Regulation of Phospholipid Synthesis

描述(申请人提供)：酵母酿酒酵母是研究脂肪合成调节的真核生物模型。PAH1编码的磷脂酸磷酸酶(PAP1)催化依赖于镁离子的磷脂酸去磷酸化生成二酰甘油和PI。这种酶产生二酰甘油，用于合成三酰甘油，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-二酰甘油途径合成磷脂的前体。对PAP1活性缺陷的酵母pah1突变体的分析表明，PAP1介导的磷脂酸含量的调控在含UASINO的磷脂合成基因的转录调控和核膜/内质网膜的生长中起主要作用。PAP1活性在脂质代谢中的重要性进一步得到强调，因为在小鼠中，这种酶(即脂蛋白1)的过度表达会导致肥胖，而它的缺失会导致脂肪营养不良和周围神经病变。此外，由PAP1酶介导的不受调控的脂质合成可能会导致代谢紊乱，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，从酵母中分离的酶中已鉴定出16个磷酸化位点。PAP1是几种调节细胞生理的蛋白激酶(如细胞周期蛋白依赖性蛋白激酶、蛋白激酶A和蛋白激酶C、酪蛋白激酶I和酪蛋白激酶II、Mpk1和Dbf2-Mob1)的靶标。PAP1的磷酸化调控其酶活性和细胞功能。在下一个授权期，我们建议进行生化和分子遗传学研究，以阐明调节PAH1编码的PAP1酶的复杂磷酸化。在特定的目标1中，我们将提出这样的假设，即PAP1是蛋白激酶的底物，预计将使酶中已识别的位点磷酸化，并检验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。建议的研究结果应该是对其他研究高等真核细胞中PAP1和脂质代谢的研究人员的极大兴趣，并补充他们的工作。公共卫生相关性：PAH1编码的磷脂酸磷酸酶(PAP1)酶催化磷脂酸脱磷生成二酰甘油和PI。二酰甘油用于合成三酰甘油和主要磷脂磷脂酰乙醇胺和磷脂酰胆碱的合成。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膜/内质网膜的生长中起着重要作用。PAP1活性在脂质代谢中的重要性进一步得到强调，因为在小鼠中，这种酶(即脂蛋白1)的过度表达会导致肥胖，而它的缺失会导致脂肪营养不良和周围神经病变。此外，由PAP1酶介导的不受调控的脂质合成可能会导致代谢紊乱，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.