RESUMO
Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein aggregates are targeted for degradation through the ubiquitin proteasome (UPS) and autophagy-lysosome systems. The ubiquitination and deubiquitinating enzymes (DUBs) have been reported to play pivotal roles in the regulation of the UPS system. However, the function of DUBs in the regulation of autophagy remain to be elucidated. In this study, we found that knockdown of Leon/USP5 caused a marked increase in the formation of autophagosomes and autophagic flux under well-fed conditions. Genetic analysis revealed that overexpression of Leon suppressed Atg1-induced cell death in Drosophila. Immunoblotting assays further showed a strong interaction between Leon/USP5 and the autophagy initiating kinase Atg1/ULK1. Depletion of Leon/USP5 led to increased levels of Atg1/ULK1. Our findings indicate that Leon/USP5 is an autophagic DUB that interacts with Atg1/ULK1, negatively regulating the autophagic process.
Assuntos
Autofagia , Proteínas de Drosophila , Animais , Autofagia/genética , Autofagossomos , Morte Celular , Drosophila , Lisossomos , Complexo de Endopeptidases do Proteassoma , Ubiquitina , Enzimas Desubiquitinantes , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas de Drosophila/genética , Proteases Específicas de Ubiquitina/genéticaRESUMO
Macroautophagy/autophagy is an evolutionarily conserved intracellular pathway for the degradation of cytoplasmic materials. Under stress conditions, autophagy is upregulated and double-membrane autophagosomes are formed by the expansion of phagophores. The ATG16L1 precursor fusion contributes to development of phagophore structures and is critical for the biogenesis of autophagosomes. Here, we discovered a novel role of the protein tyrosine phosphatase PTPN9 in the regulation of homotypic ATG16L1 vesicle fusion and early autophagosome formation. Depletion of PTPN9 and its Drosophila homolog Ptpmeg2 impaired autophagosome formation and autophagic flux. PTPN9 colocalized with ATG16L1 and was essential for homotypic fusion of ATG16L1+ vesicles during starvation-induced autophagy. We further identified the Q-SNARE VTI1B as a substrate target of PTPN9 phosphatase. Like PTPN9, the VTI1B nonphosphorylatable mutant but not the phosphomimetic mutant enhanced SNARE complex assembly and autophagic flux. Our findings highlight the important role of PTPN9 in the regulation of ATG16L1+ autophagosome precursor fusion and autophagosome biogenesis through modulation of VTI1B phosphorylation status.Abbreviations: csw: corkscrew; EBSS: Earle's balanced salt solution; ERGIC: ER-Golgi intermediate compartment; ESCRT: endosomal sorting complexes required for transport; mop: myopic; NSF: N-ethylmaleimide-sensitive factor; PAS: phagophore assembly site; PolyQ: polyglutamine; PtdIns3P: phosphatidylinositol-3-phosphate; PTK: protein tyrosine kinase; PTM: posttranslational modification; PTP: protein tyrosine phosphatase; PTPN23/HD-PTP: protein tyrosine phosphatase non-receptor type 23; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; STX7: syntaxin 7; STX8: syntaxin 8; STX17: syntaxin 17; VAMP3: vesicle associated membrane protein 3; VAMP7: vesicle associated membrane protein 7; VTI1B: vesicle transport through interaction with t-SNAREs 1B; YKT6: YKT6 v-SNARE homolog; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.