Structure and Function of HAD Phosphatase Partners Dullard and Lipin

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，由膜磷脂酸（PA）通过三种脂酶同工异构体催化的磷酸酯水解反应而得。拟建的项目将侧重于人类脂质1，它是肝脏中主要的同型异构体，是脂质代谢和脂肪组织中的中心，是三酰甘油合成的中心。脂质1在脂质稳态和膜生物生成中起着关键作用，对人体健康至关重要。脂质1突变与代谢综合征、2型糖尿病以及急性、复发性骨骼肌纤维破裂和易患他汀类药物引起的肌病（在美国有200万人患有这种疾病）有关。脂质1在两个层面上起作用：当转运到内质网膜时，脂质1催化PA水解；当转运到细胞核时，它作为转录共激活因子上调脂质代谢酶。脂质1的细胞位置取决于其磷酸化状态，这是由蛋白磷酸酶介导的。脂质1的膜结合和蛋白伴侣结合以及脂质1的磷酸化/去磷酸化是其两种功能调控的核心。三个目标将为理解人类脂素1调控和功能的复杂性提供结构和机制基础：目标1：确定脂素1膜结合、PA识别和催化周转的机制。脂素与含PA的磷脂囊泡结合及可溶性、短链PA和囊泡结合的长链PA水解的稳态动力学常数测定。野生型脂素1及其结构域将进行x射线晶体学和溶液小角x射线散射（SAXS）结构测定。目的2：描述暗质介导的脂质1去磷酸化中底物识别和催化的结构决定因素。杜拉德催化的磷脂1和磷脂1衍生的磷脂肽的去磷酸化的稳态动力学分析将确定底物特异性。钝化-底物/过渡态类似物的x射线结构测定将识别可能的底物结合和催化残基，这些残基将通过位点导向突变体的动力学分析进一步评估。目标3。确定负责脂素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¿和转录共激活剂PGC-1¿形成的Lipin1复合物将使用浆料-凝胶色谱和平衡/速度沉降技术进行分析，以定义亚单位化学计量学。蛋白质与蛋白质之间的相互作用将通过对具有修饰结合基序的结合伙伴的复合物的Kd测定来检验。在适当的情况下，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。

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.