ABCA1 cholesterol efflux & protein-protein interactions

描述（由申请人提供）：细胞将胆固醇和磷脂外排至载脂蛋白，如apoA-1，作为维持全身脂质稳态过程的一部分。脂质流出的生理重要性由患有丹吉尔病的患者证明，丹吉尔病是一种罕见的遗传性疾病，其特征在于周围神经病变、早发性心血管疾病和缺乏循环HDL。遗传作图研究将丹吉尔表型与ATP结合盒转运蛋白ABCA 1的突变相关联，随后的研究表明ABCA 1在HDL的形成中起限速作用。在一般人群中，HDL水平升高与心血管疾病的发病率呈负相关，因此增加ABCA 1活性的疗法可能有助于预防疾病进展。该提案的目的是描述定义ABCA 1外排机制的关键结构-功能关系，以及蛋白质-蛋白质相互作用在确定ABCA 1活性中发挥的作用。这一广泛的目标是通过分析天然存在的Tangier突变来实现的，该突变删除了转运蛋白的最后46个氨基酸。缺失的氨基酸是高度保守结构域的一部分，并且如该提议中所示，对于ABCA 1外排活性是必需的。在缺失区域内的其他合成突变体的分析已经定义了一种新的VFVNFA基序，其独特地识别ABCA转运蛋白的一个亚类。该基序是ABCA 1活性所必需的，并且可以反式作用以抑制外排活性。已经开发了一种蛋白质组学方法，其中ABCA 1和感兴趣的突变转运蛋白被亲和纯化，并且通过质谱分析共纯化蛋白。与VFVNFA基序相互作用的蛋白质进行了描述，并将研究其功能相关的外排机制。由于一组相互作用似乎下调外排活性，因此这种相互作用的破坏可能提供可以增加ABCA 1外排的治疗靶点。

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.