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). © 2015 by John Wiley & Sons, Inc.

New directions in poly(ADP‐ribose) polymerase biology

“…This approach is limited by the number of individual proteins used on the microarray, and the potential for false negatives if protein complexes containing PARPs are required to target substrates for ADP‐ribosylation. The analog‐sensitive approach involves the use of NAD + analogs that can only be utilized by PARPs with specifically engineered pockets within the NAD + binding site of the catalytic domain . In cells expressing these engineered PARPs, target proteins are modified with those analogs and can be selectively isolated or identified.…”

Identifying Family-Member-Specific Targets of Mono-ARTDs by Using a Chemical Genetics Approach

“…This NAD + analogue contains an alkyne at the N-6 position of the adenine ring to aid in target identification using click conjugation to a rhodamine-azide or biotin-azide. We showed that 5-Et-6-a-NAD + was used as a selective substrate for K903A (KA-), but not wild-type (WT-) ARTD1, and mutation of the ceiling lysine to an alanine in the other poly-ARTDs gave similar results (Carter-O’Connell and Cohen, 2015; Carter-O’Connell et al, 2014). …”

“…We recently adapted a sensitized enzyme-modified substrate (“bump-hole”) method for identifying the direct protein targets of poly-ARTDs (Carter-O’Connell and Cohen, 2015; Carter-O’Connell et al, 2014). This method involved mutating an active site lysine residue (Lys903 in human ARTD1, referred to here as the “ceiling” position) to an alanine to create a unique pocket for accommodating a C-5 ethyl group on the nicotinamide ring of the NAD + analogue, 5-Et-6-a-NAD + .…”

“…1 mg of total protein from either HEK 293T or HeLa lysate from cells expressing WT- or IG-tagged ARTD variants was incubated with 100 μM 5-Bn-6-a-NAD + for 2 hours at 30°C, click conjugated to biotin-PEG 3 -azide, subjected to enrichment using NeutrAvidin agarose (Pierce), and proteolysis as previously described (Carter-O’Connell and Cohen, 2015; Carter-O’Connell et al, 2014). MS experiments were performed using an Orbitrap Fusion (Thermo) equipped with a capillary HPLC system.…”

Detecting Protein ADP-Ribosylation Using a Clickable Aminooxy Probe

“… 14 To increase signal, biotinylated proteins can be enriched via immunoprecipitation using NeutrAvidin Agarose Resin as outlined previously [11, 12]. …”