Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry.
Nat Methods
; 14(5): 504-512, 2017 May.
Article
em En
| MEDLINE
| ID: mdl-28319114
ABSTRACT
Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present an approach to discovering linear polyubiquitin-modified substrates by combining a lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-REx HEK293T cells and validated several newly identified linear polyubiquitin targets. We demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for NFκB signaling.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Endopeptidases
/
Poliubiquitina
/
Ubiquitina-Proteína Ligases
/
Fator 6 Associado a Receptor de TNF
/
Ubiquitinação
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article