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Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.

ABSTRACT: 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 Ca(2+) 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.

SUBMITTER: Lu Y

PROVIDER: S-EPMC4502727 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.

Lu Yingying Y Dong Shichen S Hao Baixia B Li Chang C Zhu Kaiyuan K Guo Wenjing W Wang Qian Q Cheung King-Ho KH Wong Connie W M CW Wu Wu-Tian WT Markus Huss H Yue Jianbo J

Autophagy 20141030 11

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 po ...[more]

PMID: 25483964

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Project description:Dysfunction of macroautophagy/autophagy in macrophages contributes to atherosclerosis. Impaired autophagy-lysosomal degradation system leads to lipid accumulation, facilitating atherosclerotic plaque. ATG14 is an essential regulator for the fusion of autophagosomes with lysosomes. Whether ATG14 plays a role in macrophage autophagy dysfunction in atherosclerosis is unknown. To investigate the effects of ATG14 on macrophage autophagy, human atherosclerotic plaque, apoe-/- mice and cultured mouse macrophages were evaluated. Overexpression of ATG14 by adenovirus was used to reveal its function in autophagy, inflammation and atherosclerotic plaque formation. Results showed that impaired autophagy function with reduction of ATG14 expression existed in macrophages of human and mouse atherosclerotic plaques. Ox-LDL impaired autophagosome-lysosome fusion with reduction of ATG14 expression in macrophages. Overexpression of ATG14 in macrophages enhanced fusion of autophagosomes with lysosomes and promoted lipid degradation, decreasing Ox-LDL-induced apoptosis and inflammatory response. Augmenting ATG14 expression reversed the autophagy dysfunction in macrophages of apoe-/- mice plaque, blunted SQSTM1/p62 accumulation, inhibited inflammation, and upregulated the population of Treg cells, resulting in alleviating atherosclerotic lesions.Abbreviations: ABCC1: ATP-binding cassette, sub-family C (CFTR/MRP), member 1; ABCA1: ATP-binding cassette, sub-family A (ABC1), member 1; Ad-Atg14: adenovirus vector carrying the mouse Atg14 gene; Ad-LacZ: adenovirus vector carrying the gene for bacterial β-galactosidase; apoe-/-: apolipoprotein E knockout; ATG14: autophagy-related 14; CD68: CD68 antigen; DAPI: 4',6-diamidino-2-phenylindole; Dil-ox-LDL: Dil-oxidized low density lipoprotein; ELISA: enzyme-linked immunosorbent assay; HFD: high-fat diet (an atherogenic diet); IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LDL-C: low density lipoprotrein cholesterol; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; Ox-LDL: oxidized low density lipoprotein; PBMC: peripheral blood mononuclear cells; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; SREBF2/SREBP2: sterol regulatory element binding factor 2; STX17: syntaxin 17; TC: serum total cholesterol; TG: triglyceride; TNF: tumor necrosis factor; IFN: interferon; Treg cell: regulatory T cell.

Dataset Information

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.

Publications

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A.

Similar Datasets

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