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

Ian Carter‐O'Connell1, Michael S. Cohen1

1 Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140259
Online Posting Date:  June, 2015
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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.

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

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: In Vitro Labeling of the Direct Protein Targets of KA‐PARPs
  • Basic Protocol 2: Neutravidin Enrichment of Labeled Direct Protein Targets for Mass Spectrometry Applications
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vitro Labeling of the Direct Protein Targets of KA‐PARPs

  Materials
  • HEK 293 T cell pellet: a single 10‐cm dish of 80% to 90% confluent cells is sufficient for multiple pilot experiments (following cell harvesting, pellets can be flash frozen in liquid nitrogen and stored up to 6 months at −80°C)
  • Lysis buffer (see recipe)
  • Bradford reagent
  • BSA standards (1.0 mg/ml to 0.2 mg/ml in water)
  • 7× Protease inhibitor mix (see recipe)
  • Lysis buffer + NP‐40 (see recipe)
  • 10× R buffer (see recipe)
  • 1 μg/μl activated DNA (Sigma, cat. no. D4522)
  • Deionized water
  • Protein stock solution: 8.5 μM KA‐PARP1 (Carter‐O'Connell et al., ) in 1× R buffer
  • Analog stock solution: 1.98 mM 5‐Et‐6‐a‐NAD+ (Carter‐O'Connell et al., ) in 1× R buffer
  • 3× click buffer (see recipe)
  • 4× sample buffer (see recipe)
  • 10% Tris·Cl SDS‐PAGE gel
  • Streptavidin‐HRP buffer (see recipe)
  • 1.7‐ml microcentrifuge tubes
  • Centrifuge
  • Orbital incubator
  • SDS‐PAGE electrophoretic system
  • Nitrocellulose
  • Immunoblot transfer system

Basic Protocol 2: Neutravidin Enrichment of Labeled Direct Protein Targets for Mass Spectrometry Applications

  Materials
  • Sixteen identical HEK 293 T nuclear lysate labeling reactions generated with KA‐PARP1 and 5‐Et‐6‐a‐NAD+ from protocol 1, following click conjugation
  • Sixteen identical HEK 293 T nuclear lysate labeling reactions generated with 5‐Et‐6‐a‐NAD+ (negative control) from protocol 1, following click conjugation
  • 4× sample buffer (see recipe)
  • Methanol (HPLC grade); kept at 4°C
  • Ni‐NTA agarose (Qiagen, cat. no. 30210)
  • Deionized water
  • Dilution buffer (see recipe)
  • Ni‐NTA incubation buffer (see recipe)
  • Ice
  • 2% (w/v) sodium dodecyl sulfate (SDS) solution
  • NeutrAvidin agarose (Thermo Scientific, cat. no. 29200)
  • Wash buffer 1 (see recipe)
  • Urea wash buffer (see recipe)
  • Wash buffer 2 (see recipe)
  • 50 mM ammonium bicarbonate
  • 1× phosphate‐buffered saline (PBS), pH 7.4
  • 6 M urea
  • 0.5 M tris(2‐carboxyethyl)phosphine (TCEP)
  • 0.5 M iodoacetamide
  • 1.5× sample buffer + 1 mM biotin
  • 10 mM CaCl 2
  • 0.1 μg/μl sequencing grade trypsin in trypsin resuspension dilution buffer (Promega, cat. no V5111)
  • Formic acid
  • 10% Tris·HCl SDS‐PAGE gel
  • Nitrocellulose
  • Streptavidin‐HRP buffer (see recipe)
  • 15‐ml tubes
  • 1.7‐ml microcentrifuge tubes
  • Centrifuge
  • End‐over‐end rotisserie
  • Heating block
  • Spin columns (Pierce, cat. no. 69705)
  • SDS‐PAGE electrophoretic system
  • Immunoblot transfer system
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Figures

Videos

Literature Cited

Literature Cited
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