title: Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs.
abstract: An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process.
authors: Centre de recherche du CHU de Québec - Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada.
authors: Jean-Philippe
authors: JP
authors: Gagné
authors: Centre de recherche du CHU de Québec - Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada.
authors: Chantal
authors: C
authors: Ethier
authors: Plateforme Protéomique du Centre de Recherche du CHU de Québec - Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada.
authors: Daniel
authors: D
authors: Defoy
authors: Plateforme Protéomique du Centre de Recherche du CHU de Québec - Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada.
authors: Sylvie
authors: S
authors: Bourassa
authors: Department of Biochemistry & Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA.
authors: Marie-France
authors: MF
authors: Langelier
authors: Department of Biochemistry & Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA.
authors: Amanda A
authors: AA
authors: Riccio
authors: Department of Biochemistry & Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA.
authors: John M
authors: JM
authors: Pascal
authors: Department of Biochemistry and Molecular Biology, University of British Columbia, Centre for High-Throughput Biology, Vancouver, British Columbia, Canada.
authors: Kyung-Mee
authors: KM
authors: Moon
authors: Department of Biochemistry and Molecular Biology, University of British Columbia, Centre for High-Throughput Biology, Vancouver, British Columbia, Canada.
authors: Leonard J
authors: LJ
authors: Foster
authors: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
authors: Zhibin
authors: Z
authors: Ning
authors: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
authors: Daniel
authors: D
authors: Figeys
authors: Plateforme Protéomique du Centre de Recherche du CHU de Québec - Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada.
authors: Arnaud
authors: A
authors: Droit
authors: Centre de recherche du CHU de Québec - Pavillon CHUL, Faculté de Médecine, Université Laval, Québec, Canada. Electronic address: Guy.Poirier@crchul.ulaval.ca.
authors: Guy G
authors: GG
authors: Poirier
mesh_terms: D000818:Animals; D002417:Cattle; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D000071137:Poly (ADP-Ribose) Polymerase-1; D011064:Poly Adenosine Diphosphate Ribose / Q000032:analysis*; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D018797:Cell Cycle Proteins; D011064:Poly Adenosine Diphosphate Ribose; C499320:PARP1 protein, human; C483041:PARP2 protein, human; C480979:PARP3 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases
keywords: ADP-ribose; Hydroxylamine; Mass spectrometry; PARP-1; PARP-2; PARP-3; Poly(ADP-ribosyl)ation; Posttranslational modification; Proteomics