Association of ACAT1-Positive Vesicles with Late Endosomes/Lysosomes in Cholesterol-Rich Human Macrophages
Association of ACAT1-Positive Vesicles with Late Endosomes/Lysosomes in Cholesterol-Rich Human Macrophages
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DOI：
10.5551/jat.4416
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发表时间：
2010-01-01
影响因子：
4.4
通讯作者：
Sakashita, Naomi
中科院分区：
文献类型：
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作者：
Lei, XiaoFeng;Fujiwara, Yukio;Sakashita, Naomi
目的：酰基辅酶A：胆固醇酰基转移酶1 （ACAT1）是一种内质网（ER）常驻酶，催化胆固醇转化为胆固醇酯。我们之前的研究表明，胆固醇负载的巨噬细胞产生大量ACAT1酶活性升高的er源性囊泡；其中一些囊泡被证明与高尔基相关细胞器密切相关。本研究的目的是研究ACAT1囊泡在胆固醇负荷巨噬细胞中的易位。方法：为了证明ACAT1与晚期内体/溶酶体（LE/LS）的关联，我们对携带或不携带胆固醇的原代人巨噬细胞进行了共聚焦显微镜、免疫电镜、亚细胞分离和免疫吸附实验。此外，还进行了胆固醇酯化试验，以研究ACAT1相关LE/LS的功能。结果：共聚焦荧光显微镜显示，在无胆固醇负荷的巨噬细胞中，ACAT1信号与LE/LS标记蛋白LAMP2信号无明显关联；然而，在胆固醇负荷的巨噬细胞中，大约20%的ACAT1信号与LAMP2信号共定位。用ACAT1特异性抗体LAMP2免疫吸附分离ACAT1阳性膜，证实ACAT1与LE/LS存在关联。此外，在吞噬乳胶珠的巨噬细胞中，ACAT1与LE/LS的密切关联可以在负载胆固醇的巨噬细胞分离的吞噬体中得到证实，而非负载胆固醇的巨噬细胞分离的吞噬体中则没有。此外，即使存在U18666A（一种已知可阻止胆固醇从LE/LS中流出的试剂），装载胆固醇的巨噬细胞也会将聚集的ldl来源的胆固醇酯重新酯化。结论：我们的研究结果表明，胆固醇负荷的人巨噬细胞产生与ACAT1密切相关的LE/LS，并可能促进修饰ldl来源的游离胆固醇在LE/LS上的有效酯化。
Aim: 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.Methods: 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.Results: 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.Conclusion: 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.