Labeling Substrate Proteins of Poly(ADP‐ribose) Polymerases with Clickable NAD Analog

Hong Jiang1, Hening Lin1

1 Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110196
Online Posting Date:  March, 2012
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Abstract

Poly(ADP‐ribose) polymerases (PARPs) play important roles in various biological processes, including DNA repair, transcriptional regulation, mitosis, and RNA processing. PARP inhibitors are in clinical trials for treating human cancers. Understanding the biological function of PARPs will be important to fully realize the therapeutic potential of PARP inhibitors. We have developed a clickable analog of nicotinamide adenine dinucleotide (NAD) that can be used for in‐gel visualization, affinity purification, and identification of substrate proteins of PARPs. The protocols in this article describe a general procedure to label substrate proteins of PARPs using the clickable NAD analog. Curr. Protoc. Chem. Biol. 4:19‐34 © 2012 by John Wiley & Sons, Inc.

Keywords: clickable NAD analog; poly(ADP‐ribose) polymerase; poly(ADP‐ribosyl)ation; click chemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In‐Gel Fluorescence Visualization of Substrate Proteins Labeled by PARP‐1 and 6‐Alkyne‐NAD
  • Basic Protocol 2: Affinity Purification and Identification of Substrate Proteins of PARP‐1 Using 6‐Alkyne‐NAD
  • Support Protocol 1: Overexpression and Purification of Human Recombinant PARP‐1 from Insect Cells
  • Support Protocol 2: Synthesis of 6‐Alkyne‐NAD
  • Support Protocol 3: Preparation of Cell Lysate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In‐Gel Fluorescence Visualization of Substrate Proteins Labeled by PARP‐1 and 6‐Alkyne‐NAD

  Materials
  • 10× reaction buffer (see recipe)
  • 4 mg/ml cell lysate containing PARP‐1 substrate proteins ( protocol 5)
  • Recombinant PARP‐1 protein: 3.6 µM, overexpressed and purified as published (Jiang, ; see protocol 3), stored at −80°C (stable for up to 3 years)
  • Salmon sperm DNA: 5 mg/ml in water (Sigma, cat. no. D1626), stored at −20°C (stable for up to 3 years)
  • Clickable NAD analog (6‐alkyne‐NAD): 2 mM in water, synthesized according to published procedure (Jiang et al., ; see protocol 4), stored at −80°C (stable for up to 3 years)
  • Nicotinamide adenine dinucleotide (NAD): 2 mM in water (Sigma, cat. no. N6522), stored at −80°C (stable for up to 3 years)
  • PJ34: 0.56 mM and 0.26 mM in water (Sigma, cat. no. P4365), stored at −80°C (stable for up to 3 years)
  • Rh‐N 3 (tetramethylrhodamine 5‐carboxamido‐(6‐azidohexanyl)): 5 mM in DMF (Invitrogen, cat. no. T10182), stored at −80°C (stable for up to 3 years)
  • Ligand, Tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine: 10 mM in DMF (Sigma, cat. no. 678937), stored at −20°C (stable for up to 3 years)
  • Copper (II) sulfate (CuSO 4): 10 mM in water (Sigma‐Aldrich, cat. no. 451657), stored at 4°C (stable for up to 3 months)
  • Tris 2‐carboxyethyl phosphine (TCEP): 20 mM in water (Sigma, cat. no. 93284), prepared fresh just prior to use
  • 2× protein loading buffer (see recipe)
  • 12% acrylamide gel: 12% Mini‐PROTEAN TGX Precast Gel, Bio‐Rad, cat. no. 456‐1043
  • 10% (v/v) methanol (Sigma, cat. no. 494437)
  • Heating block
  • Mini protein gel electrophoresis system: Mini‐PROTEAN Tetra Cell, Bio‐Rad, cat. no. 165‐8001
  • Typhoon 9400 imager or other instrument that can record fluorescence image
  • Canon PowerShot S3 digital camera or other gel‐doc systems
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, )

