Expression, purification, and characterization of the TRIM49 protein.
Protein Expr Purif
; 143: 57-61, 2018 03.
Article
em En
| MEDLINE
| ID: mdl-29107100
ABSTRACT
Autophagy is the process of degradation of intracellular proteins through the lysosome. Members of the tripartite motif (TRIM) proteins have shown to directly recognize autophagic cargo and also to act as a hub for the phagophore nucleation complex. The TRIM proteins are classically characterized by the presence of an amino-terminal RING domain and a B-box domain followed by a coiled coil domain. Although regarded as ubiquitin E3 ligases, this activity has been shown only for a minor set of the 79 human TRIM proteins. Additionally, the role of each domain in the E3 ligase activity is unknown. We investigated the role of the SPRY and RING domains of the human TRIM49 protein in its E3 ubiquitin ligase activity. Wild-type and mutant constructs of tagged TRIM49 were expressed in E. coli or mammalian cells, and the autoubiquitination activity of the purified protein was assessed. The purified TRIM49 showed no ubiquitin E3 ligase activity in vitro. However, cells transfected with the wild-type or mutant protein showed increased levels of lower mass polyubiquitinated proteins and both proteins copurified with polyubiquitinated proteins. Taken together, these results indicate that the TRIM49 protein plays a role in autophagic protein degradation independently of an ubiquitin E3 ligase activity.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Proteínas de Transporte
Limite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Brasil