title: ENPP1 processes protein ADP-ribosylation in vitro.
abstract: ADP-ribosylation is a conserved post-translational protein modification that plays a role in all major cellular processes, particularly DNA repair, transcription, translation, stress response and cell death. Hence, dysregulation of ADP-ribosylation is linked to the physiopathology of several human diseases including cancers, diabetes and neurodegenerative disorders. Protein ADP-ribosylation can be reversed by the macrodomain-containing proteins PARG, TARG1, MacroD1 and MacroD2, which hydrolyse the ester bond known to link proteins to ADP-ribose as well as consecutive ADP-ribose subunits; targeting this bond can thus result in the complete removal of the protein modification or the conversion of poly(ADP-ribose) to mono(ADP-ribose). Recently, proteins containing the NUDIX domain - namely human NUDT16 and bacterial RppH - have been shown to process in vitro protein ADP-ribosylation through an alternative mechanism, converting it into protein-conjugated ribose-5'-phosphate (R5P, also known as pR). Though this protein modification was recently identified in mammalian tissues, its physiological relevance and the mechanism of generating protein phosphoribosylation are currently unknown. Here, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as the first known mammalian enzyme lacking a NUDIX domain to generate pR from ADP-ribose on modified proteins in vitro. Thus, our data show that at least two enzyme families - Nudix and ENPP/NPP - are able to metabolize protein-conjugated ADP-ribose to pR in vitro, suggesting that pR exists and may be conserved from bacteria to mammals. We also demonstrate the utility of ENPP1 for converting protein-conjugated mono(ADP-ribose) and poly(ADP-ribose) into mass spectrometry-friendly pR tags, thus facilitating the identification of ADP-ribosylation sites.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, UK.
authors.forename: Luca
authors.initials: L
authors.lastname: Palazzo
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Casey M
authors.initials: CM
authors.lastname: Daniels
authors.affiliation: OPPF-UK, The Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, UK.|Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, UK.
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Nettleship
authors.affiliation: OPPF-UK, The Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, UK.|Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, UK.
authors.forename: Nahid
authors.initials: N
authors.lastname: Rahman
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Robert Lyle
authors.initials: RL
authors.lastname: McPherson
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, WA, USA.
authors.forename: Shao-En
authors.initials: SE
authors.lastname: Ong
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Kato
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Nureki
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA. anthony.leung@jhu.edu.|Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. anthony.leung@jhu.edu.
authors.forename: Anthony K L
authors.initials: AK
authors.lastname: Leung
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, UK. ivan.ahel@path.ox.ac.uk.
authors.forename: Ivan
authors.initials: I
authors.lastname: Ahel
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
chemical_list: D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; C000603470:ARTC2.2 protein, mouse; C499320:PARP1 protein, human; C499627:PARP10 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; C549897:NUDT16 protein, mouse; D011755:Pyrophosphatases
keywords: ENPP1; PAR; PARP1; mass spectrometry; phosphodiesterase; poly(ADP-ribose); post-translational modification; protein ADP-ribosylation