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

Abstract

-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; RFP, red fluorescent protein; tfLC3, tandem fluorescence-tagged LC3.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 2C 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. IntroductionAmong 3 types of autophagy, including microautophagy, chaperone-mediated autophagy, and macroautophagy, in mammals, macroautophagy (hereafter referred as autophagy) is the most common type. Autophagy is an evolutionarily conserved catabolic degradation cellular process in which misfolded proteins or damaged organelles are first sequestered by a doublemembrane vesicle, called an autophagosome. Autophagosomes then fuse with lysosomes to form autolysosomes, inside which the sequestered contents are digested by lysosomal enzymes and recycled to maintain cellular homeostasis. Autophagy can also be markedly induced by a wide variety of stresses, e.g., nutrient starvation, infection, and aging, for cell survival. Dysfunctional autophagy has been associated with wide ranges of human diseases, e.g., cancer and neurodegenerative diseases. [1][2][3][4][5][6] Basal autophagy activity is essential for cell homeostasis, and it is tightly controlled by a complicated interplay among several key machineries, including ULK1 or ULK2 complexes and the class III phosphatidylinositol-3 kinase complexes. The MTOR (mechanistic target of rapamycin) Ser/Thr kinase suppresses autophagy by inhibiting the ULK1/2 comple...

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“…Quantitative measurements were performed using a multimode microplate reader, and the exocytosis percentages, which were defined as the ratio of FI of NPs retained in the cells to that taken by cells during the pre-incubation of 2 h, were calculated to reflect the effects of chemical inhibitors. The inhibitors used in this section included PRQ (300 μM), 25 GOL-A (50 μM), 26 VAL-1 (1 μM), 27 and WOT (33 μM), 28 which could inhibit recycling of endosome-mediated, golgi-mediated, lysosome-mediated, and MVB-mediated exocytosis, respectively.…”

“…As shown in Figure 9B, in the presence of vacuolin (VAL), which could inhibit vacuolar ATPase activity and subsequently inhibit lysosomal pH alkalinization, 27 exocytosis was significantly inhibited at 6 and 12 h, compared with the negative group, which was performed without any inhibitor. This effect indicated the contribution of the lysosomal pathway to FITC-CsNP exocytosis; however, no significant difference was observed between the VAL group and the negative control group at 24 h, suggesting the presence of a compensatory action of other pathways.…”

A Novel Bioavailable BH3 Mimetic Efficiently Inhibits Colon Cancer via Cascade Effects of Mitochondria

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“…In combination treatment, CQ also plays a role in inhibiting autophagy and enhances ch282-5-induced mitochondria-dependent apoptosis, resulting in increased DNA fragmentation and the release of mitochondrial contents. Numerous preclinical studies have found that inhibition of autophagy by CQ restores chemosensitivity and promotes tumor cell death by diverse anticancer therapies (42).…”

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“…Autophagosomes are dependent on microtubules for transport to both endosomes and lysosomes where their merging is facilitated by a variety of proteins including both endosomal sort complex required for transport (ESCRT) and soluble NSF attachment protein receptor (SNARE) proteins as well as Rab7 [ 46 -48 ]. Of peculiar interest, proper lysosomal function and acidifi cation were found to be important for autophagosome-lysosome and endosome-lysosome fusion as well [ 49 ]. Upon fusion of the autophagosome or amphisome with the lysosome, the inner membrane of the autolysosome is quickly degraded.…”