title: 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.
authors.affiliation: a Department of Biomedical Sciences ; City University of Hong Kong ; Hong Kong , China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Lu
authors.forename: Shichen
authors.initials: S
authors.lastname: Dong
authors.forename: Baixia
authors.initials: B
authors.lastname: Hao
authors.forename: Chang
authors.initials: C
authors.lastname: Li
authors.forename: Kaiyuan
authors.initials: K
authors.lastname: Zhu
authors.forename: Wenjing
authors.initials: W
authors.lastname: Guo
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: King-Ho
authors.initials: KH
authors.lastname: Cheung
authors.forename: Connie W M
authors.initials: CW
authors.lastname: Wong
authors.forename: Wu-Tian
authors.initials: WT
authors.lastname: Wu
authors.forename: Huss
authors.initials: H
authors.lastname: Markus
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yue
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001343:Autophagy; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D002738:Chloroquine / Q000737:chemistry; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D056945:Hep G2 Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry*; D006706:Homeostasis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D016086:Lentivirus / Q000235:genetics; D008247:Lysosomes / Q000378:metabolism*; D008660:Metabolism; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D010588:Phagosomes / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D006576:Heterocyclic Compounds, 4 or More Rings; C503870:vacuolin-1; D049452:Green Fluorescent Proteins; D002738:Chloroquine; D000251:Adenosine Triphosphatases; C427755:RAB5C protein, human; D020696:rab5 GTP-Binding Proteins; D002118:Calcium
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