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Action of Lipolytic Enzymes

脂肪分解酶的作用

基本信息

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
项目类别：
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

项目摘要

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.

项目总结/文摘