Molecular mechanism of ATP-dependent transporters involved in cholesterol homeostasis

ATP依赖性转运蛋白参与胆固醇稳态的分子机制

• 批准号: 14360053
• 负责人: UEDA Kazumitsu
• 金额: $ 9.41万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14360053/
• 关键词: ABC proteins; cholesterol; atherosclerosis; α1-syntrophin; lipid; HDL; transporter; membrane; 生活習慣病; リン脂質; 構造解析

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.

本研究旨在了解参与胆固醇和脂质稳态的 ATP 依赖性转运蛋白的分子机制。 ABCA1 介导细胞胆固醇和磷脂的释放，形成高密度脂蛋白 (HDL)。 ~通过使用在 HEK293 细胞中稳定表达的 ABCA1-GFP 融合蛋白，检查了与丹吉尔病相关的人 ABCA1 第一个胞外结构域中的三个不同突变体 R587W、W590S 和 Q597R 的亚细胞定位和功能。我们的研究表明，R587W 和 Q597R 中 HILL 组装的缺陷是由于 PM 定位受损所致，而 W590S 除了初始 ATP 结合和水解之外还存在功能缺陷。 ABCA1的功能在转录和转录后水平受到高度调控，合成的ABCA1蛋白周转迅速，半衰期为1-2小时。为了检查 ABCA1 的功能是否受到相关蛋白的调节，用 ABCA1 C 端 120 个氨基酸筛选了酵母双杂交文库。两种 PDZ 蛋白，α1-syntroplun 和 Lin7，被发现与 ABCA1 相互作用。我们的研究表明，α1-肌营养蛋白参与细胞内信号传导，决定了 ABCA1 的稳定性，并调节细胞胆固醇释放。

项目成果

Ikeda, Y.: "Post-transcriptional regulation of human ABCA7 and its function for the apoA-I-dependent lipid release."Biochem Biophys Res Commun. 311. 313-318 (2003)

Ikeda, Y.：“人类 ABCA7 的转录后调节及其对 apoA-I 依赖性脂质释放的功能。”Biochem Biophys Res Commun。

Suzuki, S.: "Verapamil Increases the Apolipoprotein-Mediated Release of Cellular Cholesterol by Induction of ABCA1 Expression via an LXR-Independent Mechanism."Arterioscler Thromb Vasc Biol. 24. 519-525 (2004)

Suzuki, S.：“维拉帕米通过 LXR 独立机制诱导 ABCA1 表达，增加载脂蛋白介导的细胞胆固醇释放。”动脉硬化血栓 Vasc Biol。

Hahimoto, K.: "Trafficking-defect and functional-defect by mutations of the ATP-binding domains in multidrug resistance protein 2 (MRP2, ABCC2) in patients with Dubin-Johnson syndrome"Hepatology. 36. 1246-1252 (2002)

Hahimoto, K.：“Dubin-Johnson 综合征患者多药耐药蛋白 2（MRP2、ABCC2）中 ATP 结合域突变导致的运输缺陷和功能缺陷”肝病学。

Sakai, K.: "Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein."J Exp Bot. 53. 1879-1886 (2002)

Sakai, K.：“黄连细胞中小檗碱转运的表征以及 ABC 蛋白的参与。”J Exp Bot。

Tanaka, A.R.: "Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis."J Biol Chem. 278. 8815-8819 (2003)

Tanaka, A.R.：“第一个胞外结构域中 ABCA1 突变对亚细胞运输和 ATP 结合/水解的影响。”J Biol Chem。