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Detecting and Quantifying pADPr In Vivo

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Poly(ADP-ribose) Polymerase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 780))

Abstract

Poly(ADP-ribose) polymerases (PARP) participate in diverse biological processes contributing to cellular homeostasis or exacerbating injury. PARP catalyzes the addition of ADP-ribose molecules (pADPr) to the target proteins, a process termed poly-ADP-ribosylation. Overactivation of PARP, as reflected by increased poly-ADP-ribosylation, accumulation of pADPr-modified proteins or free pADPr, contributes to the depletion of NAD+ and mitochondrial dysfunction, potentially leading to cell death via apoptosis or necrosis. Since PARP over-activation has been identified as a key contributor to the pathobiology of many diseases, monitoring PARP 1 activation by detecting and quantifying pADPr may provide valuable mechanistic insights as well as facilitating therapeutic drug monitoring for PARP inhibitors.

Several non-isotopic immunodetection methods for quantifying pADPr are discussed: western blotting of poly-ADP-ribosylated proteins, cellular localization of pADPr by immunohistochemistry, quantification of pADPr by enzyme-linked immunoassay and small-scale two-dimensional gel electrophoresis.

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References

  1. Chiarugi A, Moskowitz MA (2003) Poly(ADP-ribose) polymerase-1 activity promotes NF-kappaB-driven transcription and microglial activation: implication for neurodegenerative disorders. J Neurochem 85:306–317

    Article  PubMed  CAS  Google Scholar 

  2. de Murcia JM, Niedergang C, Trucco C, Ricoul M, Dutrillaux B, Mark M, Oliver FJ, Masson M, Dierich A, LeMeur M, Walztinger C, Chambon P, de Murcia G (1997) Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells. Proc Natl Acad Sci USA 94:7303–7307

    Article  PubMed  Google Scholar 

  3. Ferro AM, Olivera BM (1984) Poly(ADP-ribosylation) of DNA topoisomerase I from calf thymus. J Biol Chem 259:547–554

    PubMed  CAS  Google Scholar 

  4. Ha HC, Hester LD, Snyder SH (2002) Poly(ADP-ribose) polymerase-1 dependence of stress-induced transcription factors and associated gene expression in glia. Proc Natl Acad Sci USA 99:3270–3275

    Article  PubMed  CAS  Google Scholar 

  5. Oei SL, Shi Y (2001) Poly(ADP-ribosyl)ation of transcription factor Yin Yang 1 under conditions of DNA damage. Biochem Biophys Res Commun 285:27–31

    Article  PubMed  CAS  Google Scholar 

  6. Satoh MS, Lindahl T (1992) Role of poly(ADP-ribose) formation in DNA repair. Nature 356:356–358

    Article  PubMed  CAS  Google Scholar 

  7. Scovassi AI, Mariani C, Negroni M, Negri C, Bertazzoni U (1993) ADP-ribosylation of nonhistone proteins in HeLa cells: modification of DNA topoisomerase II. Exp Cell Res 206:177–181

    Article  PubMed  CAS  Google Scholar 

  8. Tulin A, Spradling A (2003) Chromatin loosening by poly(ADP)-ribose polymerase (PARP) at Drosophila puff loci. Science 299:560–562

    Article  PubMed  CAS  Google Scholar 

  9. Valenzuela MT, Guerrero R, Nunez MI, Ruiz De Almodovar JM, Sarker M, de Murcia G, Oliver FJ (2002) PARP-1 modifies the effectiveness of p53-mediated DNA damage response. Oncogene 21:1108–1116

    Article  PubMed  CAS  Google Scholar 

  10. Alano CC, Ying W, Swanson RA (2004) Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition. J Biol Chem 279:18895–18902

    Article  PubMed  CAS  Google Scholar 

  11. Cozzi A, Cipriani G, Fossati S, Faraco G, Formentini L, Min W, Cortes U, Wang ZQ, Moroni F, Chiarugi A (2006) Poly(ADP-ribose) accumulation and enhancement of postischemic brain damage in 110-kDa poly(ADP-ribose) glycohydrolase null mice. J Cereb Blood Flow Metab 26:684–695

    Article  PubMed  CAS  Google Scholar 

  12. Endres M, Wang ZQ, Namura S, Waeber C, Moskowitz MA (1997) Ischemic brain injury is mediated by the activation of poly(ADP-ribose)polymerase. J Cereb Blood Flow Metab 17:1143–1151

    Article  PubMed  CAS  Google Scholar 

  13. Lai Y, Chen Y, Watkins SC, Nathaniel PD, Guo F, Kochanek PM, Jenkins LW, Szabo C, Clark RS (2008) Identification of poly-ADP-ribosylated mitochondrial proteins after traumatic brain injury. J Neurochem 104:1700–1711

    Article  PubMed  CAS  Google Scholar 

  14. Oliver FJ, Menissier-de Murcia J, Nacci C, Decker P, Andriantsitohaina R, Muller S, de la Rubia G, Stoclet JC, de Murcia G (1999) Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice. EMBO J 18:4446–4454

