RESUMEN
ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Poli ADP Ribosilación/fisiología , ADP-Ribosilación , Adenosina Difosfato Ribosa/metabolismo , Línea Celular Tumoral , Cromatina , ADN , Daño del ADN , Fibroblastos/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/fisiología , Células HEK293 , Células HeLa , Humanos , Poli Adenosina Difosfato Ribosa/metabolismo , Cultivo Primario de CélulasRESUMEN
Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/metabolismo , Nucleosomas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , ADN Helicasas/genética , Replicación del ADN/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN/genética , Recombinación Homóloga/efectos de los fármacos , Ratones , Ratones Noqueados , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Nucleosomas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
PARP-catalysed ADP-ribosylation (ADPr) is important in regulating various cellular pathways. Until recently, PARP-dependent mono-ADP-ribosylation has been poorly understood due to the lack of sensitive detection methods. Here, we utilised an improved antibody to detect mono-ADP-ribosylation. We visualised endogenous interferon (IFN)-induced ADP-ribosylation and show that PARP14 is a major enzyme responsible for this modification. Fittingly, this signalling is reversed by the macrodomain from SARS-CoV-2 (Mac1), providing a possible mechanism by which Mac1 counteracts the activity of antiviral PARPs. Our data also elucidate a major role of PARP9 and its binding partner, the E3 ubiquitin ligase DTX3L, in regulating PARP14 activity through protein-protein interactions and by the hydrolytic activity of PARP9 macrodomain 1. Finally, we also present the first visualisation of ADPr-dependent ubiquitylation in the IFN response. These approaches should further advance our understanding of IFN-induced ADPr and ubiquitin signalling processes and could shed light on how different pathogens avoid such defence pathways.
Asunto(s)
ADP-Ribosilación , Interferones , Poli(ADP-Ribosa) Polimerasas , Ubiquitina-Proteína Ligasas , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Interferones/metabolismo , Ubiquitinación , Células HEK293 , SARS-CoV-2/metabolismo , Transducción de Señal , COVID-19/virología , COVID-19/metabolismo , Proteínas de NeoplasiasRESUMEN
The recent discovery of non-proteinaceous ubiquitylation substrates broadened our understanding of this modification beyond conventional protein targets. However, the existence of additional types of substrates remains elusive. Here, we present evidence that nucleic acids can also be directly ubiquitylated via ester bond formation. DTX3L, a member of the DELTEX family E3 ubiquitin ligases, ubiquitylates DNA and RNA in vitro and that this activity is shared with DTX3, but not with the other DELTEX family members DTX1, DTX2 and DTX4. DTX3L shows preference for the 3'-terminal adenosine over other nucleotides. In addition, we demonstrate that ubiquitylation of nucleic acids is reversible by DUBs such as USP2, JOSD1 and SARS-CoV-2 PLpro. Overall, our study proposes reversible ubiquitylation of nucleic acids in vitro and discusses its potential functional implications.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitinación , Humanos , COVID-19/virología , COVID-19/metabolismo , ADN/metabolismo , ADN/química , Ácidos Nucleicos/metabolismo , ARN/metabolismo , ARN/genética , ARN/química , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Especificidad por Sustrato , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/químicaRESUMEN
The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. After binding to genomic lesions, these enzymes use NAD+ to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors3,4. These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues5-10. Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD+-binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.
Asunto(s)
ADP-Ribosilación , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Daño del ADN , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/química , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regulación Alostérica , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Biocatálisis , Proteínas Portadoras/genética , Dominio Catalítico , Células HEK293 , Humanos , Modelos Moleculares , Mutación , NAD/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Anémonas de MarRESUMEN
Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.
Asunto(s)
ADP-Ribosilación , Ácidos Nucleicos , Ubiquitina-Proteína Ligasas , Adenosina Difosfato Ribosa/metabolismo , Ácidos Nucleicos/metabolismo , Ácido Ocadaico/análogos & derivados , Proteínas/genética , Ubiquitina-Proteína Ligasas/metabolismo , HumanosRESUMEN
ADP-ribosylation is an ancient posttranslational modification with exceptional versatility in terms of breadth of modification targets including at least seven different amino acid side chains, various moieties on nucleic acids, and a variety of small chemical compounds. The spatiotemporal signaling dynamic of the different modification variations is tightly regulated and depends on the writers, erases, and readers of each type. Among these, tyrosine ADP-ribosylation (Tyr-ADPr) has been consistently detected as a novel modification type, but systematic analysis of its potential physiological role, modification establishment, and reversal are still lacking. Here we present a re-analysis of recent ADP-ribosylome data and show that Tyr-ADPr sites are conserved and enriched among ribosome biogenesis and mRNA processing proteins and that these sites are affected by the status of the (ADP-ribosyl)hydrolase ARH3. To facilitate the study of Tyr-ADPr, we establish methodologies for the synthesis of well-defined Tyr-ADPr peptides and with these could show that Tyr-ADPr is reversed both by ARH3 and PARG enzymes. Together, our work lays the foundation for the future exploration of the Tyr-ADPr.
