A reversible autophagy inhibitor blocks autophagosome–lysosome fusion by preventing Stx17 loading onto autophagosomes (original) (raw)
Related papers
ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes
Nature, 2015
Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8)....
SNARE-mediated membrane fusion in autophagy
Seminars in cell & developmental biology, 2016
Autophagy, a conserved self-eating process for the bulk degradation of cytoplasmic materials, involves double-membrane autophagosomes formed when an isolation membrane emerges and their direct fusion with lysosomes for degradation. For the early biogenesis of autophagosomes and their later degradation in lysosomes, membrane fusion is necessary, although different sets of genes and autophagy-related proteins involved in distinct fusion steps have been reported. To clarify the molecular mechanism of membrane fusion in autophagy, to not only expand current knowledge of autophagy, but also benefit human health, this review discusses key findings that elucidate the unique membrane dynamics of autophagy.
ATG14 controls SNARE-mediated autophagosome fusion with a lysosome
Autophagy, 2015
Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for...
Molecular interplay of autophagy and endocytosis in human health and diseases
Biological Reviews, 2019
Autophagy, an evolutionarily conserved process for maintaining the physio-metabolic equilibrium of cells, shares many common effector proteins with endocytosis. For example, tethering proteins involved in fusion like Ras-like GTPases (Rabs), soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), lysosomal-associated membrane protein (LAMP), and endosomal sorting complex required for transport (ESCRT) have a dual role in endocytosis and autophagy, and the trafficking routes of these processes converge at lysosomes. These common effectors indicate an association between budding and fusion of membrane-bound vesicles that may have a substantial role in autophagic lysosome reformation, by sensing cellular stress levels. Therefore, autophagy-endocytosis crosstalk may be significant and implicates a novel endocytic regulatory pathway of autophagy. Moreover, endocytosis has a pivotal role in the intake of signalling molecules, which in turn activates cascades that can result in pathophysiological conditions. This review discusses the basic mechanisms of this crosstalk and its implications in order to identify potential novel therapeutic targets for various human diseases.
Journal of Cell Science, 2012
Autophagy is a highly regulated membrane remodeling process that allows the lysosome-mediated degradation of cytoplasmic entities by sequestrating them in double-membrane autophagosomes. Autophagy is hence highly intertwined with the endocytic trafficking pathway, sharing similar molecular machinery. Atg14L, also known as Beclin 1-associated autophagy-related key regulator (Barkor), directly interacts with Beclin 1 through its coiled-coil domain and enhances phosphatidylinositol 3-phosphate kinase class III (PI3KC3) activity to induce autophagosome membrane nucleation, highlighting its essential role in the early stage of mammalian autophagy. Here, we report a novel function of Atg14L in the endocytic trafficking pathway wherein Atg14L binds to and colocalizes with the fusogenic SNARE effector protein Snapin to facilitate endosome maturation. Atg14L specifically binds to Snapin and this interaction effectively facilitates endosomal maturation without affecting autophagic cargo degradation. Consequently, atg14l knockdown significantly delayed the late stage of endocytic trafficking, as evidenced by the retarded kinetics of internalized surface receptor degradation. This phenotype was effectively complemented by wild-type Atg14L or Beclin 1-binding mutant, but not by its Snapin-binding mutant. Taken together, our study demonstrates that Atg14L functions as a multivalent trafficking effector that regulates endosome maturation as well as autophagosome formation, reflecting the complexity of the crosstalk between autophagic and endocytic vesicle trafficking in higher eukaryotes.
Cell, 2012
The lysosome is a degradative organelle, and its fusion with other organelles is strictly regulated. In contrast to fusion with the late endosome, the mechanisms underlying autophagosome-lysosome fusion remain unknown. Here, we identify syntaxin 17 (Stx17) as the autophagosomal SNARE required for fusion with the endosome/lysosome. Stx17 localizes to the outer membrane of completed autophagosomes but not to the isolation membrane (unclosed intermediate structures); for this reason, the lysosome does not fuse with the isolation membrane. Stx17 interacts with SNAP-29 and the endosomal/ lysosomal SNARE VAMP8. Depletion of Stx17 causes accumulation of autophagosomes without degradation. Stx17 has a unique C-terminal hairpin structure mediated by two tandem transmembrane domains containing glycine zipper-like motifs, which is essential for its association with the autophagosomal membrane. These findings reveal a mechanism by which the SNARE protein is available to the completed autophagosome.
ABSTRACTEfficient mammalian autophagosome biogenesis requires coordinated input from other cellular endomembrane compartments. Such coordination includes the stimulated trafficking to autophagosome assembly sites of the essential autophagy proteins, ATG9 and ATG16L1, via distinct endosomal compartments. Protein trafficking within the endocytic network is directed by a conserved family of sorting nexins (SNXs), with previous studies implicating SNX18 (an SH3 domain-type SNX-BAR protein) in the mobilisation of ATG9A and ATG16L1 from recycling endosomes during autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that a second mammalian SNX-BAR, SNX4, is needed for efficient LC3 lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and heterodimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30, and that these associate with tubulovesicular endocytic membranes at steady state. Detailed image-based analysis during the early st...
Targeting autophagy with small molecules for cancer therapy
Journal of Cancer Metastasis and Treatment, 2019
Autophagy is a conserved lysosomal-dependent catabolic process that maintains the cellular homeostasis by recycling misfolded proteins and damaged organelles. It involves a series of ordered events (initiation, nucleation, elongation, lysosomal fusion and degradation) that are tightly regulated/controlled by diverse cell signals and stress. It is like a double-edged sword that can play either a protective or destructive role in cancer, by pro-survival or apoptotic cues. Recently, modulating autophagy by pharmacological agents has become an attractive strategy to treat cancer. Currently, a number of small molecules that inhibit autophagy initiation (e.g., ULK kinase inhibitors), nucleation (e.g., Vps34 inhibitors), elongation (e.g., ATG4 inhibitors) and lysosome fusion (e.g., chloroquine, hydroxyl chloroquine, etc .) are reported in pre-clinical and clinical study. Also a number of small molecules reported to induce autophagy by targeting mammalian target of rapamycin (e.g., rapamycin analogs) or adenosine 5'-monophosphate-activated protein kinase (e.g., sulforaphane). The study results suggest that many potential "druggable" targets exist in the autophagy pathway that could be harnessed for developing new cancer therapeutics. In this review, we discuss the reported autophagy modulators (inhibitors and inducers), their molecular mode of action and their applications in cancer therapy.