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

脂质代谢酶磷脂酶 D 和脂质的结构和功能研究

• 批准号: 10220070
• 负责人: Michael Virgil Airola
• 金额: $ 39.23万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220070
• 关键词: Anabolism; Bioinformatics; Biological; Biological Assay; Cardiovascular Diseases; Catabolism; Catalysis; Cell Proliferation; Cells; Complement; Complex; Crystallization; Data; Deuterium; Diabetes Mellitus; Diglycerides; Disease; Enzymes; Fatty Acids; Heart Diseases; Human; Hydrogen; Hydrophobicity; In Vitro; Isoenzymes; Lecithin; Lipid Biochemistry; Lipids; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane; Metabolism; Molecular; Molecular Conformation; Molecular Structure; Mus; Obesity; Pharmacology; Phosphatidic Acid; Phospholipase D; Phosphoric Monoester Hydrolases; Physiological; Play; Protein Engineering; Proteins; Regulation; Role; Second Messenger Systems; Stimulus; Structure; Tertiary Protein Structure; Therapeutic; Triglycerides; Work; Yeasts; cancer therapy; cardiovascular disorder risk; cell motility; enzyme structure; extracellular; improved; innovation; insulin sensitivity; interest; interfacial; lipid metabolism; lipine; novel; receptor; response; small molecule; therapeutic target; three dimensional structure; trafficking

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.

项目摘要：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受到调节。我们当前的重点是两个 磷脂酸 (PA) 代谢中的酶：脂质和磷脂酶 D (PLD)。脂质是 脂质磷酸酶使 PA 去磷酸化生成二酰基甘油，这是倒数第二个 甘油三酯生物合成的步骤。脂质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对肥胖、糖尿病和糖尿病的影响 心血管疾病。 PLD 水解磷脂酰胆碱产生第二类脂质 PA 对细胞外刺激作出反应的信使。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 结构未表征的蛋白质。确定三维结构 这些酶单独存在，以及与它们的脂质底物、脂质激活剂和蛋白质复合 激活剂对于理解其功能和调节至关重要。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识别内源同源物 以及更适合结构研究的小鼠/人脂质和 PLD 构建体， 证明这些构建体在细胞和体外具有完全功能，并获得了衍射 优质晶体。结构研究将得到一系列脂质生物化学和 我们熟悉的脂质-蛋白质相互作用测定，氢-氘交换质量 光谱测定法以及哺乳动物细胞和酵母的细胞研究。合作者网络 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性底物并 在界面催化过程中与膜相互作用？ （2）小说在结构和结构上起什么作用？ 功能未表征的结构域在这些酶的作用中发挥作用？ (3) 血脂如何 激活这些酶？ (4)这些酶是如何调节的？具体来说，他们是 自抑制？蛋白质效应器如何激活它们？以及发生什么构象变化 激活期间？总的来说，这项工作将提高我们对生物机制的理解 并提供有关脂质-蛋白质相互作用的生理学和药理学信息 意义。此外，这项工作将有助于我们开发和改进小型计算机的长期兴趣 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。