Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease
2013; Springer Nature; Volume: 32; Issue: 9 Linguagem: Inglês
10.1038/emboj.2013.51
ISSN1460-2075
AutoresReza Seyed Sharifi, Rosa Morra, C. Denise Appel, Michael Tallis, Barry A. Chioza, Gytis Jankevicius, Michael A. Simpson, Ivan Matić, Ege Ozkan, Barbara Golia, Matthew J. Schellenberg, Ria Weston, Jason G. Williams, Marianna Nicoletta Rossi, Hamid Galehdari, J.M. Krahn, Alexander Wan, Richard C. Trembath, Andrew H. Crosby, Dragana Ahel, Ronald T. Hay, Andreas G. Ladurner, Gyula Timinszky, R. Scott Williams, Ivan Ahel,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoArticle12 March 2013free access Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease Reza Sharifi Corresponding Author Reza Sharifi Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Rosa Morra Rosa Morra Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author C Denise Appel C Denise Appel Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Michael Tallis Michael Tallis Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Barry Chioza Barry Chioza Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Gytis Jankevicius Gytis Jankevicius Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Michael A Simpson Michael A Simpson Genetics and Molecular Medicine, King's College London, Guy's Hospital, London, UK Search for more papers by this author Ivan Matic Ivan Matic Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Scotland, UK Search for more papers by this author Ege Ozkan Ege Ozkan Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Barbara Golia Barbara Golia Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Matthew J Schellenberg Matthew J Schellenberg Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Ria Weston Ria Weston Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Jason G Williams Jason G Williams Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Marianna N Rossi Marianna N Rossi Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Hamid Galehdari Hamid Galehdari Genetics Department, Sciences Faculty, Ahvaz Shahid Chamran University, Ahvaz, Iran Search for more papers by this author Juno Krahn Juno Krahn Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Alexander Wan Alexander Wan Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Richard C Trembath Richard C Trembath Genetics and Molecular Medicine, King's College London, Guy's Hospital, London, UK Search for more papers by this author Andrew H Crosby Andrew H Crosby Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Dragana Ahel Dragana Ahel Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Ron Hay Ron Hay Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Scotland, UK Search for more papers by this author Andreas G Ladurner Corresponding Author Andreas G Ladurner Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany Center for Integrated Protein Science Munich (CIPSM), Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Gyula Timinszky Corresponding Author Gyula Timinszky Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author R Scott Williams Corresponding Author R Scott Williams Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Ivan Ahel Corresponding Author Ivan Ahel Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Reza Sharifi Corresponding Author Reza Sharifi Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Rosa Morra Rosa Morra Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author C Denise Appel C Denise Appel Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Michael Tallis Michael Tallis Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Barry Chioza Barry Chioza Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Gytis Jankevicius Gytis Jankevicius Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Michael A Simpson Michael A Simpson Genetics and Molecular Medicine, King's College London, Guy's Hospital, London, UK Search for more papers by this author Ivan Matic Ivan Matic Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Scotland, UK Search for more papers by this author Ege Ozkan Ege Ozkan Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Barbara Golia Barbara Golia Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author Matthew J Schellenberg Matthew J Schellenberg Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Ria Weston Ria Weston Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Jason G Williams Jason G Williams Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Marianna N Rossi Marianna N Rossi Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Hamid Galehdari Hamid Galehdari Genetics Department, Sciences