RESUMO
Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.
Assuntos
Moléculas de Adesão Celular Neuronais , Morte Celular , Membrana Celular , Fatores de Crescimento Neural , Animais , Humanos , Camundongos , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular Neuronais/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/ultraestrutura , Mutagênese Sítio-Dirigida , Biopolímeros/química , Biopolímeros/genética , Biopolímeros/metabolismoRESUMO
ADP-ribosylation is an ancient, highly conserved, and reversible covalent modification critical for a variety of endogenous processes in both prokaryotes and eukaryotes. ADP-ribosylation targets proteins, nucleic acids, and small molecules (including antibiotics). ADP-ribosylation signalling involves enzymes that add ADP-ribose to the target molecule, the (ADP-ribosyl)transferases; and those that remove it, the (ADP-ribosyl)hydrolases. Recently, the toxin/antitoxin pair DarT/DarG composed of a DNA ADP-ribosylating toxin, DarT, and (ADP-ribosyl)hydrolase antitoxin, DarG, was described. DarT modifies thymidine in single-stranded DNA in a sequence-specific manner while DarG reverses this modification, thereby rescuing cells from DarT toxicity. We studied the DarG homologue SCO6735 which is highly conserved in all Streptomyces species and known to be associated with antibiotic production in the bacterium S. coelicolor. SCO6735 shares a high structural similarity with the bacterial DarG and human TARG1. Like DarG and TARG1, SCO6735 can also readily reverse thymidine-linked ADP-ribosylation catalysed by DarT in vitro and in cells. SCO6735 active site analysis including molecular dynamic simulations of its complex with ADP-ribosylated thymidine suggests a novel catalytic mechanism of DNA-(ADP-ribose) hydrolysis. Moreover, a comparison of SCO6735 structure with ALC1-like homologues revealed an evolutionarily conserved feature characteristic for this subclass of macrodomain hydrolases.
RESUMO
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
ADP-ribosylation of proteins is crucial for fundamental cellular processes. Despite increasing examples of DNA ADP-ribosylation, the impact of this modification on DNA metabolism and cell physiology is unknown. Here, we show that the DarTG toxin-antitoxin system from enteropathogenic Escherichia coli (EPEC) catalyzes reversible ADP-ribosylation of single-stranded DNA (ssDNA). The DarT toxin recognizes specific sequence motifs. EPEC DarG abrogates DarT toxicity by two distinct mechanisms: removal of DNA ADP-ribose (ADPr) groups and DarT sequestration. Furthermore, we investigate how cells recognize and deal with DNA ADP-ribosylation. We demonstrate that DNA ADPr stalls replication and is perceived as DNA damage. Removal of ADPr from DNA requires the sequential activity of two DNA repair pathways, with RecF-mediated homologous recombination likely to transfer ADP-ribosylation from single- to double-stranded DNA (dsDNA) and subsequent nucleotide excision repair eliminating the lesion. Our work demonstrates that these DNA repair pathways prevent the genotoxic effects of DNA ADP-ribosylation.
Assuntos
ADP-Ribosilação , Reparo do DNA , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Recombinação Homóloga , Adenosina Difosfato Ribose/metabolismo , Viabilidade Microbiana , Modelos Biológicos , Resposta SOS em GenéticaRESUMO
ADP-ribosylation is the process of transferring the ADP-ribose moiety from NAD+ to a substrate. While a number of proteins represent well described substrates accepting ADP-ribose modification, a recent report demonstrated biological role for DNA ADP-ribosylation as well. The conserved macrodomain fold of several known hydrolyses was found to possess de-ADP-ribosylating activity and the ability to hydrolyze (reverse) ADP-ribosylation. Here we summarize the methods that can be employed to study mono-ADP-ribosylation hydrolysis by macrodomains.
Assuntos
ADP-Ribosilação , Adenosina Difosfato Ribose/química , Biologia Molecular/métodos , Proteínas/química , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Hidrólise , Modelos Moleculares , NAD/química , NAD/genética , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteínas/genéticaRESUMO
Viruses from the Coronaviridae, Togaviridae, and Hepeviridae families âall contain genes that encode a conserved protein domain, called a macrodomain; however, the role of this domain during infection has remained enigmatic. The recent discovery that mammalian macrodomain proteins enzymatically remove ADP-ribose, a common post-translation modification, from proteins has led to an outburst of studies describing both the enzymatic activity and function of viral macrodomains. These new studies have defined these domains as de-ADP-ribosylating enzymes, which indicates that these viruses have evolved to counteract antiviral ADP-ribosylation, likely mediated by poly-ADP-ribose polymerases (PARPs). Here, we comprehensively review this rapidly expanding field, describing the structures and enzymatic activities of viral macrodomains, and discussing their roles in viral replication and pathogenesis.
