Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets.
J Am Chem Soc
; 136(14): 5201-4, 2014 Apr 09.
Article
en En
| MEDLINE
| ID: mdl-24641686
Adenosine diphosphate ribosyltransferases (ARTDs; ARTD1-17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD(+)), to amino acids of target proteins. The functional redundancy and overlapping target specificities among the 17 ARTDs in humans make the identification of direct targets of individual ARTD family members in a cellular context a formidable challenge. Here we describe the rational design of orthogonal NAD(+) analogue-engineered ARTD pairs for the identification of direct protein targets of individual ARTDs. Guided by initial inhibitor studies with nicotinamide analogues containing substituents at the C-5 position, we synthesized an orthogonal NAD(+) variant and found that it is used as a substrate for several engineered ARTDs (ARTD1, -2, and -6) but not their wild-type counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear extracts highlighted the semi-complementary, yet distinct, protein targeting. Using affinity purification followed by tandem mass spectrometry, we identified 42 direct ARTD1 targets and 301 direct ARTD2 targets. This represents a powerful new technique for identifying direct protein targets of individual ARTD family members, which will facilitate studies delineating the pathway from ARTD activation to a given cellular response.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ingeniería de Proteínas
/
ADP Ribosa Transferasas
Límite:
Humans
Idioma:
En
Revista:
J Am Chem Soc
Año:
2014
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos