ABCA1 cholesterol efflux & protein-protein interactions

描述(由申请人提供)：作为维持全身脂质平衡的过程的一部分，细胞将胆雌醇和磷脂外流到载脂蛋白，如载脂蛋白A-L。丹吉尔病的患者证明了脂质外流的生理重要性，丹吉尔病是一种罕见的遗传性疾病，其特征是周围神经病变、早产性心血管疾病和循环中高密度脂蛋白的缺乏。遗传作图研究已经将Tangier表型与ATP结合盒转运体ABCA1的突变联系起来，随后的研究表明ABCA1在高密度脂蛋白的形成中起着限速作用。在普通人群中，高密度脂蛋白水平升高与心血管疾病的发生率呈负相关，因此增加ABCA1活性的治疗可能有助于防止疾病进展。这项建议的目的是描述定义ABCA1外流机制的关键结构-功能关系，以及蛋白质-蛋白质相互作用在决定ABCA1活性中所起的作用。这一广泛的目标集中在对一种自然发生的丹吉尔突变的分析上，该突变会删除转运蛋白的最后46个氨基酸。缺失的氨基酸是高度保守的结构域的一部分，如本提案所示，对于ABCA1外排活性是必不可少的。对缺失区域内其他合成突变体的分析定义了一个新的VFVNFA基序，该基序唯一地识别了ABCA转运蛋白的一个亚类。这个基序对ABCA1的活性是必不可少的，并且可以通过反式作用来抑制外排活性。建立了一种蛋白质组学方法，对ABCA1和感兴趣的突变体转运蛋白进行亲和纯化，并对共纯化的蛋白质进行了质谱分析。描述了与VFVNFA基序相互作用的蛋白质，并将研究它们与外排机制的功能相关性。由于一组相互作用似乎下调外排活性，这种相互作用的中断可能提供治疗靶点，从而可以增加ABCA1的外排。

DESCRIPTION (provided by applicant): Cells efflux cholestrol and phospholids to apolipoproteins such as apoA-l as part of a process that maintains whole body lipid homeostatsis. The physiologic importance of lipid efflux is demonstrated by patients suffering from Tangier disease, a rare genetic condition characterized by peripheral neuropathy, premature cardiovascular disease and an absence of circulating HDL. Genetic mapping studies have associated the Tangier phenotype with mutations in the ATP binding cassette transporter ABCA1 and subsequent studies have shown that ABCA1 plays a rate limiting role in the formation of HDL. In the general population elevated HDL levels are inversely correlated with the incidence of cardiovascular disease, thus therapies that increase ABCA1 activity may help prevent disease progression. The objective of this proposal is to describe key structure-function relations that define the ABCA1 efflux mechanism and what role protein-protein interactions play in determining the activity of ABCA1. This broad aim is focused by the analysis of a naturally occurring Tangier mutation that deletes the last 46 amino acids of the transporter. The deleted amino acids are part of a highly conserved domain, and as shown in this proposal, are essential for ABCA1 efflux activity. Analysis of additional synthetic mutants within the deleted region has defined a novel VFVNFA motif that uniquely identifies a subclass of ABCA transporters. This motif is essential for ABCA1 activity and can act in trans to inhibit efflux activity. A proteomic approach has been developed in which ABCA1 and interesting mutant transporters are affinity purified and the co-purify proteins are analyzed by mass spectrometry. The proteins which interact with the VFVNFA motif are described and their functional relevance to the efflux mechanism investigated will be investigated. Since a set of the interactions appear to down-regulate efflux activity disruption of such interactions may offer therapeutic targets by which ABCA1 efflux can be increased.