Faculty, Ahvaz Shahid Chamran University, Ahvaz, Iran Search for more papers by this author Juno Krahn Juno Krahn Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Alexander Wan Alexander Wan Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Richard C Trembath Richard C Trembath Genetics and Molecular Medicine, King's College London, Guy's Hospital, London, UK Search for more papers by this author Andrew H Crosby Andrew H Crosby Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK Search for more papers by this author Dragana Ahel Dragana Ahel Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Ron Hay Ron Hay Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Scotland, UK Search for more papers by this author Andreas G Ladurner Corresponding Author Andreas G Ladurner Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany Center for Integrated Protein Science Munich (CIPSM), Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Gyula Timinszky Corresponding Author Gyula Timinszky Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany Search for more papers by this author R Scott Williams Corresponding Author R Scott Williams Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Ivan Ahel Corresponding Author Ivan Ahel Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK Search for more papers by this author Author Information Reza Sharifi 1,‡, Rosa Morra2,‡, C Denise Appel3,‡, Michael Tallis2, Barry Chioza1, Gytis Jankevicius4, Michael A Simpson5, Ivan Matic6, Ege Ozkan1, Barbara Golia4, Matthew J Schellenberg3, Ria Weston2, Jason G Williams3, Marianna N Rossi2, Hamid Galehdari7, Juno Krahn3, Alexander Wan1, Richard C Trembath5, Andrew H Crosby1, Dragana Ahel2, Ron Hay6, Andreas G Ladurner 4,8,9, Gyula Timinszky 4, R Scott Williams 3 and Ivan Ahel 2 1Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London, UK 2Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK 3Laboratory of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA 4Faculty of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Munich, Germany 5Genetics and Molecular Medicine, King's College London, Guy's Hospital, London, UK 6Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Scotland, UK 7Genetics Department, Sciences Faculty, Ahvaz Shahid Chamran University, Ahvaz, Iran 8Center for Integrated Protein Science Munich (CIPSM), Munich, Germany 9Munich Cluster for Systems Neurology (SyNergy), Munich, Germany ‡These authors contributed equally to this work. *Corresponding authors. Biomedical Sciences Division, Human Genetics Research Centre, St George's University of London, London SW17 0RE, UK. Tel.:+44 2087255361; Fax:+44 2087251039; E-mail: [email protected] of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Butenandtstrasse 5, Munich 81377, Germany. Tel.:+49 89 2180 77095; Fax:+49 89 2180 77093; E-mail: [email protected] of Medicine, Butenandt Institute of Physiological Chemistry, Ludwig Maximilians University of Munich, Butenandtstrasse 5, Munich 81377, Germany. Tel.:+49 89 2180 77100; Fax:+49 89 2180 77093; E-mail: [email protected] of Structural Biology, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. Tel.:+1 9195414652; E-mail: [email protected] Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. Tel.:+44 1619187375; Fax:+44 1614463109; E-mail: [email protected] The EMBO Journal (2013)32:1225-1237https://doi.org/10.1038/emboj.2013.51 There is a Have you seen? (May 2013) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans. Introduction Adenosine diphosphate (ADP)-ribosylation is an evolutionarily conserved reversible post-translational protein modification that regulates a wide range of cellular processes, including DNA repair, transcription, telomere dynamics, cell differentiation and proliferation, the inflammatory and immune responses and apoptosis (Hassa et al, 2006). A large family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs) (Gibson and Kraus, 2012). PARPs use NAD+ as a substrate and covalently attach an ADP-ribose nucleotide, predominantly to the carboxyl group of glutamate residues on target proteins (D'Amours et al, 1999). To date, 17 members of the PARP family have been identified in humans (Gibson and Kraus, 2012), and some of these proteins (PARP1, PARP2, and tankyrases) can subsequently add additional ADP-ribose units through glycosidic ribose-ribose bonds to generate a PAR chain (D'Amours et al, 1999; Gibson and Kraus, 2012). ADP-ribosylation is a highly dynamic and reversible process. The specific hydrolysis of ribose-ribose bonds in PAR chains is catalysed by PAR glycohydrolase (PARG) (Lin et al, 1997). However, PARG is unable