Structural and Functional Studies of the Lipid Metabolizing Enzymes Phospholipase D and Lipin

项目概述：我们有兴趣了解脂质代谢酶如何 功能，并在分子和结构水平上进行调节。我们目前的重点是两个 磷脂酸（PA）代谢中的酶：Lipin和磷脂酶D（PLD）。Lipins是 使PA脱磷酸化以产生二酰基甘油的脂质磷酸酶，二酰基甘油是倒数第二个 甘油三酸酯生物合成步骤。脂类调节甘油三酯生物合成，甘油三酯催化剂， 脂肪酸合成和胰岛素敏感性与肥胖、糖尿病和 心血管疾病PLD水解磷脂酰胆碱产生脂质， 信使PA对细胞外刺激的反应。受体介导的PLD激活 调节囊泡运输、细胞增殖和细胞迁移，从而建立了它们 作为癌症的治疗靶点。Lipin和PLD都是高度调控的多结构域 结构上没有特征的蛋白质。确定的三维结构的 这些酶单独以及与它们的脂质底物、脂质活化剂和蛋白质的复合物 激活剂对于理解其功能和调节至关重要。根据初步数据，我们 利用创新的生物信息学和蛋白质工程来鉴定内源性同源物 以及更适合于结构研究的小鼠/人Lipin和PLD的构建体， 证明了这些构建体在细胞和体外都是完全功能性的，并获得了衍射 优质水晶结构研究将由一系列脂质生物化学和 我们熟悉的脂质-蛋白质相互作用分析，氢氘交换质量 光谱法和哺乳动物细胞和酵母中的细胞研究。一个合作者网络， 他们各自领域的领导者支持这些研究。我们的目标是回答几个主要问题， 问题：（1）脂质修饰酶如何识别其疏水底物， 在界面催化过程中与膜相互作用？(2)小说在结构上和 在这些酶的作用中发挥作用的功能不明的结构域？(3)脂质如何 激活这些酶？(4)这些酶是如何调节的？具体地说， 自我抑制的？蛋白质效应物如何激活它们？以及发生了什么样的构象变化 在激活？总的来说，这项工作将提高我们对生物机制的理解。 并提供有关生理和药理学的脂质-蛋白质相互作用的信息 意义此外，这项工作将有助于我们的长期利益，发展和改善小 这些酶的分子调节剂，这将具有潜在的治疗应用， 治疗癌症、心血管疾病和糖尿病。

PROJECT SUMMARY: We are interested in understanding how lipid-metabolizing enzymes function and are regulated at the molecular and structural level. Our current focus is on two enzymes in phosphatidic acid (PA) metabolism: Lipin and phospholipase D (PLD). Lipins are lipid phosphatases that dephosphorylate PA to generate diacylglycerol, which is the penultimate step in triglyceride biosynthesis. Lipins regulate triglyceride biosynthesis, triglyceride catabolism, 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. Both Lipin and PLD are highly regulated, multi-domain proteins that are structurally uncharacterized. Determining the three-dimensional structures of these enzymes alone, and in complex with their lipid substrates, lipid activators, and protein activators is essential to understanding their function and regulation. In preliminary data, we have used innovative bioinformatics and protein engineering to identify endogenous homologs and constructs of mouse/human Lipin and PLD that are more amenable for structural studies, demonstrated these constructs are fully functional in cells and in vitro, and obtained diffraction quality crystals. 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 and yeast. A network of collaborators who are leaders in their respective fields supports these studies. We aim to answer several major questions: (1) How do lipid-modifying enzymes recognize their hydrophobic substrates and interact with the membrane during interfacial catalysis? (2) What role do novel structurally and functionally uncharacterized domains play in the action of these enzymes? (3) How do lipids activate these enzymes? (4) How are these enzymes regulated? Specifically, are they autoinhibited? How do protein effectors activate them? 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. In addition, this work will aid our long-term interest to develop and improve small molecule modulators of these enzymes, which would have potential therapeutic applications for the treatment of cancer, cardiovascular disease, and diabetes.