Basic Protocol 2: Affinity Purification and Identification of Substrate Proteins of PARP‐1 Using 6‐Alkyne‐NAD

  Materials
  • 10× reaction buffer (see recipe)
  • 4 mg/ml cell lysate containing PARP‐1 substrate proteins ( protocol 5)
  • Recombinant PARP‐1 protein: 3.6 µM, overexpressed and purified as published (Jiang, ; see protocol 3), stored at −80°C (stable for up to 3 years)
  • Salmon sperm DNA: 5 mg/ml in water (Sigma, cat. no. D1626), stored at −20°C (stable for up to 3 years)
  • Clickable NAD analog (6‐alkyne‐NAD): 2 mM in water, synthesized according to published procedure (Jiang et al., ; see protocol 4), stored at −80°C (stable for up to 3 years)
  • PJ34: 0.56 mM and 0.26 mM in water (Sigma, cat. no. P4365), stored at −80°C (stable for up to 3 years)
  • Biotin‐N 3 (PEG4 carboxamide‐6‐azidohexanyl biotin): 5 mM in DMF (Invitrogen, cat. no. B10184), stored at −80°C (stable for up to 3 years)
  • Ligand, Tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine: 10 mM in DMF (Sigma, cat. no. 678937), stored at −20°C (stable for up to 3 years)
  • Copper (II) sulfate (CuSO 4): 10 mM in water (Sigma‐Aldrich, cat. no. 451657), stored at 4°C (stable for up to 3 months)
  • Tris 2‐carboxyethyl phosphine (TCEP): 20 mM in water (Sigma, cat. no. 93284), prepared fresh just prior to use
  • Acetone (Sigma, cat. no. 439126), cold
  • Methanol (Sigma, cat. no. 494437), cold
  • 2% and 0.2% (w/v) sodium dodecyl sulfate (SDS; Sigma, cat. no. L6026) in phosphate‐buffered saline (PBS), pH 7.4 (Invitrogen, cat. no. 10010‐049, store up to 3 months at 4°C)
  • Streptavidin beads (Pierce Biotechnology, cat. no. 20353)
  • Phosphate‐buffered saline (PBS), pH 7.4 (Invitrogen, cat. no. 10010‐049, store up to 3 months at 4°C)
  • 20 mM Tris⋅Cl/500 mM KCl, pH 7.4
  • 20 mM Tris⋅Cl, pH 7.4
  • 6 M urea (Sigma, cat. no. U5378) in PBS, pH 7.4, containing 9.5 mM TCEP (Sigma, cat. no. 93284)
  • 400 mM iodoacetamide (Sigma, cat. no. I1149)
  • 2 M urea (Sigma, cat. no. U5378) in PBS, pH 7.4
  • 1 µg/µl trypsin (Promega, cat. no. V5111) in PBS (pH 7.4) containing 2 M urea and 1 mM CaCl 2
  • Sep‐Pak Vac C18 cartridge (Waters Corporation, cat. no. WAT023590)
  • 10% (w/v) and 0.1% trifluoroacetic acid (TFA; Sigma, cat. no. 299537)
  • 90% (v/v) methanol/0.1% TFA/9.9% H 2O
  • 5% (v/v) methanol/0.1% TFA/94.9% H 2O
  • 50% (v/v) acetonitrile (Sigma, cat. no. 34967)/0.1% TFA/49.9% H 2O
  • 15‐ml conical centrifuge tubes
  • Refrigerated centrifuge
  • Heating block
  • 35°C water bath
  • Lyophilizer

Support Protocol 1: Overexpression and Purification of Human Recombinant PARP‐1 from Insect Cells

