Phospholipid metabolism and membrane function

磷脂酸磷酸酶（PAP）催化磷脂酸（PA）的去磷酸化，生成二酰基甘油（DAG）和Pi。反应生成的DAG用于合成三酰基甘油（TAG），并通过肯尼迪途径合成磷脂酰乙醇胺和磷脂酰胆碱。通过其反应的性质，PAP还控制PA的细胞含量，PA是通过CDP-DAG途径合成的磷脂的前体。PA也是触发磷脂合成基因表达、膜扩张、囊泡运输、分泌和内吞作用的信号分子。我们从酵母、酿酒酵母（如PAH1）和哺乳动物（如LPIN1）中发现了PAP编码基因，从而使建立PAP在脂质代谢中的作用的生化和遗传学研究成为可能。PAP在脂质代谢中的重要性通过其突变表型得到例证。在酵母中，pah1突变体在磷脂合成基因的转录调控中表现出缺陷，核/内质网膜异常扩张，在固定期细胞中TAG含量降低90%。对小鼠和人类的研究表明，脂质1和脂质2的遗传缺陷表现在几种代谢性疾病中，包括脂肪营养不良、肥胖、周围神经病变、肌红蛋白尿和炎症。在下一个资助期，我们将重点研究酵母pah1编码的PAP酶在生长过程中的调控表达及其在脂质代谢中的作用（具体目标1）。此外，我们将扩大我们对PAP的研究，包括人类LPIN1-， LPIN2-和lpin3编码PAP亚型的酶学和生化调节（具体目标2）。我们还在酵母中发现了一种新的ctp依赖性dgk1编码的DAG激酶，该激酶可以平衡PAP的活性，从而控制PA和DAG的细胞含量。dgk1突变抑制pah1突变引起的表型，而DAG激酶的过表达会减弱PAP过表达引起的表型。在酵母中，TAG水解（如脂解）生成DAG和脂肪酸是膜磷脂合成和从固定阶段恢复生长的先决条件。我们将研究dgk1编码的DAG激酶在这种代谢中的作用和调节（特定目的3）。

Phosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG) and Pi. The DAG generated in the reaction is used for the synthesis of triacylglycerol (TAG), and for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the Kennedy pathway. By the nature of its reaction, PAP also controls the cellular content of PA, which is the precursor of phospholipids synthesized via the CDP-DAG pathway. PA is also a signaling molecule that triggers phospholipid synthesis gene expression, membrane expansion, vesicular trafficking, secretion, and endocytosis. Biochemical and genetic studies to establish the roles of PAP in lipid metabolism became possible by our discoveries of the PAP-encoding genes from the yeast Saccharomyces cerevisiae (e.g., PAH1) and mammals (e.g., LPIN1). The importance PAP in lipid metabolism is exemplified by its mutant phenotypes. In yeast, pah1 mutants exhibit defects in the transcriptional regulation of phospholipid synthesis genes, the anomalous expansion of the nuclear/ER membrane, and a 90 % reduction in TAG content in stationary phase cells. Studies with mice and humans have shown that genetic defects in lipin 1 and lipin 2 are manifested in several metabolic diseases that include lipodystrophy, obesity, peripheral neuropathy, myoglobinuria, and inflammation. In the next grant period, we will focus on the regulated expression of the yeast PAH1-encoded PAP enzyme during growth and its role in lipid metabolism (specific aim 1). In addition, we will expand our studies on PAP to include the enzymology and biochemical regulation of the human LPIN1-, LPIN2-, and LPIN3-encoded PAP isoforms (specific aim 2). We also discovered a novel CTP-dependent DGK1-encoded DAG kinase in yeast that counterbalanced the activity of PAP to control the cellular contents of PA and DAG. The dgk1 mutation suppresses phenotypes caused by the pah1 mutation, whereas the overexpression of DAG kinase blunts phenotypes caused by the overexpression of PAP. In yeast, TAG hydrolysis (e.g., lipolysis) to DAG and fatty acids is a prerequisite for membrane phospholipid synthesis and resumption of growth from the stationary phase. We will examine the role and regulation of the DGK1-encoded DAG kinase enzyme in this metabolism (specific aim 3).