂分子物种。尽管我们已经开发了一种新型的PLA2特异性脂质组学测定 在体外功能，该领域进步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衡量PLA2特异性和 在培养中巨噬细胞中的抑制作用。这项工作已经并且将产生重要的广泛适用 有关生理上重要的磷脂酶和三酰基甘油脂肪酶如何相互作用的新信息 膜，胶束和脂质液滴的脂质 - 水界面，可在生理上进行竞争 他们的底物。这项工作应该使我们能够在结构层面充分解释和整合。 Pla2超家族多个成员在体外作用于特定分子物种的特异性 磷脂水解和释放的特异性脂肪酸以及与离体特异性相关的。