RESUMEN
AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments.
Asunto(s)
Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Dominios Proteicos , ADP-Ribosilación , ARN/metabolismoRESUMEN
To efficiently duplicate their genomic content, cells must overcome DNA lesions that interfere with processive DNA replication. These lesions may be removed and repaired, rather than just tolerated, to allow continuity of DNA replication on an undamaged DNA template. However, it is unclear how this is achieved at a molecular level. Here we identify a new replication-associated factor, ZRANB3 (zinc finger, RAN-binding domain containing 3), and propose its role in the repair of replication-blocking lesions. ZRANB3 has a unique structure-specific endonuclease activity, which is coupled to ATP hydrolysis. It cleaves branched DNA structures with unusual polarity, generating an accessible 3'-OH group in the template of the leading strand. Furthermore, ZRANB3 localizes to DNA replication sites and interacts with the components of the replication machinery. It is recruited to damaged replication forks via multiple mechanisms, which involve interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures. Collectively, our data support a role for ZRANB3 in the replication stress response and suggest new insights into how DNA repair is coordinated with DNA replication to maintain genome stability.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Endonucleasas/metabolismo , Inestabilidad Genómica/fisiología , Estrés Fisiológico/fisiología , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , ADN/biosíntesis , ADN/genética , ADN Helicasas/genética , Endonucleasas/genética , Células HeLa , Humanos , Hidrólisis , Poliubiquitina/genética , Poliubiquitina/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
Poly(ADP-ribosyl)ation plays a major role in DNA repair, where it regulates chromatin relaxation as one of the critical events in the repair process. However, the molecular mechanism by which poly(ADP-ribose) modulates chromatin remains poorly understood. Here we identify the poly(ADP-ribose)-regulated protein APLF as a DNA-damage-specific histone chaperone. APLF preferentially binds to the histone H3/H4 tetramer via its C-terminal acidic motif, which is homologous to the motif conserved in the histone chaperones of the NAP1L family (NAP1L motif). We further demonstrate that APLF exhibits histone chaperone activities in a manner that is dependent on its acidic domain and that the NAP1L motif is critical for the repair capacity of APLF in vivo. Finally, we identify structural analogs of APLF in lower eukaryotes with the ability to bind histones and localize to the sites of DNA-damage-induced poly(ADP-ribosyl)ation. Collectively, these findings define the involvement of histone chaperones in poly(ADP-ribose)-regulated DNA repair reactions.
Asunto(s)
Reparación del ADN , Chaperonas de Histonas/fisiología , Fosfoproteínas/fisiología , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans/genética , Línea Celular , Daño del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Eucariontes/genética , Células HeLa , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Histonas/metabolismo , Humanos , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas de Unión a Poli-ADP-Ribosa , Mapeo de Interacción de Proteínas , Proteínas/química , Proteínas/genética , Proteínas/fisiología , Homología de Secuencia , ARNt MetiltransferasasRESUMEN
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.
Asunto(s)
Glicósido Hidrolasas/fisiología , Enfermedades Neurodegenerativas/enzimología , Poli Adenosina Difosfato Ribosa/fisiología , Tioléster Hidrolasas/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Niño , Preescolar , Familia , Femenino , Glicósido Hidrolasas/genética , Células HEK293 , Células HeLa , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Enfermedades Neurodegenerativas/genética , Linaje , Poli Adenosina Difosfato Ribosa/genética , Procesamiento Proteico-Postraduccional/genética , Homología de Secuencia de Aminoácido , Tioléster Hidrolasas/genéticaRESUMEN
Post-translational modification (PTM) of proteins plays an important part in mediating protein interactions and/or the recruitment of specific protein targets. PTM can be mediated by the addition of functional groups (for example, acetylation or phosphorylation), peptides (for example, ubiquitylation or sumoylation), or nucleotides (for example, poly(ADP-ribosyl)ation). Poly(ADP-ribosyl)ation often involves the addition of long chains of ADP-ribose units, linked by glycosidic ribose-ribose bonds, and is critical for a wide range of processes, including DNA repair, regulation of chromosome structure, transcriptional regulation, mitosis and apoptosis. Here we identify a novel poly(ADP-ribose)-binding zinc finger (PBZ) motif in a number of eukaryotic proteins involved in the DNA damage response and checkpoint regulation. The PBZ motif is also required for post-translational poly(ADP-ribosyl)ation. We demonstrate interaction of poly(ADP-ribose) with this motif in two representative human proteins, APLF (aprataxin PNK-like factor) and CHFR (checkpoint protein with FHA and RING domains), and show that the actions of CHFR in the antephase checkpoint are abrogated by mutations in PBZ or by inhibition of poly(ADP-ribose) synthesis.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Reparación del ADN , Poli Adenosina Difosfato Ribosa/metabolismo , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Línea Celular , Daño del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Poli Adenosina Difosfato Ribosa/biosíntesis , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
