Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B
Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B
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DOI：
10.1091/mbc.e05-07-0659
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发表时间：
2006-06-01
影响因子：
3.3
通讯作者：
Fujimoto, Toyoshi
中科院分区：
文献类型：
--
作者：
Ohsaki, Yuki;Cheng, Jinglei;Fujimoto, Toyoshi
储存在肝细胞胞质脂滴（CLD）中的脂酯用于合成极低密度脂蛋白（VLDL），载脂蛋白B（ApoB）协同整合到VLDL中。在本研究中，通过使用来源于人肝癌并且具有VLDL分泌能力的Huh 7细胞，我们发现ApoB高度集中在CLDs周围，从而形成“ApoB新月体”。“在正常条件下，在< 10%的Huh 7细胞中观察到ApoB-新月体，但在N-乙酰基-L-亮氨酰-L-亮氨酰-L-正亮氨酸抑制蛋白酶体12小时后，该比例增加到近50%。电子显微镜显示ApoB定位于粘附于CLD的直径为50-100 nm的电子透明颗粒簇。在CLD组分中检测到ApoB、蛋白酶体亚基和泛素化蛋白，并且该ApoB是泛素化的。有趣的是，蛋白酶体抑制也引起溶酶体中自噬空泡和ApoB的增加。ApoB-新月体在蛋白酶体作用12-24 h后开始减少。自噬抑制剂3-甲基腺嘌呤可阻断这种降低。单独抑制自噬引起ApoB-新月体增加。这些观察结果表明，蛋白酶体和自噬/溶酶体的ApoB降解发生在CLD周围，CLD表面作为两种途径会聚的独特平台发挥作用。
Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in < 10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal. inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.