RESUMO
ADP-ribosylation is a ubiquitous modification of biomolecules, including proteins and nucleic acids, that regulates various cellular functions in all kingdoms of life. The recent emergence of new technologies to study ADP-ribosylation has reshaped our understanding of the molecular mechanisms that govern the establishment, removal, and recognition of this modification, as well as its impact on cellular and organismal function. These advances have also revealed the intricate involvement of ADP-ribosylation in human physiology and pathology and the enormous potential that their manipulation holds for therapy. In this review, we present the state-of-the-art findings covering the work in structural biology, biochemistry, cell biology, and clinical aspects of ADP-ribosylation.
Assuntos
ADP-Ribosilação , Humanos , Proteínas/metabolismo , DNA/metabolismo , RNA/metabolismo , Animais , Transdução de Sinais , Processamento de Proteína Pós-Traducional , ADP Ribose Transferases/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Recent developments indicate that macrodomains, an ancient and diverse protein domain family, are key players in the recognition, interpretation, and turnover of ADP-ribose (ADPr) signaling. Crucial to this is the ability of macrodomains to recognize ADPr either directly, in the form of a metabolic derivative, or as a modification covalently bound to proteins. Thus, macrodomains regulate a wide variety of cellular and organismal processes, including DNA damage repair, signal transduction, and immune response. Their importance is further indicated by the fact that dysregulation or mutation of a macrodomain is associated with several diseases, including cancer, developmental defects, and neurodegeneration. In this review, we summarize the current insights into macrodomain evolution and how this evolution influenced their structural and functional diversification. We highlight some aspects of macrodomain roles in pathobiology as well as their emerging potential as therapeutic targets.
Assuntos
Reparo do DNA , Proteínas de Escherichia coli/química , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerases/química , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/química , Viroses/enzimologia , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Animais , Dano ao DNA , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Família Multigênica , Neoplasias/química , Neoplasias/genética , Neoplasias/patologia , Filogenia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Viroses/genética , Viroses/patologia , Viroses/virologiaRESUMO
Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems.
Assuntos
Antitoxinas , Guanosina , ADP-Ribosilação , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Células Eucarióticas/metabolismo , Antitoxinas/genética , Adenosina Difosfato Ribose/metabolismoRESUMO
PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling.
Assuntos
ADP-Ribosilação , Histonas , Histonas/genética , Histonas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Cromatina , Dano ao DNA , Anticorpos/genética , Transdução de SinaisRESUMO
Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD+ onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.
Assuntos
ADP-Ribosilação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Imunidade Vegetal , Ubiquitinação , Dedos de Zinco , ADP Ribose Transferases/metabolismo , Difosfato de Adenosina/química , Arabidopsis/metabolismo , Sistemas CRISPR-Cas , Genes de Plantas , Glicosídeo Hidrolases/metabolismo , Homeostase , Humanos , Hidrólise , Mutação , Plantas Geneticamente Modificadas , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteostase , Plântula/metabolismo , Especificidade por Substrato , Tristetraprolina/química , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/químicaRESUMO
DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms.
Assuntos
Quebras de DNA de Cadeia Simples , Replicação do DNA , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Proteína-Arginina N-Metiltransferases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
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.
Assuntos
Glicosídeo Hidrolases/metabolismo , Poli ADP Ribosilação/fisiologia , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Linhagem Celular Tumoral , Cromatina , DNA , Dano ao DNA , Fibroblastos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/fisiologia , Células HEK293 , Células HeLa , Humanos , Poli Adenosina Difosfato Ribose/metabolismo , Cultura Primária de CélulasRESUMO
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.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Nucleossomos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
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.
Assuntos
ADP-Ribosilação , Interferons , Poli(ADP-Ribose) Polimerases , Ubiquitina-Proteína Ligases , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Interferons/metabolismo , Ubiquitinação , Células HEK293 , SARS-CoV-2/metabolismo , Transdução de Sinais , COVID-19/virologia , COVID-19/metabolismo , Proteínas de NeoplasiasRESUMO
ADP-ribosylation is an intricate and versatile posttranslational modification involved in the regulation of a vast variety of cellular processes in all kingdoms of life. Its complexity derives from the varied range of different chemical linkages, including to several amino acid side chains as well as nucleic acids termini and bases, it can adopt. In this review, we provide an overview of the different families of (ADP-ribosyl)hydrolases. We discuss their molecular functions, physiological roles, and influence on human health and disease. Together, the accumulated data support the increasingly compelling view that (ADP-ribosyl)hydrolases are a vital element within ADP-ribosyl signaling pathways and they hold the potential for novel therapeutic approaches as well as a deeper understanding of ADP-ribosylation as a whole.