Assuntos
Domínios Proteicos , Proteínas não Estruturais Virais/química , Replicação Viral , Vírus/genética , Vírus/patogenicidade , Adenosina Difosfato Ribose/metabolismo , Coronaviridae/genética , Coronaviridae/patogenicidade , Hepevirus/genética , Hepevirus/patogenicidade , Histonas , Poli(ADP-Ribose) Polimerases , Processamento de Proteína Pós-Traducional , Togaviridae/genética , Togaviridae/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Vírus/enzimologiaRESUMO
ADP-ribosylation is a dynamic post-translation modification that regulates the early phase of various DNA repair pathways by recruiting repair factors to chromatin. ADP-ribosylation levels are defined by the activities of specific transferases and hydrolases. However, except for the transferase PARP1/ARDT1 little is known about regulation of these enzymes. We found that MacroD2, a mono-ADP-ribosylhydrolase, is exported from the nucleus upon DNA damage, and that this nuclear export is induced by ATM activity. We show that the export is dependent on the phosphorylation of two SQ/TQ motifs, suggesting a novel direct interaction between ATM and ADP-ribosylation. Lastly, we show that MacroD2 nuclear export temporally restricts its recruitment to DNA lesions, which may decrease the net ADP-ribosylhydrolase activity at the site of DNA damage. Together, our results identify a novel feedback regulation between two crucial DNA damage-induced signaling pathways: ADP-ribosylation and ATM activation.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Hidrolases/genética , Poli(ADP-Ribose) Polimerases/genética , Processamento de Proteína Pós-Traducional , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Retroalimentação Fisiológica , Células HeLa , Humanos , Hidrolases/metabolismo , Osteoblastos , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de SinaisRESUMO
The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.
Assuntos
ADP Ribose Transferases/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , DNA de Cadeia Simples/metabolismo , Mycobacterium tuberculosis/genética , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Difosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Antitoxinas/química , Antitoxinas/genética , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Timidina/metabolismoRESUMO
ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3's de-ADP-ribosylation activity in vitro Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs. IMPORTANCE: The macrodomain is a ubiquitous structural domain that removes ADP-ribose from proteins, reversing the activity of ADP-ribosyltransferases. All coronaviruses contain a macrodomain, suggesting that ADP-ribosylation impacts coronavirus infection. However, its function during infection remains unknown. Here, we found that the macrodomain is an important virulence factor for a highly pathogenic human CoV, SARS-CoV. Viruses with macrodomain mutations that abrogate its ability to remove ADP-ribose from protein were unable to cause lethal disease in mice. Importantly, the SARS-CoV macrodomain suppressed the innate immune response during infection. Our data suggest that an early innate immune response can protect mice from lethal disease. Understanding the mechanism used by this enzyme to promote disease will open up novel avenues for coronavirus therapies and give further insight into the role of macrodomains in viral pathogenesis.
Assuntos
Coronavirus/imunologia , Coronavirus/patogenicidade , Imunidade Inata , Domínios Proteicos , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Proteínas não Estruturais Virais/genética , Difosfato de Adenosina/metabolismo , Animais , Brônquios/citologia , Brônquios/imunologia , Brônquios/virologia , Linhagem Celular , Coinfecção/imunologia , Coinfecção/virologia , Coronavirus/química , Coronavirus/genética , Citocinas/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Mutação , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Carga Viral , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , VirulênciaRESUMO
ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Streptomyces coelicolor/metabolismo , Adenosina Difosfato Ribose/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dano ao DNA , Processamento de Proteína Pós-Traducional , Streptomyces coelicolor/química , Streptomyces coelicolor/genéticaRESUMO
UNLABELLED: ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD(+) to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE: Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are demodified by macro domains from members of these virus families. In the case of hepatitis E virus, the adjacent viral helicase domain dramatically increases the binding of the macro domain to PAR and simulates the demodification activity.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Vírus da Hepatite E/fisiologia , Poliproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Virais/metabolismo , Humanos , HidróliseRESUMO
The poly(ADP-ribose) polymerases (PARPs) are a major family of enzymes capable of modifying proteins by ADP-ribosylation. Due to the large size and diversity of this family, PARPs affect almost every aspect of cellular life and have fundamental roles in DNA repair, transcription, heat shock and cytoplasmic stress responses, cell division, protein degradation, and much more. In the past decade, our understanding of the PARP enzymatic mechanism and activation, as well as regulation of ADP-ribosylation signals by the readers and erasers of protein ADP-ribosylation, has been significantly advanced by the emergence of new structural data, reviewed herein, which allow for better understanding of the biological roles of this widespread post-translational modification.
Assuntos
Domínio Catalítico , Glicosídeo Hidrolases/química , Poli Adenosina Difosfato Ribose/química , Poli(ADP-Ribose) Polimerases/química , Estrutura Terciária de Proteína , Tioléster Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Tioléster Hidrolases/metabolismoRESUMO
The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylation by DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET and RING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity of DNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1 and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruption of the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domain led to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1 for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicated DNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNA methylation.