to cleave the ester bond between the terminal ADP-ribose unit and the ADP-ribosylated glutamate (Slade et al, 2011). The de(ADP-ribosyl)ation of glutamate residues would be of critical importance for cells to complete the reversal of ADP-ribosylation signalling or for the recycling of the covalently linked ADP-ribose. Although the enzymes that catalyse the removal of this type of protein modification have not yet been identified, analogous enzymatic activity has been detected in rat cell extracts (Oka et al, 1984). Macrodomains are evolutionarily conserved structural protein modules of ∼150 amino acids that bind NAD metabolites, including ADP-ribose/poly(ADP-ribose) and the sirtuin by-product O-acetyl-ADP-ribose (Karras et al, 2005; Ahel et al, 2009; Till and Ladurner, 2009; Timinszky et al, 2009). Some of the macrodomain proteins have been shown to possess catalytic activity on ADP-ribosylated substrates (Chen et al, 2011; Slade et al, 2011; Zaja et al, 2013). In this work, we identify the c6orf130 gene as a defective gene in patients with severe neurodegeneration. We show that the protein product of this gene is a PARP-interacting macrodomain protein with the ability to cleave the mono(ADP-ribose) from PARP-modified proteins. Our X-ray structures of C6orf130 and supporting solution biochemical studies suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Finally, we demonstrate that the function of C6orf130 protein is important for normal cellular proliferation and cellular response to DNA damage. Results C6orf130 gene is mutated in patients with severe neurodegeneration We studied an extended family with an autosomal recessive trait presenting with a severe form of progressive neurodegenerative and seizure disorder without dysmorphic features (Figure 1A and B; Supplementary Table 1). The autozygosity mapping revealed a homozygous region on chromosome 6p21 (20 cM) and linkage analysis produced a significant multipoint LOD score of 7.4 for the mapped region (Figure 1C). Subsequent refinement mapping defined a 6.54-Mb interval flanked by markers D6S1610 and D6S459 and containing a total of 30 labelled genes and 5 open-reading frames (Supplementary Figure 1). To exclude the possible existence of a pathogenic mutation in the linked interval, we performed whole-exome sequencing in one individual from the family and verified three non-sense novel variants within the extended 8.51 Mb linkage region (Supplementary Table 2). In the kindred, we identified a distinct homozygous sequence variant (NC_000006.11:g.41037831G>A; NM_145063.2:c.227C>T) within exon 4 of the c6orf130 gene that segregates with the phenotype and predicts the formation of a truncated C6orf130 protein lacking the C-terminal half of the protein due to a premature stop codon (NP_659500.1:p.R76X) (Figures 1D and E). No likely disease-causing sequence variants were detected in the other genes analysed by direct sequencing (Supplementary Table 3). We did not detect the c.227C>T variant in over 1200 chromosomes assayed from unrelated ethnic matched and European origin control subjects. Figure 1.The genetic and clinical data. (A) Pedigree diagram of family. (B) Photographs of individuals VI:1,VI:10, VII:4 affected by neurodegeneration. (C) EasyLinkage Plus v.5.08 output of parametric analysis of chromosome 6 under an autosomal recessive model by Simwalk2.91. (D) Electropherograms showing the identified mutation in c6orf130 gene (NC_000006.11:g.41037831G>A; NM_145063.2:c.227C>T; NP_659500.1:p.R76X). (E) Structure-based sequence alignment of C6orf30 homologues. The conserved residues K84 and G123/D125 are highlighted in blue and green, respectively. The predicted C6orf130 protein truncation in the patients analysed in this study is marked with red arrow. Download figure Download PowerPoint The c6orf130 gene encodes a macrodomain-containing protein of unknown physiological significance that is ubiquitously expressed in different tissues (Supplementary Figure 2). It was recently demonstrated that the C6orf130 protein can hydrolyse O-acetyl-ADP-ribose in vitro (Peterson et al, 2011). Given the similarity of the chemical bond between the glutamate and ADP-ribose in mono(ADP-ribosyl)ated proteins and the bond in the acetylated ADP-ribose, we postulated that C6orf130 could function as a long-sought protein that reverses the protein mono(ADP-ribosyl)ation synthesized by PARPs. This possibility is further substantiated by another case of severe neurodegeneration that has been described previously (Williams et al, 1984). For this patient, who died after 6 years of progressive neurologic deterioration, it was