Novel Roles for Lysophospholippids in Eukaryotic Membrane Biogenesis and Turnover

描述(申请人提供)：溶血磷脂是一类膜甘油磷脂，可被从酵母到人类的许多真核细胞快速转运和代谢。我们最近鉴定了两种酵母转运蛋白，它们是真核生物中发现的主要溶血磷脂，即溶血磷脂酰乙醇胺(lyso-PtdEtn)和溶血磷脂酰胆碱(lyso-PtdCho)。酵母lyso-PtdEtn和lyso-PtdCho转运蛋白也是参与调节磷脂酰丝氨酸和磷脂酰乙醇胺质膜不对称性的分子。这些转运蛋白的后生动物形式在向吞噬细胞发出靶细胞的凋亡状态信号方面起着至关重要的作用。哺乳动物转运蛋白功能缺陷与人类原发性家族性肝内胆汁淤积症和良性复发性肝内胆汁淤积症有关。酵母中的遗传和生化研究表明，其他的lyso-PtdCho和lyso-PtdEtn转运蛋白仍有待鉴定。这项提案的第一个具体目标将集中在识别剩余酵母溶血磷脂转运蛋白的基因，并对它们进行生化表征。第二个具体目标将利用酵母遗传学的强大工具，从结构上定义酵母和哺乳动物转运蛋白识别其底物并将其引入运输通道的机制基础。在被酵母和哺乳动物细胞摄取后，lyso-PtdCho和lyso-PtdEtn通过酰基转移酶代谢成它们的双酰基对应物PtdCho和PtdEtn。虽然酶的活性已经知道40多年了，但我们最近在酵母上的工作首次定义了执行这些反应的基因。酵母基因ALE1是多种真核生物中的一个家族的创始成员，该家族参与了溶血磷脂与二不饱和和多不饱和脂肪酸的酰化反应。在第三个特定目标中，我们将利用酵母遗传学的力量来阐明属于ALE1家族的后生动物酰基转移酶的生化活性和功能。遗传学研究已经表明，果蝇ALE1家族基因的损伤会导致发育缺陷和雄性不育。对人类的研究表明，ALE1家族基因的缺陷会导致手和骨骼的发育缺陷，也会导致男性不育。酵母中的生化研究将通过在哺乳动物细胞中的详细研究来检验ALE1家族成员作为花生四烯酸动员合成二十烷基酸的调节因子的作用。总之，这项提案中的工作将定义溶血磷脂运输和代谢的主要遗传和生化方面，这些方面与细胞生物学的基本过程密切相关，细胞生物学的功能障碍会导致严重的人类疾病。公共卫生相关性：这项提案的重点是涉及一类被称为溶血磷脂的脂类的运输和新陈代谢的基因和酶。溶血磷脂转运相关基因的功能障碍被认为是导致肝病、进展性家族性肝内胆汁淤积症和良性复发性肝内胆汁淤积症的主要原因。参与溶血磷脂代谢的基因功能障碍与人类手的发育异常和不育有关。这个项目的目标是了解溶血磷脂运输如何发生以及脂类如何代谢的机械性生物化学。

DESCRIPTION (provided by applicant): Lysophospholipids constitute a class of membrane glycerophospholipids that are rapidly transported and metabolized by many eukaryotic cells ranging from yeast to humans. We have recently identified two yeast transporters for the major lysophospholipids found throughout the eukaryotic world, lyso- phosphatidylethanolamine (lyso-PtdEtn) and lyso-phosphatidylcholine (lyso-PtdCho). The yeast lyso-PtdEtn and lyso-PtdCho transporters are also the same molecules implicated in regulating the plasma membrane asymmetry of phosphatidylserine and phosphatidylethanolamine. The metazoan forms of these transporters play crucial roles in signaling the apoptotic status of target cells to phagocytes. Defects in the function of the mammalian transporters are linked to primary familial intrahepatic cholestasis and benign recurrent intrahepatic cholestasis in humans. Genetic and biochemical work in yeast reveals that additional lyso-PtdCho and lyso-PtdEtn transporters remain to be identified. The first specific aim of this proposal will focus on identifying the genes for the remaining yeast lysophospholipid transporters and characterizing them biochemically. The second specific aim will capitalize upon the powerful tools of yeast genetics to structurally define the mechanistic basis by which the yeast and mammalian transporters recognize their substrates and introduce them into the transport channel. Subsequent to uptake by yeast and mammalian cells, lyso-PtdCho and lyso-PtdEtn are metabolized to their diacyl counterparts, PtdCho and PtdEtn, by acyltransferase enzymes. Although the enzyme activities have been known for more than 40 years, our recent work in yeast provided the first definition of the genes executing these reactions. The yeast gene ALE1 is the founding member of a family represented in multiple eukaryotes that is involved in acylating lysophospholipids with di-unsaturated and polyunsaturated fatty acids. In the third specific aim we will use the power of yeast genetics to elucidate the biochemical activities and functions of the metazoan acyltransferases belonging to the ALE1-family. Genetic studies already reveal that lesions in Drosophila ALE1-family genes cause developmental defects and male sterility. Studies in humans reveal defects in ALE1-family genes cause developmental defects of the hands and skeleton and also produce male sterility. The biochemical studies in yeast will be complemented by detailed studies in mammalian cells examining the role of ALE1-family members as regulators of arachidonic acid mobilization for the synthesis of eicosanoids. In summary, the work in this proposal will define major genetic and biochemical aspects of lysophospholipid transport and metabolism that are intimately associated with fundamental processes of cell biology whose dysfunction leads to serious human disease. PUBLIC HEALTH RELEVANCE: The focus of this proposal is upon genes and enzymes involved in transport and metabolism of a class of lipids known as lysophospholipids. Dysfunction of genes involved in lysophospholipid transport is known to cause the liver diseases Progressive Familial Intrahepatic Cholestasis and Benign Recurrent Intrahepatic Cholestasis. Dysfunction of genes involved in the metabolism of lysophospholipids is linked to developmental abnormalities of the hands and sterility in humans. The goals of this project are to understand the mechanistic biochemistry of how lysophospholipid transport occurs and how the lipids are metabolized.