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Autophagy. 2014;10(11):1895-905. doi: 10.4161/auto.32200. Epub 2014 Oct 30.

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

Author information

1
a Department of Biomedical Sciences ; City University of Hong Kong ; Hong Kong , China.

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.

KEYWORDS:

ATG, autophagy-related; BAF, bafilomycin A1; CQ, chloroquine; CTSB, cathepsin B; CTSL, cathepsin L; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; GPN, glycyl-l-phenylalanine 2-naphthylamide; LAMP1, lysosomal-associated membrane protein 1; Leup, leupeptin; MAP1LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin; RAB5A; RFP, red fluorescent protein; autophagosomes; endosomes; lysosomes; pH; tfLC3, tandem fluorescence-tagged LC3; vacuolin-1

PMID:
25483964
PMCID:
PMC4502727
DOI:
10.4161/auto.32200
[Indexed for MEDLINE]
Free PMC Article
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