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，可被从酵母到人类的许多真核细胞快速运输和代谢。我们最近确定了真核世界中发现的两种主要溶血磷脂的酵母转运体，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转运体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相同分子。这些转运蛋白的后生形式在向吞噬细胞发送靶细胞凋亡信号中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。酵母的遗传和生化工作表明，其他lyso-PtdCho和lyso-PtdEtn转运蛋白仍有待鉴定。本提案的第一个具体目标将集中于鉴定剩余酵母溶血磷脂转运蛋白的基因并对其进行生物化学表征。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，通过酰基转移酶将lyso-PtdCho和lyso-PtdEtn代谢为它们的二酰基对应物PtdCho和PtdEtn。虽然这种酶的活性已经被发现了40多年，但我们最近在酵母中的工作首次为执行这些反应的基因提供了定义。酵母基因ALE1是一个家族的创始成员，在多个真核生物中都有代表，参与溶血磷脂与双不饱和脂肪酸和多不饱和脂肪酸的酰化。在第三个具体目标中，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。在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ale1家族成员作为花生四烯酸动员的调节剂的作用，以合成类二十烷酸。总之，本提案中的工作将确定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，其功能障碍导致严重的人类疾病。公共卫生相关性：本提案的重点是涉及一类被称为溶血磷脂的脂质的运输和代谢的基因和酶。参与溶血磷脂转运的基因功能障碍可导致肝脏疾病进行性家族性肝内胆汁淤积和良性复发性肝内胆汁淤积。参与溶血磷脂代谢的基因功能障碍与手部发育异常和人类不育有关。这个项目的目标是了解溶血磷脂运输发生的机制生化以及脂质是如何代谢的。

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.