demonstrated that the primary defect was a genetic abnormality in an unidentified enzyme involved in the cleavage of the bond between glutamate and ADP-ribose. C6orf130 demodifies mono(ADP-ribosyl)ated PARP substrates To analyse the ability of C6orf130 to cleave ADP-ribosylated peptides in vitro, we employed an automodified PARP1 E988Q mutant as a substrate. Previous studies demonstrated that the PARP1 E988Q mutant is incapable of poly(ADP-ribosyl)ation activity (Marsischky et al, 1995), but instead mono(ADP-ribosyl)ates itself at two glutamate and one aspartate residues (Tao et al, 2009). We further characterized this substrate and identified several additional modification sites exclusively on glutamate residues clustering in the automodification and the second zinc-finger domains (Figure 2A; Supplementary Dataset 1). Our data showed that the recombinant human C6orf130 protein efficiently cleaved a radioactively labelled PARP1 peptide, whereas another macrodomain protein, GDAP2, was unable to perform the same reaction (Figure 2B, C). Figure 2.C6orf130 protein is a mono(ADP-ribosyl) protein hydrolase. (A) Schematic representation of PARP1 E988Q mutant protein. Residues found to be mono(ADP-ribosyl)ated by mass spectrometry analysis of automodified recombinant PARP1 E988Q protein are shown and marked with an asterisk. (B) SDS–PAGE based assay showing that purified recombinant human C6orf130 de(ADP-ribosyl)ates 32P-labelled mono(ADP-ribosyl)ated PARP1 E988Q protein. Macrodomain-containing protein GDAP2 served as a negative control. (C) The time curve showing the activity of C6orf130 on ADP-ribosylated PARP1 E988Q protein. (D) Hydrolytic activity of C6orf130 wild-type and mutated proteins on mono(ADP-ribosyl) PARP1 peptide derived from the activity of PARG protein on [32P]-labelled poly(ADP-ribosyl)ated wt PARP1. Mono(ADP-ribosyl)ated PARP1 species is indicated by green asterisk. (E) Analysis of the product of the C6orf130 reaction on mono(ADP-ribosyl)ated PARP E988Q by thin-layer chromatography (TLC). (F) Catalytic activity of C6orf130 proteins carrying point mutations in the indicated residues (top panel). The band appearing above 97 kDa in the SDS–PAGE in the C6orf130 D125A mutant lane (top panel, red asterisk) contains a crosslinked C6orf130 protein as revealed by western blotting (bottom panel). (G) De(ADP-ribosyl)ation of the PARP10 substrate by C6orf130. (H) Analysis of the reaction products of C6orf130 on ADP-ribosylated PARP10 substrate by TLC (top panel) and mass spectrometry (bottom panel). Download figure Download PowerPoint We next analysed the ability of C6orf130 to act on mono(ADP-ribosyl)ated substrates that are the products of PARG activity on poly(ADP-ribosyl)ated wild-type PARP1 protein. While PARG is unable to remove the terminal ADP-ribose directly linked to PARP1, the sequential addition of C6orf130 removed most of the remaining peptide ADP-ribosylation (Figure 2D). Analysis of the products of the C6orf130 reaction by thin-layer chromatography (TLC) revealed that C6orf130 released a product with a mobility aligned to the mono(ADP-ribose) marker (Figure 2E), suggesting that C6orf130 acts as an ADP-ribose hydrolase. C6orf130 similarly efficiently de(ADP-ribosyl)ates automodified PARP10, a PARP family member capable of only mono(ADP-ribosyl)ation (Kleine et al, 2008) (Figure 2G and H). A previously proposed hydrolytic mechanism for the deacylation of O-acetyl-ADP-ribose by C6orf130 suggested a conserved aspartate in C6orf130 (Asp125 in human C6orf130; Figure 1E) participates in general acid base catalysis (Peterson et al, 2011). We therefore analysed the importance of Asp125 for protein de(ADP-ribosyl)ation. The D125A mutation mutant abolished the de(ADP-ribosyl)ation of the automodified PARP1 (Figure 2F). Unexpectedly, however, the D125A mutant protein also formed a stable covalently bound complex with the mono(ADP-ribosyl)ated PARP1 E988Q peptide, as observed by the appearance of a supershifted band above 97 kDa (Figure 2F, top panel) that is recognized by a C6orf130-specific antibody (Figure 2E, bottom panel). Because the D125A mutant was catalytically defective, we hypothesized that this covalent linkage is a reaction intermediate with ADP-ribose. Consistent with this proposal, we also detected covalent adduct formation with the wild-type C6orf130 protein when incubated with free ADP-ribose (instead of ADP-ribosylated peptide) (Supplementary Dataset 2). MS/MS analysis of this adduct showed that the absolutely conserved Lys84 residue reacts with free ADP-ribose, and is conjugated to a dehydrated ADP-ribose adduct. Isothermal titration