Structure and Function of HAD Phosphatase Partners Dullard and Lipin

描述(申请人提供)：二酰基甘油酯是三酰甘油酯和磷脂的前体，由膜磷脂酸(PA)通过三种不同类型的脂酶催化的磷酸酯水解反应而得。拟议的计划将专注于人类脂蛋白1，肝脏中的主要异构体，脂类代谢的中心，以及脂肪组织中三酰甘油合成的中心。脂蛋白1是脂类动态平衡和膜生物发生的关键分子，对人类健康至关重要。Lipin1基因突变与代谢综合征、2型糖尿病、骨骼肌纤维急性反复受损以及他汀类药物引起的肌病的易感性有关(美国有200万人患有这种病)。脂蛋白1在两个水平上发挥作用：当转位到内质网膜上时，它催化PA的水解；当转位到细胞核时，它作为转录辅助激活因子来上调脂质代谢酶。Lipin1的细胞定位取决于它的磷酸化状态，而磷酸化状态是由蛋白质磷酸酶DULLAD介导的。脂蛋白1的膜结合和蛋白伴侣结合以及脂蛋白1的磷酸化/去磷酸化是其两个功能调节的中心。三个目标将为理解人类脂蛋白1调控和功能的复杂性提供结构和机制基础：目标1：确定脂蛋白1的机制-膜结合、PA识别和催化周转。脂类与含有PA的磷脂囊泡的结合以及可溶性、短链PA和囊泡结合的长链PA的稳态动力学常数的测定将被测定。野生型脂蛋白1和结构域结构将接受X射线结晶学和溶液小角X射线散射(SAXS)结构测定。目的2：描述大头鸭介导的脂蛋白1去磷酸化中底物识别和催化的结构决定因素。对杜鹃催化的磷脂1和脂蛋白1衍生的磷酸肽的脱磷的稳态动力学分析将确定底物的专一性。对杜鹃-底物/过渡态类似物的X射线结构测定将确定可能的底物结合和催化残基，这将通过定点突变的动力学分析来进一步评估。目的3.确定脂蛋白1介导的转录激活的蛋白质-蛋白质相互作用。与转录因子PPAR和转录共激活因子PGC-1形成的Lipin1复合体将使用尺寸凝胶层析和平衡/速度沉淀技术进行分析，以确定亚基的化学计量比。蛋白质之间的相互作用将通过对具有修改的结合基序的结合伙伴的络合物的Kd测定来检查。在适当的情况下，X射线结晶学、溶液SAXS和蛋白质氢-氢交换研究将确定复杂的结构。 与公共健康相关：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，拟议的跨学科努力将提供对细胞脂代谢复杂性的重要洞察。这一发现将为合理设计治疗药物以治疗与糖尿病相关的疾病和临床上发现的脂肪代谢缺陷奠定基础。

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.