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1.
Nature ; 584(7820): 279-285, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760005

RESUMO

In pathophysiology, reactive oxygen species oxidize biomolecules that contribute to disease phenotypes1. One such modification, 8-oxoguanine2 (o8G), is abundant in RNA3 but its epitranscriptional role has not been investigated for microRNAs (miRNAs). Here we specifically sequence oxidized miRNAs in a rat model of the redox-associated condition cardiac hypertrophy4. We find that position-specific o8G modifications are generated in seed regions (positions 2-8) of selective miRNAs, and function to regulate other mRNAs through o8G•A base pairing. o8G is induced predominantly at position 7 of miR-1 (7o8G-miR-1) by treatment with an adrenergic agonist. Introducing 7o8G-miR-1 or 7U-miR-1 (in which G at position 7 is substituted with U) alone is sufficient to cause cardiac hypertrophy in mice, and the mRNA targets of o8G-miR-1 function in affected phenotypes; the specific inhibition of 7o8G-miR-1 in mouse cardiomyocytes was found to attenuate cardiac hypertrophy. o8G-miR-1 is also implicated in patients with cardiomyopathy. Our findings show that the position-specific oxidation of miRNAs could serve as an epitranscriptional mechanism to coordinate pathophysiological redox-mediated gene expression.


Assuntos
Cardiomegalia/genética , Cardiomegalia/patologia , Inativação Gênica , MicroRNAs/química , MicroRNAs/metabolismo , Animais , Pareamento de Bases , Linhagem Celular , Modelos Animais de Doenças , Guanina/análogos & derivados , Guanina/análise , Guanina/química , Guanina/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxirredução , Ratos , Transcrição Genética/genética , Transcriptoma/genética
2.
Mem Inst Oswaldo Cruz ; 115: e190469, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32638832

RESUMO

BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.


Assuntos
Antimônio/toxicidade , Proteínas de Escherichia coli/genética , Escherichia coli , Guanina/análogos & derivados , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Pirofosfatases , Superóxido Dismutase/metabolismo , Animais , Antiprotozoários/farmacologia , Proteínas de Escherichia coli/metabolismo , Guanina/farmacologia , Humanos , Peróxido de Hidrogênio/toxicidade , Leishmania braziliensis/enzimologia , Leishmania infantum/enzimologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Coelhos , Ratos , Superóxido Dismutase/genética
3.
Medicine (Baltimore) ; 99(27): e21032, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32629728

RESUMO

BACKGROUND: Chronic hepatitis B is often complicated with different degrees of hepatic fibrosis, which affects the quality of life. Nucleoside analogs are recommended by almost all guidelines in the world for the treatment of chronic hepatitis B. At present, there is no specific and effective chemical and biological agents for hepatic fibrosis. In China, Chinese compound prescription combined with nucleoside analogs have been used to treat hepatic fibrosis of chronic hepatitis B patients in more and more cases, and good results have been achieved. Several Chinese compound prescriptions that have been made into proprietary Chinese medicine for the convenience of use. This article aims to systematically evaluate the efficacy and safety of Chinese medicine compounds assisting nucleoside analogs in the treatment of hepatic fibrosis in chronic hepatitis B patients. METHOD: The following databases will be searched from their inception to September 2019: PubMed, EMBASE, EBSCOhost, The Cochrane Library, China National Knowledge Infrastructure (CNKI), Chinese Biomedical literature Database (CBM), VIP Database, Wanfang Database. Languages are limited to Chinese and English. The study includes randomized controlled trials using Chinese compound prescription combined with entecavir and Chinese compound prescription combined with tenofovir disoproxil fumarate to treat hepatic fibrosis of chronic hepatitis B patients. The primary outcomes including effective rate and biochemical parameters (levels of hyaluronic acid, laminin, pre-type-III collagen and type IV collagen will be tested. Additional outcomes include liver function indexes (levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin) and levels of hepatitis B virus DNA. Stata14.0 software will be used for meta-analysis. RESULT: The efficacy and safety of Chinese compound prescriptions assisting nucleoside analogs for hepatic fibrosis of chronic hepatitis B patients will be assessed from the effective rate, biochemical parameters, liver function indexes, and levels of hepatitis B virus DNA. CONCLUSION: The conclusion of this study will be used to evaluate the efficacy and safety of Chinese compound prescriptions assisting nucleoside analogs in the treatment of hepatic fibrosis of chronic hepatitis B patients, as well as the adjuvant effectiveness of Chinese compound prescriptions in combined therapy. PROSPERO REGISTRATION NUMBER: CRD42020156859.


