Regulation of Phospholipid Synthesis

描述（由申请人提供）：酵母酿酒酵母是研究脂质合成调控的真核生物模型。pah1编码的磷脂酸磷酸酶（PAP1）催化磷脂酸的Mg2+依赖性去磷酸化，生成二酰基甘油和Pi。该酶产生二酰基甘油，用于合成三酰基甘油，并通过肯尼迪途径合成磷脂酰乙醇胺和磷脂酰胆碱。PAP1活性还控制其底物磷脂酸的细胞浓度，磷脂酸是通过CDP-二酰基甘油途径合成的磷脂的前体。对PAP1活性缺陷酵母pah1突变体的分析表明，PAP1介导的磷脂酸含量控制在含有uasino的磷脂合成基因的转录调控和核/内质网膜的生长中起主要作用。在小鼠中，PAP1酶（即脂质1）的过表达导致肥胖，而其缺失导致脂肪营养不良和周围神经病变，这一事实进一步强调了PAP1活性在脂质代谢中的重要性。此外，由PAP1酶介导的不受调节的脂质合成可能导致代谢紊乱，如胰岛素抵抗、糖尿病和心脏病。pah1编码的PAP1已被鉴定为一种磷酸化蛋白，并从酵母分离的酶中鉴定出16个磷酸化位点。PAP1是几种调节细胞生理的蛋白激酶（如周期蛋白依赖性蛋白激酶、蛋白激酶a和C、酪蛋白激酶I和II、Mpk1和Dbf2-Mob1）的靶标。PAP1的磷酸化控制着它的酶活性和细胞功能。在接下来的资助期内，我们计划进行生化和分子遗传学研究，以阐明调控pah1编码的PAP1酶的复杂磷酸化。在具体的目标1中，我们将解决PAP1是蛋白激酶的底物的假设，预测磷酸化酶中已确定的位点，并检查这些磷酸化的相互依赖性。在目标2中，我们将研究磷酸化对PAP1活性及其细胞位置的影响，并研究这些磷酸化的生理相关性。提出的研究结果应该引起人们的极大兴趣，并补充其他研究人员在高等真核细胞中研究PAP1和脂质代谢的工作。公共卫生相关性：pah1编码的磷脂酸磷酸酶（PAP1）酶催化磷脂酸的去磷酸化，生成二酰基甘油和Pi。二酰基甘油用于合成三酰基甘油，并用于合成主要磷脂磷脂酰乙醇胺和磷脂酰胆碱。pap1介导的磷脂酸含量调控在磷脂合成基因的转录调控和核/内质网膜的生长中起主要作用。在小鼠中，PAP1酶（即脂质1）的过表达导致肥胖，而其缺失导致脂肪营养不良和周围神经病变，这一事实进一步强调了PAP1活性在脂质代谢中的重要性。此外，由PAP1酶介导的不受调节的脂质合成可能导致代谢紊乱，如胰岛素抵抗、糖尿病和心脏病。

DESCRIPTION (provided by applicant): The yeast Saccharomyces cerevisiae serves as a model eukaryotic organism to study the regulation of lipid synthesis. The PAH1-encoded phosphatidic acid phosphatase (PAP1) catalyzes the Mg2+dependent dephosphorylation of phosphatidic acid to yield diacylglycerol and Pi. This enzyme generates the diacylglycerol used for the synthesis of triacylglycerol and for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the Kennedy pathway. PAP1 activity also controls the cellular concentration of its substrate phosphatidic acid, which is the precursor for phospholipids that are synthesized via the CDP- diacylglycerol pathway. The analysis of yeast pah1 mutants defective in PAP1 activity has shown that the PAP1-mediated control of phosphatidic acid content plays a major role in the transcriptional regulation of UASINO-containing phospholipid synthesis genes and the growth of the nuclear/ endoplasmic reticulum membrane. The importance of PAP1 activity in lipid metabolism is further emphasized by the fact that overexpression of the enzyme (i.e., lipin 1) in mice leads to obesity while its loss leads to lipodystrophy and peripheral neuropathy. Moreover, unregulated lipid synthesis, as mediated by the PAP1 enzyme, may cause metabolic disorders such as insulin resistance, diabetes, and heart disease. The PAH1-encoded PAP1 has been identified as a phosphoprotein, and sixteen phosphorylation sites have been identified from the enzyme isolated from yeast. PAP1 is a target for several protein kinases (e.g., cyclin-dependent protein kinase, protein kinases A and C, casein kinases I and II, Mpk1, and Dbf2-Mob1) that regulate cell physiology. The phosphorylation of PAP1 governs its enzymatic activity and its cellular functions. In the next grant period, we propose biochemical and molecular genetic studies to elucidate the complex phosphorylation that regulates the PAH1-encoded PAP1 enzyme. In specific aim 1, we will address the hypothesis that PAP1 is a substrate for protein kinases predicted to phosphorylate the identified sites in the enzyme, and to examine the interdependencies of these phosphorylations. In aim 2, we will examine the effects of the phosphorylations on PAP1 activity and its cellular location, and examine the physiological relevance of these phosphorylations. The results of the proposed studies should be of great interest to, and complement the work of other investigators who study PAP1 and lipid metabolism in higher eukaryotic cells. PUBLIC HEALTH RELEVANCE: The PAH1-encoded phosphatidic acid phosphatase (PAP1) enzyme catalyzes the dephosphorylation of phosphatidic acid to yield diacylglycerol and Pi. The diacylglycerol is used for the synthesis of triacylglycerol and for the synthesis of the major phospholipids phosphatidylethanolamine and phosphatidylcholine. The PAP1-mediated control of phosphatidic acid content plays a major role in the transcriptional regulation of phospholipid synthesis genes and the growth of the nuclear/endoplasmic reticulum membrane. The importance of PAP1 activity in lipid metabolism is further emphasized by the fact that overexpression of the enzyme (i.e., lipin 1) in mice leads to obesity while its loss leads to lipodystrophy and peripheral neuropathy. Moreover, unregulated lipid synthesis, as mediated by the PAP1 enzyme, may cause metabolic disorders such as insulin resistance, diabetes, and heart disease.