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

项目摘要：我们感兴趣的是了解脂肪代谢酶如何 功能，并在分子和结构水平上受到调节。我们目前的重点是两个 磷脂酸代谢的酶：脂类和磷脂酶D(PLD)。脂类是 使PA去磷酸化以生成倒数第二个甘油的脂磷酸酶 参与甘油三酯的生物合成。脂类调节甘油三酯的生物合成，甘油三酯的分解代谢， 脂肪酸合成和胰岛素敏感性与肥胖、糖尿病和 心血管疾病。磷脂酰胆碱水解磷脂酰胆碱生成第二脂 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。脂类和PLD都是高度受调控的、多领域的 结构上没有特征的蛋白质。确定物体的三维结构 这些酶单独存在，并与它们的脂质底物、脂质激活剂和蛋白质形成复合体 激活剂是理解其功能和调节的关键。在初步数据中，我们 使用创新的生物信息学和蛋白质工程来鉴定内源性同源物 以及更适合结构研究的鼠/人脂类和PLD的构建， 证明了这些构建物在细胞和体外都具有完全的功能，并获得了衍射 优质水晶。结构研究将得到一系列脂质生物化学和 脂质-蛋白质相互作用分析表明，我们对氢-重离子交换质量非常熟悉 在哺乳动物细胞和酵母中的光谱分析和细胞研究。一个合作者网络， 是各自领域的领军人物，为这些研究提供支持。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和 在界面催化过程中与膜的相互作用？(2)新颖的结构和 功能未知的结构域在这些酶的作用中发挥作用？(3)脂类如何 激活这些酶？(4)这些酶是如何调节的？具体地说，他们是 自闭症？蛋白质效应器是如何激活它们的？以及发生了什么构象变化 在激活期间？总体而言，这项工作将提高我们对生物机制的理解 并提供了有关生理和药理作用的脂质-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远利益，发展和改进小 这些酶的分子调节剂，这将具有潜在的治疗应用 癌症、心血管疾病和糖尿病的治疗。

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.