Identifying Direct Protein Targets of Poly-ADP-Ribose Polymerases (PARPs) Using Engineered PARP Variants-Orthogonal Nicotinamide Adenine Dinucleotide (NAD+) Analog Pairs.
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).
Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon.
Ian
I
Carter-O'Connell
Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon.
Michael S
MS
Cohen
D057809:HEK293 Cells; D006801:Humans; D009243:NAD / Q000031:analogs & derivatives*; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D020033:Protein Isoforms / Q000378:metabolism
D020033:Protein Isoforms; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases
ADP-ribose; ADP-ribosylation; ADPr; PARP; click chemistry; poly-ADP-ribose polymerase; post-translational modification; proteins