Structure and regulation of lipid metabolism and transport

项目概述：我们有兴趣了解脂质代谢酶和脂质转运 蛋白质在分子和结构水平上起作用并受到调节。之前，我们关注了两种酶 在磷脂酸（PA）代谢中：脂蛋白和磷脂酶D（PLD）。脂蛋白是PA磷酸酶， PA脱磷酸化以产生二酰基甘油，这是甘油三酯生物合成中的倒数第二步。利平 调节磷脂和脂蛋白的合成，脂肪储存如甘油三酯，脂肪酸合成，和胰岛素 对肥胖、糖尿病和心血管疾病的敏感性。PLD水解 磷脂酰胆碱产生脂质第二信使PA响应细胞外刺激。受体- 介导的PLD激活调节囊泡运输、细胞增殖和细胞迁移， 使它们成为癌症的治疗靶点。尽管对这些高度管制的，多- 虽然我们对结构域酶的研究还处于起步阶段，但我们对其分子作用机制的认识仍存在重大空白。期间 在过去的资助期间，我们确定了lipin和PLD的第一个结构，确定了一种新型的膜- 结合结构域的脂质，并提供了深入了解PLD激活脂质和蛋白质效应。这些 这些发现为这一应用提供了良好的基础，我们的主要目标包括：1）继续 了解PLD是如何被蛋白质效应物和脂质激活的，以及2）检查分子机制 调节脂蛋白PA磷酸酶活性和PA底物特异性。我们还将从生物化学的角度， 在结构上表征核膜蛋白磷酸酶复合物CTDNEP 1-NEP 1 R1，其 研究Nir脂质的结构和功能 转移蛋白，其在膜接触位点之间转移PA和磷脂酰肌醇以补充池 用PLC水解后的质膜磷酸肌醇。结构研究将由一个 我们熟知的一系列脂质生物化学和脂质-蛋白质相互作用测定，氢氘 交换质谱和哺乳动物细胞中的细胞研究。一个合作者网络， 他们各自领域的领导人支持这些研究。我们旨在回答几个主要问题：（1）如何 脂质修饰酶的结构是否允许它们识别它们的脂质底物并与脂质底物相互作用？ 膜？(2)结构上和/或功能上未表征的结构域在这些结构域的作用中起什么作用？ 蛋白质？(3)这些酶/蛋白质是如何调节的？具体来说，它们是自我抑制的吗？蛋白质如何 效应物、脂质、离子和翻译后修饰调节活性？什么样的构象变化 在激活期间发生？总的来说，这项工作将提高我们对生物机制的理解，并提供 具有生理学和药理学意义的脂质-蛋白质相互作用的信息。

PROJECT SUMMARY: We are interested in understanding how lipid-metabolizing enzymes and lipid transport proteins function and are regulated at the molecular and structural level. Previously, we focused on two enzymes in phosphatidic acid (PA) metabolism: lipin and phospholipase D (PLD). Lipins are PA phosphatases that dephosphorylate PA to generate diacylglycerol, which is the penultimate step in triglyceride biosynthesis. Lipins regulate phospholipid and lipoprotein synthesis, fat storage as triglycerides, fatty acid synthesis, and insulin sensitivity with implications to obesity, diabetes, and cardiovascular disease. PLDs hydrolyze phosphatidylcholine to produce the lipid second messenger PA in response to extracellular stimuli. Receptor- mediated activation of PLD regulates vesicular trafficking, cell proliferation, and cell migration, which has established them as therapeutic targets for cancer. Despite extensive studies on these highly regulated, multi- domain enzymes, significant gaps remain in our knowledge of their molecular mechanisms of action. During the past funding period, we determined the first structures of lipin and PLD, identified a new type of membrane- binding domain in lipins, and provided insight into PLD activation by lipids and protein effectors. These discoveries provide an excellent foundation for this application, where our main goals include 1) continuing to understand how PLD is activated by protein effectors and lipids, and 2) examining the molecular mechanisms regulating lipin PA phosphatase activity and PA substrate specificity. We will also 3) biochemically and structurally characterize the nuclear envelope protein phosphatase complex CTDNEP1-NEP1R1, which dephosphorylates lipin to control lipin PA phosphatase activity; and 4) study the structure and function of Nir lipid transfer proteins, which transfer PA and phosphatidylinositol between membrane contact sites to replenish pools of plasma membrane phosphoinositides after hydrolysis by PLC. Structural studies will be complemented by an array of lipid biochemistry and lipid-protein interaction assays that we are well versed in, hydrogen-deuterium exchange mass spectrometry, and cellular studies in mammalian cells. A network of collaborators who are leaders in their respective fields supports these studies. We aim to answer several major questions: (1) How does the structure of lipid-modifying enzymes allow them to recognize their lipid substrates and interact with the membrane? (2) What role do structurally and/or functionally uncharacterized domains play in the action of these proteins? (3) How are these enzymes/proteins regulated? Specifically, are they autoinhibited? how do protein effectors, lipids, ions, and post-translational modifications regulate activity? and what conformational changes occur during activation? Overall, this work will improve our understanding of biological mechanisms and provide information on lipid-protein interactions of physiological and pharmacological significance.