Structure and Function of HAD Phosphatase Partners Dullard and Lipin

HAD 磷酸酶伙伴 Dullard 和 Lipin 的结构和功能

• 批准号: 8668084
• 负责人: Karen N. Allen
• 金额: $ 31.51万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8668084
• 关键词: Acute; Address; Adipose tissue; Affinity; Binding; Binding Proteins; Biochemical; Biological Assay; Catalysis; Cell Membrane Proteins; Cell Nucleus; Cells; Cellular biology; Complex; Computer Simulation; DNA Binding; Defect; Deuterium; Diabetes Mellitus; Disease; Enzymes; Equilibrium; Foundations; Gel; Gene Targeting; Genetic Engineering; Goals; Health; Homeostasis; Human; Hydrogen; Hydrolysis; Insulin; Kinetics; Ligands; Link; Lipids; Lipoproteins; Liver; Location; Measures; Mediating; Membrane; Metabolic syndrome; Muscle Fibers; Mutagenesis; Mutation; Myopathy; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Envelope; Obesity; Peptides; Phosphatidic Acid; Phospholipid Metabolism; Phospholipids; Phosphopeptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Predisposition; Property; Protein Dephosphorylation; Protein Isoforms; Protein phosphatase; Proteins; Reaction; Recruitment Activity; Recurrence; Regulation; Reporting; Roentgen Rays; Role; Site; Solutions; Structural Models; Structure; Substrate Specificity; Techniques; Testing; Therapeutic Agents; Tissues; Transcription Coactivator; Transcriptional Activation; Triglyceride Metabolism; Triglycerides; United States; Vesicle; analog; base; design; in vitro Assay; insight; lipid metabolism; lipine; membrane biogenesis; mutant; phosphate ester; programs; protein protein interaction; receptor; research study; response; sedimentation velocity; stoichiometry; transcription factor

DESCRIPTION (provided by applicant): Diacylglycerides, the precursors to triacylglycerides and phospholipids, are derived from membrane phosphatidic acids (PA) through the phosphate ester hydrolysis reaction catalyzed by three isoforms of the enzyme lipin. The proposed program will focus on human lipin1, the predominant isoform in the liver, the center of lipid metabolism and in adipose tissue, the center for triacylglyceride synthesis. Lipin1, a key player in lipid homeostasis and membrane biogenesis, is essential to human health. Lipin1 mutations are linked to metabolic syndrome and type-2 diabetes as well as acute, recurrent breakdown of skeletal muscle fibers and susceptibility to statin-induced myopathy (suffered by 2 million people in the United States). Lipin1 functions on two levels: when translocated to the ER membrane lipin1 catalyzes PA hydrolysis and when translocated to the nucleus it acts as a transcriptional co-activator to up-regulate lipid-metabolizing enzymes. Lipin1 cellular location depends on its phosphorylation state, which is mediated by the protein phosphatase, dullard. Lipin1 membrane binding and protein-partner binding as well as lipin1 phosphorylation/ dephosphorylation are central to the regulation of its two functions. Three aims will provide a structural and mechanisti basis for understanding the complexities of human lipin1 regulation and function: Aim 1: Determine the mechanism of lipin1-membrane binding, PA recognition and catalytic turnover. Lipin binding to PA-containing phospholipid vesicles and steady-state kinetic constant determination of hydrolysis of soluble, short-chain PA and vesicle-bound long-chain PA will be determined. Wild-type lipin1 and domain constructs will be subjected to X-ray crystallographic and solution small angle X-ray scattering (SAXS) structure determination. Aim 2: Delineate the structural determinants of substrate recognition and catalysis in dullard-mediated lipin1 dephosphorylation. The steady-state kinetic analysis of dullard-catalyzed dephosphorylation of phospholipin1 and lipin1-derived phosphopeptides will define substrate specificity. X-ray structure determination of dullard-substrate/transition state analog complexes will identify possible substrate-binding and catalytic residues which will be further evaluated through kinetic analysis of site-directed mutants. Aim 3. Identify the protein-protein interactions responsible fo lipin1-mediated transcriptional activation. Lipin1 complexes formed with the transcription factor PPAR¿ and transcriptional co-activator PGC-1¿ will be analyzed using sizing-gel chromatographic and equilibrium/velocity sedimentation techniques to define subunit stoichiometry. Protein-protein interactions will be examined by Kd determinations of complexes of binding partners having modified binding motifs. Where appropriate, X-ray crystallographic, solution SAXS and protein deuterium-hydrogen exchange studies will define complex structures.

描述（由申请人提供）：二酰基甘油酯是三酰基甘油酯和磷脂的前体，是通过脂质酶的三种亚型催化的磷酸酯水解反应衍生自膜磷脂酸（PA）。肝脏（脂质代谢中心）和脂肪组织（脂肪组织）中的主要异构体三酰基甘油酯合成是脂质稳态和膜生物发生的关键因素，对人类健康至关重要，Lipin1 突变与代谢综合征和 2 型糖尿病以及骨骼肌纤维急性、复发性损伤和对他汀类药物的易感性有关。肌病（美国有 200 万人患有这种疾病）。Lipin1 在两个层面上发挥作用：当转移到 ER 膜时，lipin1 会催化 PA。水解，当转移到细胞核时，它作为转录共激活剂上调脂质代谢酶，其细胞位置取决于其磷酸化状态，这是由蛋白质磷酸酶、Lipin1 膜结合和蛋白质伴侣介导的。结合以及 lipin1 磷酸化/去磷酸化是其两个功能调节的核心，三个目标将为理解复杂性提供结构和机制基础。人类 lipin1 调节和功能的研究： 目标 1：确定 lipin1 膜结合、PA 识别和催化周转的机制，Lipin 与含 PA 的磷脂囊泡结合，并测定可溶性短链 PA 水解的稳态动力学常数。将对囊泡结合的长链 PA 进行测定，并对野生型 lipin1 和结构域构建体进行 X 射线晶体学和溶液小角度 X 射线分析。目标 2：描述 dullard 介导的 lipin1 去磷酸化中底物识别和催化的结构决定因素 dullard 催化的磷脂 1 和 lipin1 衍生磷酸肽的去磷酸化的稳态动力学分析将定义特定的底物性。迪拉德底物/过渡态类似复合物的射线结构测定将识别可能的底物结合和催化残基，并将对其进行进一步评估通过定点突变体的动力学分析。目标 3. 确定与转录因子 PPAR 形成的 Lipin1 介导的转录激活相关的蛋白质-蛋白质相互作用。和转录共激活因子 PGC-1¿将使用尺寸凝胶色谱和平衡/速度沉降技术进行分析，以定义亚基化学计量，将通过具有修饰的结合基序的结合配偶体的Kd测定来检查（在适当情况下，X射线晶体学、溶液SAXS和）。蛋白质氘氢交换研究将定义复杂的结构。