  Materials
  • SF9 insect cells (Invitrogen, cat. no. 12659‐017)
  • Sf‐900 III SFM (1×): Invitrogen, cat. no. 12658‐019, stored at 4°C (stable for up to 1 year)
  • Baculovirus vector containing full‐length human PARP‐1 with C‐terminal FLAG tag (can be ordered from Invitrogen Baculovirus Expression Services)
  • Phosphate buffered saline (PBS, 1×, pH 7.4): Invitrogen, cat. no. 10010‐049, stored at 4°C (stable for up to 1 year)
  • Lysis buffer 1 (see recipe)
  • Dilution buffer (see recipe)
  • ANTI‐FLAG M2 affinity resin: (Sigma, cat. no. A2220), stored at ‐20°C (stable for up to one year)
  • 15‐cm culture plates
  • Culture incubator (no CO 2)
  • Cell scraper (Fisher Scientific, cat. no. 08‐100‐242)
  • 50‐ml conical centrifuge tubes
  • Refrigerated centrifuge
  • 7‐ml Dounce Homogenizer
  • 2.0‐ml microcentrifuge tubes
  • Additional reagents and equipment for culture of insect cells (Murphy et al., )

Support Protocol 2: Synthesis of 6‐Alkyne‐NAD

  Materials
  • Absolute ethanol (Sigma, cat. no. 459836)
  • 6‐Chloropurine nucleoside (Sigma, cat. no. C8276)
  • Calcium carbonate (Sigma, cat. no. C4830)
  • Propargylamine (Sigma, cat. no. P50900)
  • N 2 source
  • Ether (dry; Sigma, cat. no. 673811)
  • Trimethyl phosphate (Sigma, cat. no. 241024)
  • Phosphoryl chloride (Sigma, cat. no. 201170)
  • Formic acid (Sigma, cat. no. 56302)
  • Acetonitrile (Sigma, cat. no. 34967)
  • Trifluoroacetic acid (Sigma, cat. no. 302031)
  • Triethylamine (Sigma, cat. no. T0886)
  • Methanol (Sigma, cat. no. 322415)
  • Nicotinamide mononucleotide trimethylammonium salt (Sigma, cat. no. N3501)
  • Dioxane (dry; Sigma, cat. no. 296309)
  • N,N‐dimethylformamide (DMR, dry; Sigma, cat. no. 227056)
  • Hexamethyl phosphoramide (Sigma, cat. no. H11602)
  • Diphenyl phosphochloridate (Sigma, cat. no. D206555)
  • Tri‐n‐butylamine (Sigma, cat. no. 90780)
  • Benzyltributylammonium chloride (Sigma, cat. no. 193771)
  • Pyridine (dry; Sigma, cat. no. 270970)
  • 25‐, 50‐, and 100‐ml round bottom flasks
  • Septum stoppers
  • Reflux condenser
  • Stirring bar and plate
  • Filter paper and funnel
  • Oil bath
  • Syringe and needle
  • −20°C freezer
  • Vacuum pump
  • LC‐MS system: SHIMADZU LCMS‐QP8000α with a Sprite TARGA C18 column (40 × 2.1 mm, 5 µm, Higgins Analytical, Inc.) monitoring at 215 and 260 nm with positive mode for mass detection
  • HPLC system: Beckman Coulter System Gold 125p solvent module and 168 detector with a TARGA C18 column (250 × 20 mm, 10 µm, Higgins Analytical, Inc.) monitoring at 215 and 260 nm
  • Rotary evaporator

Support Protocol 3: Preparation of Cell Lysate

  Materials
  • MCF‐7 cells (ATCC, cat. no. HTB‐22)
  • Dulbecco's modified eagle medium (DMEM; Invitrogen, cat. no. 11965‐118, store up to 1 year at 4°C) containing 10% fetal bovine serum (FBS; Invitrogen, cat. no. 10438‐034, store up to 1 year at −20°C)
  • Trypsin (Invitrogen, cat. no. 15400‐054; store up to 1 year at 4°C)
  • Phosphate buffered saline (PBS, 1×, pH 7.4; Invitrogen, cat. no. 10010‐049, store up to 1 year at 4°C)
  • Lysis buffer 2 (see recipe)
  • Protease inhibitor cocktail (Sigma, cat. no. P8340, store up to 1 year at −20°C
  • 10% (v/v) NP‐40: (Sigma, cat. no. 74385; store up to 3 years at 4°C)
  • 10‐cm culture plates
  • Refrigerated centrifuge
  • 20‐ml Dounce homogenizer
  • Refrigerated centrifuge
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Figures

