Molecular mechanism of ATP-dependent transporters involved in cholesterol homeostasis

本研究旨在了解ATP依赖性转运蛋白参与胆固醇和脂质稳态的分子机制。ABCA 1介导细胞胆固醇和磷脂的释放以形成高密度脂蛋白（HDL）。本研究利用在HEK 293细胞中稳定表达的ABCA 1-GFP融合蛋白，对Tangier病相关的ABCA 1第一胞外区的三种不同突变体R587 W、W590 S和Q597 R进行了亚细胞定位和功能研究。我们的研究表明，在R587 W和Q597 R中，HILL组装的缺陷是由于在PM上的定位受损，而W590 S除了初始的ATP结合和水解之外还存在功能缺陷。ABCA 1的功能在转录和转录后水平受到高度调节，合成的ABCA 1蛋白质迅速转变，半衰期为1-2小时。为了检测ABCA 1的功能是否受相关蛋白的调节，用ABCA 1的C-末端120个氨基酸筛选酵母双杂交文库。发现两种PDZ蛋白，α1-syntroplun和Lin 7，与ABCA 1相互作用。我们的研究表明，α1-syntrophin参与细胞内信号传导，决定ABCA 1的稳定性，并调节细胞胆固醇的释放。

This study was done to understand the molecular mechanism of ATP-dependent transporters involved in cholesterol and lipid homeostasis. ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). ~The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. Our study suggested that the defect of HILL assembly in R587W and Q597R is due to the impaired localization to PM, while W590S have functional defect other than the initial ATP binding and hydrolysis. Functions of ABCA1 are highly regulated at he transcriptional and post-transcriptional levels, and the synthesized ABCA1 protein turns over rapidly with a half-life of 1-2 hours. To examine if functions of ABCA1 are modulated by associated proteins, a yeast two-hybrid library was screened with the C-terminal 120 amino acids of ABCA1. Two PDZ proteins, α1-syntroplun and Lin7, were found to interact with ABCA1. Our -study suggested that α1-syntrophin is involved in intracellular signaling, which determines the stability of ABCA1, and modulates cellular cholesterol release.