Structurally complex genomic regions, such as centromeres, are inherently difficult to duplicate. The mechanism behind centromere inheritance is not well understood, and one of the key questions relates to the reassembly of centromeric chromatin following DNA replication. Here, we define ERCC6L2 as a key regulator of this process. ERCC6L2 accumulates at centromeres and promotes deposition of core centromeric factors. Interestingly, ERCC6L2-/- cells show unrestrained replication of centromeric DNA, likely caused by the erosion of centromeric chromatin. Beyond centromeres, ERCC6L2 facilitates replication at genomic repeats and non-canonical DNA structures. Notably, ERCC6L2 interacts with the DNA-clamp PCNA through an atypical peptide, presented here in a co-crystal structure. Finally, ERCC6L2 also restricts DNA end resection, acting independently of the 53BP1-REV7-Shieldin complex. We propose a mechanistic model, which reconciles seemingly distinct functions of ERCC6L2 in DNA repair and DNA replication. These findings provide a molecular context for studies linking ERCC6L2 to human disease.
Asunto(s)
Centrómero , Cromatina , Humanos , Centrómero/metabolismo , ADN/química , Replicación del ADN , Reparación del ADN , ADN Helicasas/metabolismoRESUMEN
The timely removal of ADP-ribosylation is crucial for efficient DNA repair. However, much remains to be discovered about ADP-ribosylhydrolases. Here, we characterize the physiological role of TARG1, an ADP-ribosylhydrolase that removes aspartate/glutamate-linked ADP-ribosylation. We reveal its function in the DNA damage response and show that the loss of TARG1 sensitizes cells to inhibitors of topoisomerase II, ATR, and PARP. Furthermore, we find a PARP1-mediated synthetic lethal interaction between TARG1 and PARG, driven by the toxic accumulation of ADP-ribosylation, that induces replication stress and genomic instability. Finally, we show that histone PARylation factor 1 (HPF1) deficiency exacerbates the toxicity and genomic instability induced by excessive ADP-ribosylation, suggesting a close crosstalk between components of the serine- and aspartate/glutamate-linked ADP-ribosylation pathways. Altogether, our data identify TARG1 as a potential biomarker for the response of cancer cells to PARP and PARG inhibition and establish that the interplay of TARG1 and PARG protects cells against genomic instability.
Asunto(s)
Ácido Aspártico , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ácido Aspártico/metabolismo , ADP-Ribosilación , Inestabilidad Genómica , Glutamatos/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.
Asunto(s)
COVID-19 , Transferasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Antivirales , Hidrolasas , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
Ubiquitylation had been considered limited to protein lysine residues, but other substrates have recently emerged. Here, we show that DELTEX E3 ligases specifically target the 3' hydroxyl of the adenosine diphosphate (ADP)-ribosyl moiety that can be linked to a protein, thus generating a hybrid ADP-ribosyl-ubiquitin modification. Unlike other known hydroxyl-specific E3s, which proceed via a covalent E3~ubiqutin intermediate, DELTEX enzymes are RING E3s that stimulate a direct ubiquitin transfer from E2~ubiquitin onto a substrate. However, DELTEXes follow a previously unidentified paradigm for RING E3s, whereby the ligase not only forms a scaffold but also provides catalytic residues to activate the acceptor. Comparative analysis of known hydroxyl-ubiquitylating active sites points to the recurring use of a catalytic histidine residue, which, in DELTEX E3s, is potentiated by a glutamate in a catalytic triad-like manner. In addition, we determined the hydrolase specificity profile of this modification, identifying human and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enzymes that could reverse it in cells.
RESUMEN
Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.
Asunto(s)
Adenosina Difosfato/metabolismo , Proteínas Portadoras/metabolismo , Glicósido Hidrolasas/metabolismo , Proteínas Nucleares/metabolismo , ADP-Ribosilación , Proteínas Portadoras/genética , Línea Celular Tumoral , Daño del ADN , Técnicas de Inactivación de Genes , Glicósido Hidrolasas/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Proteómica , Serina/metabolismoRESUMEN
Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.
Asunto(s)
Proteínas Portadoras/metabolismo , Daño del ADN , Reparación del ADN , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Serina/metabolismo , ADP-Ribosilación , Línea Celular , Línea Celular Tumoral , Humanos , Neoplasias/enzimología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.