Videos

Literature Cited

Literature Cited
   Ahel, I., Ahel, D., Matsusaka, T., Boulton, S.J., and West, S.C. 2008. Poly(ADP‐ribose)‐binding zinc finger motifs in DNA repair/checkpoint proteins. Nature 451:81‐85.
   Amé, J.C., Spenlehauer, C., and de Murcia, G. 2004. The PARP superfamily. Bioessays 26:882‐893.
   Belenky, P., Bogan, K.L., and Brenner, C. 2007. NAD+ metabolism in health and disease. Trends Biochem. Sci. 32:12‐19.
   Gagné, J.P., Isabelle, M., Lo, K.S., Bourassa, S., Hendzel, M.J., Dawson, V.L., Dawson, T.M., and Poirier, G.G. 2008. Proteome‐wide identification of poly(ADP‐ribose) binding proteins and poly(ADP‐ribose)‐associated protein complexes. Nucl. Acids Res. 36:6959‐6976.
   Gallagher, S.R. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1‐10.2A.37.
   Jiang, H., Kim, J.H., Frizzell, K.M., Kraus, W.L., and Lin, H. 2010. Clickable NAD analogs for labeling substrate proteins of poly(ADP‐ribose) polymerases. J. Am. Chem. Soc. 132:9363‐9372.
   Lai, Y., Chen, Y., Watkins, S.C., Nathaniel, P.D., Guo, F., Kochanek, P.M., Jenkins, L.W., Szabó, C., and Clark, R.S. 2008. Identification of poly‐ADP‐ribosylated mitochondrial proteins after traumatic brain injury. J. Neurochem. 104:1700‐1711.
   Mendoza‐Alvarez, H. and Alvarez‐Gonzalez, R. 1993. Poly(ADP‐ribose) polymerase is a catalytic dimer and the automodification reaction is intermolecular. J. Biol. Chem. 268:22575‐22580.
   Mendoza‐Alvarez, H. and Alvarez‐Gonzalez, R. 2001. Regulation of p53 sequence‐specific DNA‐binding by covalent poly(ADP‐ribosyl)ation. J. Biol. Chem. 276:36425‐36430.
   Murphy, C.I., Piwnica‐Worms, H., Grünwald, S., Romanow, W.G., Francis, N., and Fan, H.‐Y. 2004. Maintenance of insect cell cultures and generation of recombinant baculoviruses. Curr. Protoc. Mol. Biol. 65:16.10.1‐16.10.19.
   Rawling, J. M. and Alvarez‐Gonzalez, R. 1997. TFIIF, a basal eukaryotic transcription factor, is a substrate for poly(ADP‐ribosyl)ation. Biochem. J. 324:249‐253.
   Rostovtsev, V.V., Green, L.G., Fokin, V.V., and Sharpless, K.B. 2002. A stepwise huisgen cycloaddition process: copper(I)‐catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41:2596‐2599.
   Sala, A., La Rocca, G., Burgio, G., Kotova, E., Di Gesù, D., Collesano, M., Ingrassia, A.M., Tulin, A.V., and Corona, D.F. 2008. The nucleosome‐remodeling ATPase ISWI is regulated by poly‐ADP‐ribosylation. PLoS Biol. 6:e252.
   Schreiber, V., Dantzer, F., Ame, J.C., and de Murcia, G. 2006. Poly(ADP‐ribose): Novel functions for an old molecule. Nat. Rev. Mol. Cell Biol. 7:517‐528.
   Weerapana, E., Speers, A.E., and Cravatt, B.F. 2007. Tandem orthogonal proteolysis‐activity‐based protein profiling (TOP‐ABPP)—A general method for mapping sites of probe modification in proteomes. Nat. Protoc. 2:1414‐1425.
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