Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes.
Nat Commun
; 9(1): 1162, 2018 03 21.
Article
em En
| MEDLINE
| ID: mdl-29563501
ABSTRACT
Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Coloração e Rotulagem
/
Processamento de Proteína Pós-Traducional
/
Proteômica
/
Proteína de Replicação A
/
Enzimas Desubiquitinantes
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Nat Commun
Assunto da revista:
BIOLOGIA
/
CIENCIA
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Estados Unidos