著作: Lei XiaoFeng/Fujiwara Yukio/Chang C. Y. Catherine/Chang Ta-Yuan/Takeya Motohiro/[坂下 直実]/Association of ACAT1-positive vesicles with late endosomes/ lysosomes in cholesterol-rich human macrophages./[Journal of Atherosclerosis and Thrombosis]
(英) Association of ACAT1-positive vesicles with late endosomes/ lysosomes in cholesterol-rich human macrophages.
(英) Acyl-coenzyme A: cholesterol acyltransferase1 (ACAT1) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the conversion of cholesterol into cholesteryl esters. We previously showed that cholesterol-loaded macrophages produce numerous ER-derived vesicles with elevated ACAT1 enzyme activity; some of these vesicles were shown to be closely associated with Golgi-related organelle(s). The aim of this study was to investigate the translocation of ACAT1 vesicle in cholesterol-loaded macrophages. To demonstrate association of ACAT1 with late endosomes/lysosomes (LE/LS), primary human macrophages with or without cholesterol-loading was subjected to confocal microscopy, immunoelectron microscopy, subcellular fractionation, and immunoadsorption assay. Furthermore, cholesterol esterification assay was also carried out to investigate function of ACAT1 associated LE/LS. Confocal fluorescence microscopy revealed that no significant ACAT1 signal was associated with the signal for LAMP2, a marker protein for LE/LS, in cholesterol non-loaded macrophages; however, approximately 20% of the total ACAT1 signals colocalized with the LAMP2 signal in cholesterol-loaded macrophages. ACAT1-positive membranes isolated by immunoadsorption using ACAT1-specific antibody contained LAMP2, demonstrating the association of ACAT1 and LE/LS. In addition, in macrophages phagocytosing latex beads, the close association of ACAT1 with LE/LS can be demonstrated in phagosomes isolated from cholesterol-loaded macrophages, not from non-loaded macrophages. Furthermore, cholesterol-loaded macrophages re-esterified aggregated LDL-derived cholesteryl ester even in the presence of U18666A, a reagent known to block egression of cholesterol from LE/LS. Our results indicated that cholesterol-loaded human macrophages produce LE/LS in close association with ACAT1, and may promote efficient esterification of modified LDL-derived free cholesterol on LE/LS.
Journal of Atherosclerosis and Thrombosis(日本動脈硬化学会)
|年月日||必須||2010年 6月 2日|