    Article  PubMed  CAS  Google Scholar 

  15. Satchell MA, Zhang X, Kochanek PM, Dixon CE, Jenkins LW, Melick J, Szabo C, Clark RS (2003) A dual role for poly-ADP-ribosylation in spatial memory acquisition after traumatic brain injury in mice involving NAD+ depletion and ribosylation of 14-3-3gamma. J Neurochem 85:697–708

    Article  PubMed  CAS  Google Scholar 

  16. Zingarelli B, Hake PW, O’Connor M, Denenberg A, Wong HR, Kong S, Aronow BJ (2004) Differential regulation of activator protein-1 and heat shock factor-1 in myocardial ischemia and reperfusion injury: role of poly(ADP-ribose) polymerase-1. Am J Physiol Heart Circ Physiol 286:H1408–H1415

    Article  PubMed  CAS  Google Scholar 

  17. Szabo C, Dawson VL (1998) Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion. Trends Pharmacol Sci 19:287–298

    Article  PubMed  CAS  Google Scholar 

  18. Zhang J, Dawson VL, Dawson TM, Snyder SH (1994) Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 263:687–689

    Article  PubMed  CAS  Google Scholar 

  19. Andrabi SA, Kim NS, Yu SW, Wang H, Koh DW, Sasaki M, Klaus JA, Otsuka T, Zhang Z, Koehler RC, Hurn PD, Poirier GG, Dawson VL, Dawson TM (2006) Poly(ADP-ribose) (PAR) polymer is a death signal. Proc Natl Acad Sci USA 103:18308–18313

    Article  PubMed  CAS  Google Scholar 

  20. Yu SW, Andrabi SA, Wang H, Kim NS, Poirier GG, Dawson TM, Dawson VL (2006) Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death. Proc Natl Acad Sci USA 103:18314–18319

    Article  PubMed  CAS  Google Scholar 

  21. Besson VC, Margaill I, Plotkine M, Marchand-Verrecchia C (2003) Deleterious activation of poly(ADP-ribose)polymerase-1 in brain after in vivo oxidative stress. Free Radic Res 37:1201–1208

    Article  PubMed  CAS  Google Scholar 

  22. Kanai Y, Miwa M, Matsushima T, Sugimura T (1974) Studies on anti-poly(adenosine ­diphosphate ribose) antibody. Biochem Biophys Res Commun 59:300–306

    Article  PubMed  CAS  Google Scholar 

  23. Sairanen T, Szepesi R, Karjalainen-Lindsberg ML, Saksi J, Paetau A, Lindsberg PJ (2009) Neuronal caspase-3 and PARP-1 correlate differentially with apoptosis and necrosis in ischemic human stroke. Acta Neuropathol 118:541–552

    Article  PubMed  CAS  Google Scholar 

  24. Fink EL, Lai Y, Zhang X, Janesko-Feldman K, Adelson PD, Szabo C, Berger RP, Sarnaik AA, Kochanek PM, Clark RS (2008) Quantification of poly(ADP-ribose)-modified proteins in cerebrospinal fluid from infants and children after traumatic brain injury. J Cereb Blood Flow Metab 28:1523–1529

    Article  PubMed  CAS  Google Scholar 

  25. Sarnaik AA, Conley YP, Okonkwo DO, Barr TL, Fink EL, Szabo C, Kochanek PM, Clark RS (2010) Influence of PARP-1 polymorphisms in patients after traumatic brain injury. J Neurotrauma 27:465–471

    Article  PubMed  Google Scholar 

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Acknowledgment

This work was supported by P50 NS30318 (RSC) and T32 HD40686 (YL, MAS).

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Authors and Affiliations

  1. Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

    Yi-Chen Lai

  2. Kravis Childrens Hospital at Mount Sinai, New York, NY, USA

    Margaret A. Satchell

  3. Safar Center for Resuscitation Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Robert S. B. Clark

  4. Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Rajesh K. Aneja

Authors
  1. Yi-Chen Lai
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  2. Rajesh K. Aneja
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  3. Margaret A. Satchell
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  4. Robert S. B. Clark
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Editor information

Editors and Affiliations

  1. Keystone Program in Epigenetics &, Progenitor Cells, Fox Chase Cancer Center, Cottman Avenue 333, Philadelphia, 19111, Pennsylvania, USA

    Alexei V. Tulin

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Lai, YC., Aneja, R.K., Satchell, M.A., Clark, R.S.B. (2011). Detecting and Quantifying pADPr In Vivo. In: Tulin, A. (eds) Poly(ADP-ribose) Polymerase. Methods in Molecular Biology, vol 780. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-270-0_8

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  • DOI: https://doi.org/10.1007/978-1-61779-270-0_8

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-269-4

  • Online ISBN: 978-1-61779-270-0

  • eBook Packages: Springer Protocols

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