RESUMO
Nuclear magnetic resonance (NMR) methods that quantitatively probe motions on molecular and atomic levels have propelled the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. In this work, we studied the structure and dynamics of the essential 100-kDa eukaryotic 5'â3' exoribonuclease Xrn2. A combination of complementary fluorine and methyl-TROSY NMR spectroscopy reveals that the apo enzyme is highly dynamic around the catalytic center. These observed dynamics are in agreement with a transition of the enzyme from the ground state into a catalytically competent state. We show that the conformational equilibrium in Xrn2 shifts substantially toward the active state in the presence of substrate and magnesium. Finally, our data reveal that the dynamics in Xrn2 correlate with the RNA degradation rate, as a mutation that attenuates motions also affects catalytic activity. In that light, our results stress the importance of studies that go beyond static structural information.
Assuntos
Exorribonucleases , Flúor , Catálise , Exorribonucleases/genética , Magnésio , Ressonância Magnética Nuclear BiomolecularRESUMO
Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Interferon-alfa/farmacologia , RNA Viral/metabolismo , SARS-CoV-2/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Exorribonucleases/genética , Vetores Genéticos , Células HeLa , Humanos , Interferon-alfa/administração & dosagem , Luciferases/genética , Luciferases/metabolismo , Naftiridinas/administração & dosagem , Naftiridinas/farmacologia , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacologia , RNA Viral/efeitos dos fármacos , RepliconRESUMO
The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Capuzes de RNA/metabolismo , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/química , Clorobenzenos/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/genética , Exorribonucleases/isolamento & purificação , Exorribonucleases/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Indazóis/farmacologia , Indenos/farmacologia , Indóis/farmacologia , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Nitrilas/farmacologia , Fenotiazinas/farmacologia , Purinas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato , Trifluperidol/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
SARS-CoV-2is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. The nsp14 protein of SARS-CoV-2 houses a 3' to 5' exoribonuclease activity responsible for removing mismatches that arise during genome duplication. A homology model of nsp10-nsp14 complex was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp14. This exercise showed that ritonavir might bind to the exoribonuclease active site of the nsp14 protein. A model of the SARS-CoV-2-nsp10-nsp14 complex bound to substrate RNA showed that the ritonavir binding site overlaps with that of the 3' nucleotide of substrate RNA. A comparison of the calculated energies of binding for RNA and ritonavir suggested that the drug may bind to the active site of nsp14 with significant affinity. It is, therefore, possible that ritonavir may prevent association with substrate RNA and thus inhibit the exoribonuclease activity of nsp14. Overall, our computational studies suggest that ritonavir may serve as an effective inhibitor of the nsp14 protein. nsp14 is known to attenuate the inhibitory effect of drugs that function through premature termination of viral genome replication. Hence, ritonavir may potentiate the therapeutic properties of drugs such as remdesivir, favipiravir and ribavirin.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Exorribonucleases/antagonistas & inibidores , Ritonavir/farmacologia , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/administração & dosagem , Antivirais/química , COVID-19/virologia , Domínio Catalítico , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Exorribonucleases/química , Exorribonucleases/genética , Genoma Viral/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Pandemias , Ritonavir/administração & dosagem , Ritonavir/química , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
Organellar DNAs in mitochondria and plastids are present in multiple copies and make up a substantial proportion of total cellular DNA despite their limited genetic capacity. We recently demonstrated that organellar DNA degradation occurs during pollen maturation, mediated by the Mg(2+) -dependent organelle exonuclease DPD1. To further understand organellar DNA degradation, we characterized a distinct mutant (dpd2). In contrast to the dpd1 mutant, which retains both plastid and mitochondrial DNAs, dpd2 showed specific accumulation of plastid DNAs. Multiple abnormalities in vegetative and reproductive tissues of dpd2 were also detected. DPD2 encodes the large subunit of ribonucleotide reductase, an enzyme that functions at the rate-limiting step of de novo nucleotide biosynthesis. We demonstrated that the defects in ribonucleotide reductase indirectly compromise the activity of DPD1 nuclease in plastids, thus supporting a different regulation of organellar DNA degradation in pollen. Several lines of evidence provided here reinforce our previous conclusion that the DPD1 exonuclease plays a central role in organellar DNA degradation, functioning in DNA salvage rather than maternal inheritance during pollen development.
