title: Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets.
abstract: 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.
authors.affiliation: Program in Chemical Biology and Department of Physiology and Pharmacology, and †Department of Biochemistry, Oregon Health & Science University , Portland, Oregon 97210, United States.
authors.forename: Ian
authors.initials: I
authors.lastname: Carter-O'Connell
authors.forename: Haihong
authors.initials: H
authors.lastname: Jin
authors.forename: Rory K
authors.initials: RK
authors.lastname: Morgan
authors.forename: Larry L
authors.initials: LL
authors.lastname: David
authors.forename: Michael S
authors.initials: MS
authors.lastname: Cohen
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D036002:ADP Ribose Transferases