calorimetry (ITC) demonstrates that the K84A mutation ablates C6orf130 ADP-ribose binding (Supplementary Figure 3), and the mutation of Lys84 to Ala or Met blocked ADP-ribose adduction as monitored by mass spectrometry (Supplementary Dataset 2). Moreover, the K84A mutant ablated de(ADP-ribosyl)ation activity, but unlike the D125A mutant, K84A did not yield a stable covalent adduct with the ADP-ribosylated peptide (Figure 2F). Altogether, these results demonstrate that C6orf130 catalyses the hydrolysis of ADP-ribosylated peptides, and that its catalytic mechanism involves the formation of a transient covalent Lys84-(ADP-ribose) intermediate. Activity of C6orf130 on poly(ADP-ribosyl)ated PARP substrates Next, we tested the ability of C6orf130 to directly hydrolyse PAR. While C6orf130 protein exhibited activity in removing PAR chains from poly(ADP-ribosyl)ated PARP1, this activity was lower than the activity observed with PARG protein (Figure 3A). Importantly, we could show that C6orf130 is unable to act on ribose-ribose bonds in PAR chains to release ADP-ribose as a reaction product (Figure 3B), but that this macrodomain protein rather removes the whole PAR chain acting specifically at the glutamate-ADP-ribose ester bonds. Figure 3.Activities of C6orf130 on poly(ADP-ribosyl)ated PARP1 substrates. (A) Hydrolysis of PAR by C6orf130 and PARG analysed by SDS–PAGE and (B) by thin-layer chromatography-based assays. (C) C6orf130 inhibits automodification activity of PARP1 in vitro. Download figure Download PowerPoint To assess how C6orf130 activities alter PARP1 function in vitro, we performed a PARP1 automodification assay in the presence of wild-type or catalytically inactive C6orf130 proteins. Expectedly, PARP1 activity is greatly suppressed in these conditions, most likely due to the catalytic action of C6orf130 on mono- and poly(ADP-ribosyl)ated PARP1 species as the C6orf130 K84A mutant fails to block PARP1 automodification (Figure 3C). C6orf130 structure To understand the molecular basis for C6orf130 catalytic activities, we determined high-resolution X-ray crystal structures of C6orf130 in: (1) a ligand-free form at 1.35 Å, (2) bound to the ADP-ribose analogue ADP-HPD at 1.25 Å and (3) as a covalent ADP-ribose complex at 1.55 Å (Figures 4 and 5; Supplementary Table 4). The C6orf130 macrodomain fold adopts a compact 6-stranded β-sheet flanked by four α-helices and two 310-helical elements. Four surface loops coalesce to form the electropositive ligand-binding pocket (L1–L4, Figures 4A–C). The premature truncation of the C6orf130 open reading frame (ORF) in patients (Figure 1) is predicted to generate a non-functional peptide with a stop codon near the β3-β4 junction (Figures 1E and 4D). Figure 4.Crystal structure of the C6orf130 ADP-HPD complex. (A) Orthogonal views of C6orf130 (blue and tan) bound to ADP-HPD (spheres). (B) Surface charge representation of C6orf130 (blue, positive; red, negative; grey, neutral or hydrophobic) showing a positively charged binding site for ADP-HPD. (C) ADP-HPD binding site is composed of four sequence motifs, L1–L4. (D) The C6orf130 protein truncation (R76X) in the patients analysed in this study. Download figure Download PowerPoint Figure 5.C6orf130 active site architecture and ligand interactions. (A) Stereo view of the active site of C6orf130. A chloride ion (green) interacts with the 2′′- and 3′′-hydroxyls of the ADP-HPD. (B) Final 1.25 Å sigma-A weighted 2Fo-Fc electron density map (contoured at 1.0σ) showing ADP-HPD bound in the C6orf130 active site. Lys84 and Asp125 and a bound chloride ion are in close proximity to the pyrrolidine ring of ADP-HPD. (C) Stereo view of the active site of C6orf130 (ADP-ribose) complex. Molecule D (Supplementary Figure 5) is displayed. (D) Final 1.55 Å sigma-A weighted 2Fo-Fc electron density map (contoured at 1.0σ) showing the covalent lysyl-ADP-ribose adduct of chain D. Download figure Download PowerPoint Clamping by the L1 and L3 elements grasps the ADP-HPD and ADP-ribose upon binding. Gly123 undergoes a peptide flipping rearrangement, and together with Leu124 secures the pyrophosphate moiety of bound ligands. In the ADP-HPD complex, the pyrophosphate and pyrrolidine groups are well defined, and the adenine base displays two alternate binding conformations in the base interaction groove that is capped by the C6orf130 C-terminal Leu152 (Figures 5A and B). Within the ligand-binding grooves, the structures reveals the active centre comprised of Asp125, Lys84, and Ser35. Notably, although the overall fold of C6orf130 in our X-ray structures i
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