Neufeld Edward B, Stonik John A, Demosky Stephen J, Knapper Catherine L, Combs Christian A, Cooney Adele, Comly Marcella, Dwyer Nancy, Blanchette-Mackie Joan, Remaley Alan T, Santamarina-Fojo Silvia, Brewer H Bryan
Molecular Disease Branch, NHLBI, NHLBI Light Microscopy Core Facility, and Laboratory for Cellular Biology and Biochemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2004 Apr 9;279(15):15571-8. doi: 10.1074/jbc.M314160200. Epub 2004 Jan 27.
We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.
我们之前已经证实，ABCA1转运蛋白在细胞外载脂蛋白受体的脂化过程中起关键作用，它在晚期内吞小泡和细胞表面之间穿梭运输（Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584 - 27590）。本研究提供了证据表明晚期内吞小泡中的ABCA1在细胞脂质流出中起作用。在缺乏功能性ABCA1的丹吉尔病成纤维细胞中，晚期内吞运输存在缺陷。与晚期内吞蛋白运输缺陷一致，疏水胺U18666A将NPC1保留在异常管状、胆固醇含量低的丹吉尔病晚期内体中，而不是像野生型成纤维细胞那样保留在富含胆固醇的溶酶体中。与脂质运输缺陷一致，丹吉尔病晚期内吞小泡积累了胆固醇和鞘磷脂，并固定在核周定位。丹吉尔病晚期内吞小泡中过量的胆固醇保留了大量的NPC1，NPC1将溶酶体胆固醇运输到细胞的其他部位。外源性载脂蛋白A-I消除了野生型晚期内体中胆固醇诱导的NPC1保留，但在丹吉尔病晚期内体中没有作用。腺病毒介导的ABCA1 - GFP在丹吉尔病成纤维细胞中的表达纠正了晚期内吞运输缺陷，并恢复了载脂蛋白A-I介导的胆固醇流出。野生型成纤维细胞中ABCA1 - GFP的表达也减少了与晚期内体相关的NPC1，诱导荧光载脂蛋白A-I显著摄取到含有ABCA1 - GFP的内体中（这些内体在晚期内体和细胞表面之间穿梭），并增强了载脂蛋白A-I介导的胆固醇流出。本研究的综合结果表明，ABCA1将保留NPC1的晚期内吞脂质池转化为可以与内吞的载脂蛋白A-I结合，并以新生高密度脂蛋白的形式从细胞中释放的脂质池。