Assuntos
Proteínas de Arabidopsis/genética , DNA de Plantas/genética , Exorribonucleases/genética , Ribonucleotídeo Redutases/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA de Plantas/metabolismo , Exorribonucleases/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Plastídeos/genética , Pólen/genética , Pólen/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleotídeo Redutases/metabolismoRESUMO
Organelle DNA in plastids and mitochondria is present in multiple copies and undergoes degradation developmentally. For example, organelle DNA that is detectable cytologically using DNA-fluorescent dye disappears during pollen development. Nevertheless, nucleases involved in this degradation process remain unknown. Our recent study identified the organelle nuclease, DPD1, which has Mg2+ -dependent exonuclease activity in vitro. The discovery of DPD1 emerged from Arabidopsis mutant screening and concomitant isolation of dpd1 mutants that retain organelle DNA in mature pollen. DPD1 is conserved only in angiosperms: not in other photosynthetic organisms. Despite these findings, the physiological significance of organelle DNA degradation during pollen development remains unclear because dpd1 exhibits no apparent defects in pollen viability or in the maternal inheritance of organelle DNA. We discuss a possible role of organelle DNA degradation mediated by DPD1, based on a DPD1 expression profile studied using in silico analyses.
Assuntos
Proteínas de Arabidopsis/metabolismo , DNA/metabolismo , Exorribonucleases/metabolismo , Proteínas de Arabidopsis/genética , Exorribonucleases/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Magnoliopsida/enzimologia , Magnoliopsida/metabolismo , Organelas/enzimologia , Pólen/metabolismoRESUMO
In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg²âº-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , DNA de Plantas/metabolismo , Exonucleases/metabolismo , Exorribonucleases/metabolismo , Magnésio/metabolismo , Organelas/genética , Pólen/crescimento & desenvolvimento , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada/genética , DNA de Cloroplastos/metabolismo , DNA Mitocondrial/metabolismo , Exorribonucleases/genética , Genes de Plantas/genética , Teste de Complementação Genética , Germinação , Padrões de Herança/genética , Células do Mesofilo/citologia , Células do Mesofilo/metabolismo , Mitocôndrias/metabolismo , Proteínas Mutantes/isolamento & purificação , Mutação/genética , Especificidade de Órgãos , Fenótipo , Plastídeos/metabolismo , Pólen/citologia , Pólen/metabolismo , Pólen/ultraestrutura , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , ReproduçãoRESUMO
Unconventional mRNA splicing by an endoplasmic reticulum stress-inducible endoribonuclease, IRE1, is conserved in all known eukaryotes. It controls the expression of a transcription factor, Hac1p/XBP-1, that regulates gene expression in the unfolded protein response. In yeast, the RNA fragments generated by Ire1p are ligated by tRNA ligase (Trl1p) in a process that leaves a 2'-PO4(2-) at the splice junction, which is subsequently removed by an essential 2'-phosphotransferase, Tpt1p. However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin the cleaved Xbp-1 mRNA fragments. We report that inactivation of the Trpt1 gene, encoding the only known mammalian homolog of Tpt1p, eliminates all detectable 2'-phosphotransferase activity from cultured mouse cells but has no measurable effect on spliced Xbp-1 translation. Furthermore, the relative translation rates of tyrosine-rich proteins is unaffected by the Trpt1 genotype, suggesting that the pool of (normally spliced) tRNA(Tyr) is fully functional in the Trpt1-/- mouse cells. These observations argue against the presence of a 2'-PO4(2-) at the splice junction of ligated RNA molecules in Trpt1-/- cells, and suggest that Xbp-1 and tRNA ligation proceed by distinct pathways in yeast and mammals.
Assuntos
Proteínas de Ligação a DNA/genética , Exorribonucleases/metabolismo , Proteínas Nucleares/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Biossíntese de Proteínas/genética , Splicing de RNA/genética , Animais , Exorribonucleases/genética , Variação Genética , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Filogenia , Dobramento de Proteína , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismo , RNA de Transferência de Tirosina/metabolismo , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição , Tirosina/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenous Pseudomonas plasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3' to 5' exoribonuclease specific for small oligoribonucleotides. The deduced amino acid sequence of the plasmid-derived orn (orn(pl)) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes. Deletion of orn(pl) generated no observable phenotype, but inactivation of the chromosomal copy caused slow growth in Pseudomonas putida KT2440. This defect was fully restored by complementation with orn from Escherichia coli (orn(E.coli)). Plasmid-derived orn(pl) was capable of partially complementing the P. putida orn mutant, demonstrating functionality of orn(pl). Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria. A survey of orn(pl) from related Pseudomonas plasmids showed a sporadic distribution but no sequence diversity. These data suggest that the orn(pl) was acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of the Proteobacteria.