Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 136, Issue 14, Pages 5201-5204Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja412897a
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Funding
- NCI NIH HHS [P30 CA069533] Funding Source: Medline
- NCRR NIH HHS [S10 RR025571] Funding Source: Medline
- NEI NIH HHS [P30 EY010572] Funding Source: Medline
- NINDS NIH HHS [R01 NS088629] Funding Source: Medline
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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.
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