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Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

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11 pages
Poly(ADP-ribose) polymerases (PARPs) catalyze the formation of poly(ADP-ribose) (pADPr), a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose) glycohydrolase (PARG), on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosyl)ation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS) aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions. Results PARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1. Conclusions This study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose) metabolism.
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Isabelleet al.Proteome Science2010,8:22 http://www.proteomesci.com/content/8/1/22
R E S E A R C HOpen Access Research Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry
1 11,2 11 1 Maxim Isabelle, Xavier Moreel, Jean-Philippe Gagné, Michèle Rouleau, Chantal Ethier, Pierre Gagné, 3 1,4 Michael J Hendzeland Guy G Poirier*
Background14, -15, and -16) or catalytically inactive members (PARP-Poly(ADP-ribose) polymerases (PARPs) catalyze the for-9 and -13). Poly(ADP-ribose) glycohydrolase (PARG), on mation of poly(ADP-ribose) (pADPr), a protein post-the other hand, catabolizes pADPr and thereby accounts translational modification involved in several importantfor the transient nature of poly(ADP-ribosyl)ation. biological processes, namely surveillance of genomeIn this study, we chose to investigate PARP-1 and integrity, cell cycle progression, initiation of the DNAPARP-2 because of their pivotal role in the maintenance damage response, apoptosis, and regulation of transcrip-of genome integrity, and PARG to cover both the synthe-tion (reviewed in [1]). Recently Kleineet al.sis and degradation components of pADPr metabolism.[2] limited the PARP family to PARPs possessing the HYE catalyticThrough their strand break-dependent PARP activity, core motif as well as a long β4/β5 loop, namely PARP-1, -both PARP-1 and PARP-2 are able to initiate a rapid 2, -3, tankyrase-1 and -2, and vault-PARP. All other puta-response to DNA damage via pADPr synthesis on them-tive PARP family members were re-classified as mono-selves (automodification) and on other nuclear acceptors ADP-ribosyltransferases (PARP-6, -7, -8, -10, -11, -12, -such as histones. This DNA damage response facilitates base-excision repair (BER) [3,4] and contributes to non-* Correspondence: guy.poirier@crchul.ulaval.ca 1homologous end joining (NHEJ) [5,6]. Empirical evidence Axe cancer, CHUQ Research Center, Faculty of Medicine, Laval University, 2705 Boulevard Laurier, Québec, Canada, G1V 4G2however indicates that the functions of PARP-2 do not Full list of author information is available at the end of the article © 2010 Isabelle et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons BioMedCentral Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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