Identifying Direct Protein Targets of Poly-ADP-Ribose Polymerases (PARPs) Using Engineered PARP Variants-Orthogonal Nicotinamide Adenine Dinucleotide (NAD+) Analog Pairs

Abstract

Poly-ADP-ribose polymerases (PARPs) comprise a family of 17 distinct enzymes that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to acceptor sites on protein targets. PARPs have been implicated in a number of essential signaling pathways regulating both normal cell function and pathophysiology. To understand the physiological role of each PARP family member in the cell we need to identify the direct targets for each unique PARP in a cellular context. PARP-family member-specific target identification is challenging because of their shared catalytic mechanism and functional redundancy. To address this challenge, we have engineered a PARP variant that efficiently uses an orthogonal NAD+ analog, an analog that endogenous PARPs cannot use, as a substrate for ADP-ribosylation. The protocols in this unit describe a general procedure for using engineered PARP variants-orthogonal NAD+ analog pairs for labeling and identifying the direct targets of the poly-subfamily of PARPs (PARPs 1-3, 5, and 6).

Keywords: ADP-ribose; ADP-ribosylation; ADPr; PARP; click chemistry; poly-ADP-ribose polymerase; post-translational modification; proteins.

Figure 1

(A) Schematic for the modification of direct PARP protein targets using engineered KA-PARP and 5-Et-6-a-NAD⁺ analogues. Labeled direct protein targets are modified with a 6-a-ADPr adduct that is conjugated to an azide-linked probe using copper catalyzed azide-alkyne cycloaddition. Modified targets can then be visualized or enriched for downstream LC-MS/MS applications. P: phosphate. (B) 5-Et-6-a-NAD⁺ (1) used in this study. Adapted with permission from Carter-O’Connell, I., Jin, H., Morgan, R.K., David, L.L., and Cohen, M.S. 2014. Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets. Journal of the American Chemical Society 136:5201–5204. Copyright 2014 American Chemcial Society.

Figure 2

Lysate labeling by PARPs and modified NAD⁺ analogues. The faint bands observed in the 5-Et-6-a-NAD⁺ only lane correspond to endogenous biotinylated proteins. The membrane stained with Ponceau S serves as a loading control. The asterisks mark the KA-PARP1 (upper) and KA-PARP2 (lower) bands. Adapted with permission from Carter-O’Connell, I., Jin, H., Morgan, R.K., David, L.L., and Cohen, M.S. 2014. Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets. Journal of the American Chemical Society 136:5201–5204. Copyright 2014 American Chemcial Society.

Figure 3

Immunoblot analysis of the LC-MS/MS sample preparation. The indicated fractions from the NeutrAvidin enrichment protocol were fractionated by SDS-PAGE and imaged using streptavidin-HRP. The modified NAD⁺ analogue was spiked into HEK 293T nuclear extract (6-a-NAD⁺: 100 μM; 5-Et-6-a-NAD⁺: 250 μM) both without any additional source of exogenous PARP and with either KA-PARP1 or KA-PARP2, samples were labeled, conjugated to biotin, enriched, and submitted for LC-MS/MS analysis. Positions of the molecular-weight markers are indicated on the left of the blot. Reprinted with permission from Carter-O’Connell, I., Jin, H., Morgan, R.K., David, L.L., and Cohen, M.S. 2014. Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets. Journal of the American Chemical Society 136:5201–5204. Copyright 2014 American Chemcial Society.