Assuntos
ADP-Ribosilação/fisiologia , Difosfato de Adenosina/metabolismo , Hidrolases/química , Hidrolases/metabolismo , Humanos , Hidrolases/classificação , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.
Assuntos
ADP-Ribosilação , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , Timina/química , Timina/metabolismo , Adenosina Difosfato Ribose/metabolismo , Antitoxinas , Proteínas de Bactérias/química , Toxinas Bacterianas , Sequência de Bases , Biocatálise , DNA/genética , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA , Reparo do DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Modelos Moleculares , Mycobacterium/enzimologia , Mycobacterium/genética , Nitrogênio/química , Nitrogênio/metabolismo , Poli(ADP-Ribose) Polimerases/química , Origem de Replicação/genética , Especificidade por Substrato , Thermus/enzimologia , Timidina/química , Timidina/metabolismoRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) has emerged as a central target for cancer therapies due to the ability of PARP inhibitors to specifically kill tumors deficient for DNA repair by homologous recombination. Upon DNA damage, PARP1 quickly binds to DNA breaks and triggers ADP-ribosylation signaling. ADP-ribosylation is important for the recruitment of various factors to sites of damage, as well as for the timely dissociation of PARP1 from DNA breaks. Indeed, PARP1 becomes trapped at DNA breaks in the presence of PARP inhibitors, a mechanism underlying the cytotoxitiy of these inhibitors. Therefore, any cellular process influencing trapping is thought to impact PARP inhibitor efficiency, potentially leading to acquired resistance in patients treated with these drugs. There are numerous ADP-ribosylation targets after DNA damage, including PARP1 itself as well as histones. While recent findings reported that the automodification of PARP1 promotes its release from the DNA lesions, the potential impact of other ADP-ribosylated proteins on this process remains unknown. Here, we demonstrate that histone ADP-ribosylation is also crucial for the timely dissipation of PARP1 from the lesions, thus contributing to cellular resistance to PARP inhibitors. Considering the crosstalk between ADP-ribosylation and other histone marks, our findings open interesting perspectives for the development of more efficient PARP inhibitor-driven cancer therapies.
Assuntos
ADP-Ribosilação , Histonas , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Histonas/metabolismo , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1-5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6-8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2-HPF1 bound to a nucleosome. This showed that PARP2-HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12-16.
Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Biocatálise , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica , DNA/metabolismo , Reparo do DNA , Ativação Enzimática , Humanos , Modelos Moleculares , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Nucleossomos/química , Nucleossomos/ultraestrutura , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/ultraestrutura , Domínios ProteicosRESUMO
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.
Assuntos
ADP-Ribosilação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biocatálise , Proteínas de Transporte/genética , Domínio Catalítico , Células HEK293 , Humanos , Modelos Moleculares , Mutação , NAD/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Anêmonas-do-MarRESUMO
Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.
Assuntos
Núcleo Celular/enzimologia , Dano ao DNA , Enzimas Desubiquitinantes/metabolismo , Instabilidade Genômica , Poliubiquitina/metabolismo , Sítios de Ligação , Sobrevivência Celular , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Lisina , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato , UbiquitinaçãoRESUMO
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.
Assuntos
ADP-Ribosilação , Ácidos Nucleicos , Ubiquitina-Proteína Ligases , Adenosina Difosfato Ribose/metabolismo , Ácidos Nucleicos/metabolismo , Ácido Okadáico/análogos & derivados , Proteínas/genética , Ubiquitina-Proteína Ligases/metabolismo , HumanosRESUMO
The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Cristalografia , Pandemias , Ligantes , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Antivirais/farmacologia , Antivirais/químicaRESUMO
ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteínas de Transporte/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Instabilidade Genômica , Humanos , Proteínas Nucleares/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , Proteômica/métodos , Serina , TransfecçãoRESUMO
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.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos , ADP-Ribosilação , RNA/metabolismoRESUMO
Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate, the reason for the lack of N2 fixation in the mutant has remained unclear. During our investigations into the role of AatA, we found that it catalyses an additional transamination reaction between aspartate and pyruvate, forming alanine. This secondary reaction runs at around 60â% of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate-pyruvate transaminase activity in R. leguminosarum, which is common in eukaryotic and many prokaryotic genomes. However, the aspartate-to-pyruvate transaminase reaction is not needed for N2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2 fixation, rather than biosynthetic transamination to form an amino acid. Hence, the enzyme aspartase, which catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2 fixation in pea nodules.