Structure and Function of HAD Phosphatase Partners Dullard and Lipin

描述（由申请人提供）：二酰基甘油酯（三酰基甘油酯和磷脂的前体）通过由酶lipin的三种亚型催化的磷酸酯水解反应从膜磷脂酸（PA）衍生而来。拟议的计划将集中在人类lipin 1，在肝脏中的主要亚型，脂质代谢的中心和脂肪组织中，三酰甘油合成的中心。Lipin 1是脂质稳态和膜生物合成的关键参与者，对人类健康至关重要。Lipin 1突变与代谢综合征和2型糖尿病以及骨骼肌纤维的急性、复发性断裂和他汀类药物诱导的肌病易感性（美国有200万人患有这种疾病）有关。Lipin 1在两个水平上起作用：当移位到ER膜时，Lipin 1催化PA水解，当移位到细胞核时，它作为转录共激活剂上调脂质代谢酶。Lipin 1的细胞定位取决于其磷酸化状态，磷酸化状态由蛋白磷酸酶dullard介导。lipin 1膜结合和蛋白质伴侣结合以及lipin 1磷酸化/去磷酸化是其两种功能调节的核心。三个目标将提供一个结构和mechanisti的基础，了解人类lipin 1的调控和功能的复杂性：目标1：确定lipin 1的膜结合，PA识别和催化周转的机制。将测定Lipin与含PA磷脂囊泡的结合以及可溶性短链PA和囊泡结合长链PA水解的稳态动力学常数。野生型lipin 1和结构域构建体将进行X射线晶体学和溶液小角X射线散射（SAXS）结构测定。 目的2：阐明dullard介导的lipin 1去磷酸化中底物识别和催化的结构决定因素。磷脂1和脂1衍生的磷酸肽的Dullard催化的去磷酸化的稳态动力学分析将定义底物特异性。X-射线结构测定dullard底物/过渡态类似物复合物将确定可能的底物结合和催化残基，这将通过定点突变体的动力学分析进一步评估。 目标3。确定负责lipin 1介导的转录激活的蛋白质-蛋白质相互作用。Lipin 1与转录因子PPAR和转录辅激活因子PGC-1形成的复合物将使用胶凝色谱和平衡/速度沉降技术进行分析，以确定亚基化学计量。蛋白质-蛋白质相互作用将通过具有修饰的结合基序的结合配偶体的复合物的Kd测定来检查。在适当的情况下，X射线晶体学，溶液SAXS和蛋白质氘-氢交换研究将确定复杂的结构。

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.