Assuntos
Hepatite B Crônica/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Nucleosídeos/análogos & derivados , Antivirais/uso terapêutico , China/epidemiologia , Vírus de DNA/efeitos dos fármacos , Bases de Dados Factuais , Quimioterapia Combinada/métodos , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Guanina/análogos & derivados , Guanina/uso terapêutico , Hepatite B Crônica/complicações , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/psicologia , Testes de Função Hepática/métodos , Masculino , Nucleosídeos/uso terapêutico , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto , Tenofovir/uso terapêutico
4.
Pediatrics ; 146(2)2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32680878

RESUMO

Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening syndrome classified into primary HLH and secondary HLH. Secondary HLH is always caused by autoimmune disease, infections, or cancer. The first-line therapy for secondary HLH is the HLH 2004 protocol, including dexamethasone, etoposide, and supportive therapy. However, up to 30% of patients, especially pediatric patients, remain unresponsive to first-line treatment, and the mortality rate reaches 50% in children with HLH. Furthermore, some children who have special conditions, such as an active virus infection, are not suitable for immunosuppressants treatment. Recently, several HLH-promoting cytokines have been identified, including interferon-γ, interleukin-2, and interleukin-6. Janus kinase 1 and 2 control the signaling of many cytokines, notably interferon-γ, interleukin-2, and interleukin-6. Janus kinase 1 and 2 inhibitors, such as ruxolitinib, have been successfully used to treat HLH in mice. Here, we report that a boy, diagnosed with HLH and high titer of hepatitis B virus-DNA copies, improved quickly, and the cytokine storm of HLH was alleviated after receiving ruxolitinib. Five days after ruxolitinib treatment, entecavir was introduced and serum titer results of hepatitis B virus-DNA returned negative. With 3 months of ruxolitinib treatment and following-up 1 year, the boy's situation maintained sustained remission. In this study, it is suggested that ruxolitinib might be a first-line drug, which could alleviate the cytokine storm of HLH. This treatment may be ushering in the age of glucocorticosteroid-free HLH treatment, which is particularly meaningful for children because it avoids the side effects of glucocorticosteroid.


Assuntos
Síndrome da Liberação de Citocina/tratamento farmacológico , Linfo-Histiocitose Hemofagocítica/etiologia , Pirazóis/uso terapêutico , Antivirais/efeitos adversos , Antivirais/uso terapêutico , Criança , Síndrome da Liberação de Citocina/etiologia , Quimioterapia Combinada , Febre/etiologia , Guanina/análogos & derivados , Guanina/uso terapêutico , Hepatite B Crônica/complicações , Hepatite B Crônica/congênito , Hepatite B Crônica/tratamento farmacológico , Humanos , Interferon-alfa/efeitos adversos , Interferon-alfa/uso terapêutico , Janus Quinases/antagonistas & inibidores , Masculino , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/uso terapêutico , Pirazóis/farmacologia , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Carga Viral
6.
Medicine (Baltimore) ; 99(22): e20330, 2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32481407

RESUMO

The renal protective effect of telbivudine (LdT) was verified by a previous meta-analysis. It was left unclear, however if this effect offsets the associated risk of virological breakthrough in hepatitis B e-antigen-negative (HBeAg-) patients receiving chemotherapy (C/T).Records of 260 HBeAg-, non-cirrhotic cancer patients undergoing systemic C/T with prophylactic LdT or entecavir (ETV) were retrospectively investigated. The investigation was conducted 6 months after completion of C/T, patient death from cancer, or antiviral modification. Treatment duration, outcome, change of renal function, and reason for antiviral modification were analyzed. The primary endpoint was the occurrence of virological breakthrough during prophylaxis C/T and the change in renal function.Of the 126 HBeAg- patients treated with LdT, 3 (2.38%) experienced HBV virological breakthroughs, whereas none of the patients treated with ETV (P = .07) did. The estimated glomerular filtration rate for the patients treated with LdT was essentially unaltered, decreasing only slightly from 87.5 ±â€Š23.1 to 87.3 ±â€Š21.3 ml/minute/1.73 m (P = .55), while the rate for the ETV-treated patients was significantly lowered from 95.7 ±â€Š32.2 to 85.5 ±â€Š85.7 ml/minute/1.73 m (P = .0009).The absolute risk reduction ARR is 27.8% - 21.2% = 6.6%, comparing ETV with LdT for reduction of renal function impairment and the absolute risk increase for virological breakthrough during C/T, the absolute risk increase (ARI) is 2.38% - 0% = 2.38%. The overall likelihood of being helped over being harmed was 2.77. With careful selection of patients with the criteria of HBeAg-status and non-hematologic cancer, it is feasible that telbivudine raise lower probability of virological breakthroughs during prophylaxis treatment.


