High-throughput screening of functional deubiquitinating enzymes in autophagy.
Autophagy
; 17(6): 1367-1378, 2021 06.
Article
en En
| MEDLINE
| ID: mdl-32453962
ABSTRACT
Macroautophagy/autophagy, a eukaryotic homeostatic process that sequesters cytoplasmic constituents for lysosomal degradation, is orchestrated by a number of autophagy-related (ATG) proteins tightly controlled by post-translational modifications. However, the involvement of reversible ubiquitination in the regulation of autophagy remains largely unclear. Here, we performed a single-guide RNA-based screening assay to investigate the functions of deubiquitinating enzymes (DUBs) in regulating autophagy. We identified previously unrecognized roles of several DUBs in modulating autophagy at multiple levels by targeting various ATG proteins. Mechanistically, we demonstrated that STAMBP/AMSH (STAM-binding protein) promotes the stabilization of ULK1 by removing its lysine 48 (K48)-linked ubiquitination, whereas OTUD7B mediates the degradation of PIK3 C3 by enhancing its K48-linked ubiquitination, thus positively or negatively affects autophagy flux, respectively. Together, our study elaborated on the broad involvement of DUBs in regulating autophagy and uncovered the critical roles of the reversible ubiquitination in the modification of ATG proteins.Abbreviations ATG autophagy-related; Baf A1 bafilomycin A1; DUB deubiquitinating enzyme; EBSS Earle's balanced salt solution; KO knockout; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; OTUD7B OTU domain-containing protein 7B; PIK3C3 phosphatidylinositol 3-kinase catalytic subunit type 3; sgRNA single-guide RNA; SQSTM1/p62 sequestosome 1; STAMBP/AMSH STAM-binding protein; ULK1 unc-51 like autophagy activating kinase 1; USP ubiquitin specific peptidase.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Autofagia
/
Ubiquitinación
/
Ensayos Analíticos de Alto Rendimiento
/
Proteasas Ubiquitina-Específicas
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Límite:
Humans
Idioma:
En
Revista:
Autophagy
Año:
2021
Tipo del documento:
Article
País de afiliación:
China