Assuntos
Ilhas de CpG/fisiologia , DNA (Citosina-5-)-Metiltransferases/química , Metilação de DNA/fisiologia , Replicação do DNA/fisiologia , DNA/química , Proteínas Nucleares/química , Regulação Alostérica/fisiologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT , DNA/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Ubiquitina-Proteína LigasesRESUMO
Poly(ADP-ribosyl)ation is involved in the regulation of a variety of cellular pathways, including, but not limited to, transcription, chromatin, DNA damage and other stress signalling. Similar to other tightly regulated post-translational modifications, poly(ADP-ribosyl)ation employs 'writers', 'readers' and 'erasers' to confer regulatory functions. The generation of poly(ADP-ribose) is catalyzed by poly(ADP-ribose) polymerase enzymes, which use NAD(+) as a cofactor to sequentially transfer ADP-ribose units generating long polymers, which, in turn, can affect protein function or serve as a recruitment platform for additional factors. Historically, research has focused on poly(ADP-ribose) generation pathways, with knowledge about PAR recognition and degradation lagging behind. Over recent years, several discoveries have significantly furthered our understanding of poly(ADP-ribose) recognition and, even more so, of poly(ADP-ribose) degradation. In this review, we summarize current knowledge about the protein modules recognizing poly(ADP-ribose) and discuss the newest developments on the complete reversibility of poly(ADP-ribosyl)ation.
Assuntos
Poli Adenosina Difosfato Ribose/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Dano ao DNA/fisiologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Poli Adenosina Difosfato Ribose/química , Domínios e Motivos de Interação entre Proteínas , Proteínas/químicaRESUMO
ADP-ribosylation is a reversible post-translational modification with wide-ranging biological functions in all kingdoms of life. A variety of enzymes use NAD(+) to transfer either single or multiple ADP-ribose (ADPr) moieties onto distinct amino acid substrates, often in response to DNA damage or other stresses. Poly-ADPr-glycohydrolase readily reverses poly-ADP-ribosylation induced by the DNA-damage sensor PARP1 and other enzymes, but it does not remove the most proximal ADPr linked to the target amino acid. Searches for enzymes capable of fully reversing cellular mono-ADP-ribosylation back to the unmodified state have proved elusive, which leaves a gap in the understanding of this modification. Here, we identify a family of macrodomain enzymes present in viruses, yeast and animals that reverse cellular ADP-ribosylation by acting on mono-ADP-ribosylated substrates. Our discoveries establish the complete reversibility of PARP-catalyzed cellular ADP-ribosylation as a regulatory modification.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Biocatálise , Modelos Moleculares , Dados de Sequência Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Glicosídeo Hidrolases/fisiologia , Doenças Neurodegenerativas/enzimologia , Poli Adenosina Difosfato Ribose/fisiologia , Tioléster Hidrolases/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Criança , Pré-Escolar , Família , Feminino , Glicosídeo Hidrolases/genética , Células HEK293 , Células HeLa , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Doenças Neurodegenerativas/genética , Linhagem , Poli Adenosina Difosfato Ribose/genética , Processamento de Proteína Pós-Traducional/genética , Homologia de Sequência de Aminoácidos , Tioléster Hidrolases/genéticaRESUMO
The maintenance methylation of hemimethylated CpG sites by the DNA methyltransferase Dnmt1 is the molecular basis of the inheritance of DNA methylation patterns. Based on structural data and kinetics obtained with a truncated form of Dnmt1, an autoinhibition model for the specificity of Dnmt1 was proposed in which unmethylated DNA binds to Dnmt1's CXXC domain, which prevents its methylation. We have prepared CXXC domain variants that lost DNA binding. Corresponding full-length Dnmt1 variants did not display a reduction in specificity, indicating that the autoinhibition model does not apply in full-length Dnmt1. Furthermore, we show that the Dnmt1 M1235S variant, which carries an exchange in the catalytic domain of the enzyme, has a marked reduction in specificity, indicating that the recognition of the hemimethylated state of target sites resides within the catalytic domain.
Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferases/genética , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Our understanding of poly-ADP-ribosylation as a posttranslational modification was limited by the lack of structural information on poly-ADP-ribose (PAR) hydrolysing enzymes. A recent study in Nature (Slade et al., 2011) reports the structure of PAR glycohydrolase (PARG), revealing unexpected similarity to the ubiquitous ADP-ribose-binding macrodomains.
RESUMO
Major histocompatibility complex (MHC) class I molecules present cell internally derived peptides at the plasma membrane for surveillance by cytotoxic T lymphocytes. The surface expression of most class I molecules at least partially depends on the endoplasmic reticulum protein, tapasin, which helps them to bind peptides of the right length and sequence. To determine what makes a class I molecule dependent on support by tapasin, we have conducted in silico molecular dynamics (MD) studies and laboratory experiments to assess the conformational state of tapasin-dependent and -independent class I molecules. We find that in the absence of peptide, the region around the F pocket of the peptide binding groove of the tapasin-dependent molecule HLA-B*44:02 is in a disordered conformational state and that it is converted to a conformationally stable state by tapasin. This novel chaperone function of tapasin has not been described previously. We demonstrate that the disordered state of class I is caused by the presence of two adjacent acidic residues in the bottom of the F pocket of class I, and we suggest that conformational disorder is a common feature of tapasin-dependent class I molecules, making them essentially unable to bind peptides on their own. MD simulations are a useful tool to predict such conformational disorder of class I molecules.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Proteínas de Membrana Transportadoras/farmacologia , Conformação Proteica/efeitos dos fármacos , Linhagem Celular , Antígeno HLA-B44/imunologia , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.