Assuntos
Antineoplásicos/uso terapêutico , Antivirais/uso terapêutico , Guanina/análogos & derivados , Antígenos E da Hepatite B/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Telbivudina/uso terapêutico , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/prevenção & controle , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Feminino , Taxa de Filtração Glomerular , Guanina/administração & dosagem , Guanina/efeitos adversos , Guanina/uso terapêutico , Humanos , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Telbivudina/administração & dosagem , Telbivudina/efeitos adversos
7.
Science ; 368(6498): 1465-1468, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32587016

RESUMO

DNA and proteins are chiral: Their three-dimensional structures cannot be superimposed with their mirror images. Circular dichroism spectroscopy is widely used to characterize chiral compounds, but data interpretation is difficult in the case of mixtures. We recorded the electronic circular dichroism spectra of DNA helices separated in a mass spectrometer. We studied guanine-rich strands having various secondary structures, electrosprayed them as negative ions, irradiated them with an ultraviolet nanosecond optical parametric oscillator laser, and measured the difference in electron photodetachment efficiency between left and right circularly polarized light. The reconstructed circular dichroism ion spectra resembled those of their solution-phase counterparts, thereby allowing us to assign the DNA helical topology. The ability to measure circular dichroism directly on biomolecular ions expands the capabilities of mass spectrometry for structural analysis.


Assuntos
Dicroísmo Circular/métodos , DNA/química , Guanina/química , Espectrometria de Massas/métodos , Guanina/análise , Conformação de Ácido Nucleico
8.
Antiviral Res ; 180: 104857, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32562705

RESUMO

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH2-dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/virologia , Nucleotídeos/farmacologia , Pneumonia Viral/virologia , RNA Replicase/antagonistas & inibidores , Vírus da SARS/enzimologia , Síndrome Respiratória Aguda Grave/virologia , Antivirais/química , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Betacoronavirus/genética , Cidofovir/química , Cidofovir/farmacologia , Cidofovir/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Didesoxinucleosídeos/química , Didesoxinucleosídeos/farmacologia , Didesoxinucleosídeos/uso terapêutico , Ganciclovir/química , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Guanina/análogos & derivados , Guanina/química , Guanina/farmacologia , Guanina/uso terapêutico , Nucleotídeos/química , Nucleotídeos/uso terapêutico , Pandemias , Pneumonia Viral/tratamento farmacológico , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Estavudina/química , Estavudina/farmacologia , Estavudina/uso terapêutico , Valganciclovir/química , Valganciclovir/farmacologia , Valganciclovir/uso terapêutico
11.
PLoS One ; 15(5): e0232724, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374749

RESUMO

DNA damage in the A549 human lung cancer cell line treated with cold plasma irradiation was investigated. We confirmed that cold atmospheric plasma generated reactive oxygen and nitrogen species (RONS) in a liquid, and the intracellular RONS level was increased in plasma-irradiated cells. However, a notable decrease in cell viability was not observed 24 hours after plasma irradiation. Because RONS induce oxidative damage in cells, strand breaks and chemical modification of DNA in the cancer cells were investigated. We found that 8-oxoguanine (8-oxoG) formation as well as DNA strand breaks, which have been thoroughly investigated, were induced by plasma irradiation. In addition, up-regulation of 8-oxoG repair enzyme was observed after plasma irradiation.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , DNA Glicosilases/metabolismo , Reparo do DNA , Guanina/análogos & derivados , Gases em Plasma/farmacologia , Células A549 , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Guanina/biossíntese , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
12.
Toxicol Lett ; 331: 75-81, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32434050

RESUMO

Fungi of the genus Alternaria infest many agricultural crops and produce numerous mycotoxins, of which altertoxin II (ATX II) is one of the most mutagenic metabolites. ATX II carries an epoxide group but the formation of DNA adducts has not been demonstrated to date. We report now that ATX II gives rise to two covalent adducts with guanine when incubated with DNA under cell-free conditions. These adducts were demonstrated by LC-high resolution MS after enzymatic degradation of the incubated DNA to deoxynucleosides. The major adduct results from the covalent binding of ATX II, presumably through the epoxide group, to guanine, whereas the minor guanine adduct is derived from the major one by the elimination of two equivalents of water. In addition, a third adduct was detected, formed through covalent binding of ATX II to cytosine followed by the loss of two equivalents of water. The direct DNA reactivity of ATX II may explain its high mutagenicity.


Assuntos
Benzo(a)Antracenos/toxicidade , Adutos de DNA/análise , DNA/química , Guanina/química , Mutagênicos/toxicidade , Alternaria/química , Animais , Benzo(a)Antracenos/isolamento & purificação , Cromatografia Líquida , DNA/isolamento & purificação , Masculino , Espectrometria de Massas , Salmão , Testículo
14.
Nucleic Acids Res ; 48(11): 5907-5925, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32383760

RESUMO

Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.


Assuntos
Elementos Facilitadores Genéticos , Genoma Humano , Guanina , Conformação de Ácido Nucleico , Pareamento de Bases , Quadruplex G , Rearranjo Gênico , