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: 8373199
• 负责人: Karen N. Allen
• 金额: $ 31.21万
• 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/8373199
• 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; Engineering; Enzymes; Equilibrium; Foundations; Gel; Gene Targeting; 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; Peroxisome Proliferator-Activated Receptors; 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. PUBLIC HEALTH RELEVANCE: By defining the structural features of enzymes that allow recognition of specific proteins and cell membrane components, the proposed interdisciplinary effort will provide significant insight into the complexities of cell lipid metabolism. The finding will lay the foundation for the rational design of therapeutic agents to treat the diseases associated with diabetes and clinically identified defects in fat metabolism.

描述（由适用提供）：二酰基甘油酸酯，三酰基甘油三酸酯和磷脂的前体是通过磷酸酯的水解反应源自膜磷脂酸（PA）的，这些水解反应由酶lipin的三种同工型催化。拟议的程序将重点介绍肝脏中的主要同工型，脂质代谢中心和脂肪组织（三酰基甘油酸甘油三酸酯合成中心）。 Lipin1, a key player Lipin1 functions on two levels: when translocated to the ER membrane lipin1 catalyzes PA hydrolysis and 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细胞位置取决于其磷酸化态，该磷酸化状态是由蛋白质磷酸酶Dullard介导的。 Lipin1膜结合和蛋白质合作蛋白结合以及Lipin1磷酸化/去磷酸化对于其两种功能的调节至关重要。三个目标将为理解人Lipin1调节和功能的复杂性提供结构和机制基础：目标1：确定Lipin1-Mmbrane结合，PA识别和催化转让的机理。将确定将确定与含有PA的磷脂蔬菜的Lipin结合，并确定溶液水解，短链PA和结合蔬菜的长链PA的稳态动力学恒定确定。野生型Lipin1和域构建体将经受X射线晶体学和溶液小角度X射线散射（SAXS）结构的测定。 AIM 2：描述钝化介导的Lipin1去磷酸化中底物识别和催化的结构决定剂。磷脂素1和lipin1衍生的磷酸肽的暗催化性去磷酸化的稳态动力学分析将定义底物特异性。 X射线结构的测定钝 - 基底/过渡状态模拟复合物将确定可能的底物结合和催化残差，这些残留物将通过对位置定向突变体的动力学分析进一步评估。 AIM 3。确定蛋白质 - 蛋白质相互作用负责Lipin1介导的转录激活。由转录因子PPAR形成的LIPIN1复合物和转录共激活因子PGC-1¿蛋白质 - 蛋白质相互作用将通过KD测定具有修饰结合基序的结合伴侣的复合物来检查。在适当的情况下，X射线晶体学，溶液SAX和蛋白质氘化交换研究将定义复杂的结构。 公共卫生相关性：通过定义允许识别特定蛋白质和细胞膜成分的酶的结构特征，拟议的跨学科工作将为细胞脂质代谢的复杂性提供重大见解。这一发现将为治疗与糖尿病和临床鉴定出脂肪代谢缺陷有关的治疗剂的理性设计奠定基础。