Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.
Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.
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作者：
Lu Y;Dong S;Hao B;Li C;Zhu K;Guo W;Wang Q;Cheung KH;Wong CW;Wu WT;Markus H;Yue J
自噬是一种分解代谢的溶酶体降解过程，对细胞内稳态和细胞存活至关重要。功能障碍性自噬与多种人类疾病有关，例如，癌症和神经退行性疾病。大量调节自噬的小分子已被广泛用于剖析这一过程，其中一些，例如，氯喹（CQ），可能最终应用于治疗各种自噬相关的人类疾病。在这里，我们发现vacuolin-1有效地和可逆地抑制哺乳动物细胞中自噬体和溶酶体之间的融合，从而诱导自噬体的积累。有趣的是，vacuolin-1的毒性较低，但与CQ相比，其抑制自噬的效力至少高出10倍。乙酰胆碱-1处理还阻断了内体和溶酶体之间的融合，导致一般内体-溶酶体降解的缺陷。用vacuolin-1处理细胞使溶酶体pH碱化并降低溶酶体Ca 2+含量。除了轻微抑制液泡ATP酶活性外，vacuolin-1处理还显着激活RAB 5A GTPase活性。RAB 5A或RAB 5A敲低的显性负突变体的表达显著抑制vacuolin-1诱导的自噬体-溶酶体融合阻断，而RAB 5A的组成性活性形式的表达抑制自噬体-溶酶体融合。这些数据表明vacuolin-1激活RAB 5A以阻断自噬体-溶酶体融合。拟南芥素-1及其类似物提供了一类新型药物，可以有效且可逆地调节自噬。
Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca2+ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.