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.

Figure 1

(a) Schematic for the design of modified NAD⁺ analogues that are preferentially utilized by engineered ARTDs (KA-ARTD). P = phosphate. (b) 5-Et-6-a-NAD⁺ (1) used in this study.

Figure 2

(a) The key residue, K903, in ARTD1 (green), near the C-5 position of 3-methoxybenzamide (yellow) (PDB ID: 3PAX). (b) Sequence alignment of the nicotinamide binding site of the poly-ARTDs. (c) Comparative inhibition of WT-ARTD1 and KA-ARTD1 by select nicotinamide analogues. IC₅₀ values (mM) for each analogue are plotted comparing the WT-ARTD1 (y-axis) and KA-ARTD1 (x-axis) variants.

Figure 3

Orthogonal auto-ADPr of sensitized ARTD(s) using modified NAD⁺ variants. The concentration of the NAD⁺ analogue is indicated. Samples were subjected to immunoblot detection with streptavidin (biotin) to detect modified protein. His-tag (His) detection served as a loading control. Poly-ADPr and mono-ADPrmodified fractions are indiciated: (a) ARTD1, (b) ARTD2, and (c) ARTD6_cat.

Figure 4

(a) Lysate labeling by ARTDs and modified NAD⁺ analogues. The faint bands observed in the 5-Et-NAD⁺-only lane correspond to endogenous biotinylated proteins. The membrane stained with Ponceau S serves as a loading control. The asterisks mark the KA-ARTD1 (upper) and KA-ARTD2 (lower) bands. (b) Immunoblot detection of the LC-MS/MS-identified targets (XRCC5, Catenin-δ-1, hnRNP Q/R) following NeutrAvidin enrichment.