Action of Lipolytic Enzymes

项目摘要/摘要 近年来，这项资助的总体目标一直是从分子上详细描述 生理上重要的人类形式磷脂酶A2(PLA2)的作用。在这个过程中 研究发现，这一超家族酶的活性严重依赖于 两个大分子(蛋白质和大的脂质聚集体)的相互作用，其中 相对于脂质-水界面平面的酶可以对活性有显著的影响。这个 这种相互作用的性质一直很难探索，但我们现在已经证明了 膜或胶束与酶的界面导致变构激活，通过产生 构象变化。这一更新应用将扩展我们目前对纯重组的研究 人胞浆组IVA cPLA2，分泌组V sPLA2，通过iPLA2的钙非依赖性组，以及 脂蛋白相关PLA2/PAF(血小板激活因子)乙酰水解酶组VIIA LpPLA2。在.期间 在续期期间，我们将重点关注三个新方向。首先，我们将探索额外的作用 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，我们将把我们所学到的磷脂酶应用于甘油三酯脂肪酶。 从PNPLA3开始，它包含一个Patatin样结构域，并与 IPLA2。PNPLA3之所以引起人们的极大兴趣，是因为Gwas的研究表明，一种自然突变(I148M) 拉美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，纯的 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 催化位采用三酰甘油底物，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。 A2在相关完整细胞中，其中实际特异性将取决于最佳的接近和可获得性 磷脂分子种类。尽管我们已经开发出一种新的PLA2特异性的脂质组学分析方法 和体外功能，这一领域取得进展的一个障碍是缺乏测定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的特异性和 体外抑制巨噬细胞培养。这项工作已经并将产生重要的广泛应用 生理上重要的磷脂酶和三酰甘油脂肪酶如何相互作用的新信息 膜、胶束和脂滴的脂水界面在选择中的生理竞争 它们的底物。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 PLA2超家族多个成员与特定分子物种体外作用的特异性 磷脂的降解和特异性脂肪酸的释放以及与体外特异性的相关性。

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.