Action of Lipolytic Enzymes

项目总结/摘要 近年来，这项资助的总体目标是在分子水平上详细描述 生理上重要的人类形式的磷脂酶A2（PLA 2）的作用。在此期间， 研究中，我们发现，这个超家族的酶的活性关键取决于 两个大的大分子（蛋白质和大的脂质聚集体）的相互作用，其中 相对于脂-水界面平面的酶可对活性具有显著影响。的 这种相互作用的性质一直是具有挑战性的探索，但我们现在已经表明， 膜或胶束与酶的界面通过所产生的 构象变化这一更新申请将扩展我们目前对纯重组的研究 人胞质组IVA cPLA 2、分泌组V sPLA 2、Ca 2+非依赖性组VIA iPLA 2，和 脂蛋白相关PLA 2/PAF（血小板活化因子）乙酰水解酶组VIIA LpPLA 2。期间 更新期间，我们将重点关注三个新方向。首先，我们将探讨进一步的作用， iPLA 2（用于ATP和钙调蛋白）和cPLA 2（用于PIP 2）上的变构位点，用于酶调节和作为药物 目标的其次，我们将扩大和应用我们学到的磷脂酶甘油三酯脂肪酶 从PNPLA 3开始，PNPLA 3含有马铃薯糖蛋白样结构域，并且与 iPLA 2。PNPLA 3引起了极大的兴趣，因为GWAS研究表明，一种天然突变（I148 M） 在西班牙裔人群中富集导致非酒精性脂肪性肝炎（NASH）增加， 晚期非酒精性脂肪性肝病（NAFLD）。我们的脂质组学分析表明， 重组人突变体PNPLA 3具有降低的甘油三酯水解酶活性，并且我们的MD研究显示 催化位点已经适应了三酰甘油酯底物而不是磷脂底物， iPLA 2。第三，我们将探讨各种细胞内磷脂酶的功能和生理作用， A2在相关完整细胞中的表达，其中实际特异性将取决于最佳表达的邻近性和可用性。 磷脂分子种类。虽然我们已经开发了一种新的磷脂酶A2特异性的脂质组学检测方法， 和功能，在该领域的进展的一个障碍是缺乏用于确定PLA 2活性的方法， 活细胞。为了解决这个问题，我们开发了一种新的平台来测量PLA 2特异性， 在培养的巨噬细胞中的离体抑制。这项工作已经并将产生重要的广泛适用 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， 它们的基质。这项工作应使我们能够充分解释和整合在结构层面上产生的 PLA 2超家族的多个成员在体外与特定分子种类的特异性 磷脂水解和释放的特定脂肪酸以及与离体特异性相关。

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.