Abe-Dohmae Sumiko, Ikeda Yuika, Matsuo Michinori, Hayashi Michi, Okuhira Kei-ichiro, Ueda Kazumitsu, Yokoyama Shinji
Biochemistry, Molecular Biology and Metabolism 1, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
J Biol Chem. 2004 Jan 2;279(1):604-11. doi: 10.1074/jbc.M309888200. Epub 2003 Oct 21.
Apolipoprotein-mediated release of cellular cholesterol and phospholipids was induced in HEK293 cells by expressing human ATP-binding cassette transporter A7 (ABCA7) and ABC transporter A1 (ABCA1) proteins, whether transient or stable, to generate cholesterol-rich high density lipoprotein (HDL). Green fluorescent protein (GFP) attached at their C termini did not influence the lipid release reactions. Transfected ABCA7-GFP induced apolipoprotein-mediated assembly of cholesterol-containing HDL also in L929 cells, which otherwise generate only cholesterol-deficient HDL with their endogenous ABCA1. Time-dependent release of cholesterol and phospholipid by apolipoprotein A (apoA)-I was parallel both with ABCA1 and with ABCA7 when highly expressed in HEK293 cells, but dose-dependent profiles of lipid release on apoA-I and apoA-II were somewhat different between ABCA1 and ABCA7. Analyses of the stable clones with ABCA1-GFP (293/2c) and ABCA7-GFP (293/6c) by using the same vector indicated some differences in regulation of their activities by protein kinase modulators. Dibutyryl cyclic AMP increased ABCA1-GFP and the release of cholesterol and phospholipid in 293/2c but increased neither ABCA7-GFP nor the lipid release in 293/6c. Expression of ABCA1-GFP- and apoA-I-mediated lipid release were enhanced in parallel by phorbol 12-myristate 13-acetate (PMA) in 293/2c cells. In contrast, the same treatment of 293/6c increased ABCA7-GFP, but apoA-I-mediated lipid release was significantly suppressed. Despite these different responses to PMA, all of the effects of PMA were reversed by a specific protein kinase C inhibitor Gö6976, suggesting that the changes were in fact due to protein kinase C activation. A thiol protease inhibitor, N-acetyl-Leu-Leu-norleucinal, increased the protein levels of ABCA1-GFP in 293/2c and ABCA7-GFP in 293/6c, indicating their common degradation pathway. The data indicated that human ABCA7 would compensate the function of ABCA1 for release of cell cholesterol in a certain condition(s), but post-transcriptional regulation of their activity is different.
通过表达人ATP结合盒转运蛋白A7（ABCA7）和ABC转运蛋白A1（ABCA1），无论是瞬时表达还是稳定表达，均可在HEK293细胞中诱导载脂蛋白介导的细胞胆固醇和磷脂释放，以生成富含胆固醇的高密度脂蛋白（HDL）。连接在其C末端的绿色荧光蛋白（GFP）不影响脂质释放反应。转染的ABCA7-GFP在L929细胞中也诱导了载脂蛋白介导的含胆固醇HDL的组装，否则L929细胞仅利用其内源性ABCA1生成缺乏胆固醇的HDL。当在HEK293细胞中高表达时，载脂蛋白A（apoA）-I介导的胆固醇和磷脂的时间依赖性释放与ABCA1和ABCA7均平行，但ABCA1和ABCA7之间apoA-I和apoA-II的脂质释放剂量依赖性曲线有所不同。使用相同载体对ABCA1-GFP（293/2c）和ABCA7-GFP（293/6c）稳定克隆的分析表明蛋白激酶调节剂对其活性的调节存在一些差异。二丁酰环磷酸腺苷增加了293/2c中的ABCA1-GFP以及胆固醇和磷脂的释放，但在293/6c中既未增加ABCA7-GFP也未增加脂质释放。在293/2c细胞中，佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）使ABCA1-GFP和apoA-I介导的脂质释放平行增强。相反，对293/6c进行相同处理可增加ABCA7-GFP，但apoA-I介导的脂质释放被显著抑制。尽管对PMA有这些不同反应，但PMA的所有作用均被特异性蛋白激酶C抑制剂Gö6976逆转，这表明这些变化实际上是由于蛋白激酶C激活所致。一种巯基蛋白酶抑制剂N-乙酰-Leu-Leu-正亮氨酸增加了293/2c中ABCA1-GFP的蛋白水平以及293/6c中ABCA7-GFP的蛋白水平，表明它们有共同的降解途径。数据表明，在某些条件下，人ABCA7可补偿ABCA1在细胞胆固醇释放方面的功能，但它们活性的转录后调节有所不同。
