Novel Roles for Lysophospholippids in Eukaryotic Membrane Biogenesis and Turnover

描述（由申请人提供）：溶血磷脂构成一类膜甘油磷脂，其被从酵母到人类的许多真核细胞快速转运和代谢。我们最近已经鉴定了两种酵母转运蛋白，用于在真核世界中发现的主要溶血磷脂，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的质膜不对称性的相同分子。这些转运蛋白的后生动物形式在向吞噬细胞发出靶细胞凋亡状态的信号中起着至关重要的作用。哺乳动物转运蛋白的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。酵母中的遗传和生物化学工作揭示了另外的溶血-PtdCho和溶血-PtdEtn转运蛋白仍有待鉴定。该提案的第一个具体目标将集中在确定剩余的酵母溶血磷脂转运蛋白的基因和生化特性。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转运通道的机制基础。在被酵母和哺乳动物细胞摄取后，溶血-PtdCho和溶血-PtdEtn通过酰基转移酶代谢成其二酰基对应物PtdCho和PtdEtn。虽然酶的活性已经知道了40多年，我们最近在酵母中的工作提供了执行这些反应的基因的第一个定义。酵母基因ALE 1是在多种真核生物中代表的家族的创始成员，其涉及用二不饱和和多不饱和脂肪酸酰化溶血磷脂。在第三个具体的目标，我们将利用酵母遗传学的力量来阐明生化活动和功能的后生动物酰基转移酶属于ALE 1家族。遗传学研究已经揭示果蝇ALE 1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，ALE 1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补充，研究ALE 1家族成员作为花生四烯酸动员合成类花生酸的调节剂的作用。总之，本提案中的工作将定义溶血磷脂转运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导致严重的人类疾病。公共卫生相关性：这项建议的重点是基因和酶参与运输和代谢的一类脂质称为溶血磷脂。已知参与溶血磷脂转运的基因功能障碍会引起肝脏疾病进行性家族性肝内胆汁淤积症和良性复发性肝内胆汁淤积症。参与溶血磷脂代谢的基因功能障碍与人类手部发育异常和不育有关。这个项目的目标是了解溶血磷脂转运发生的机制和脂质如何代谢。

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.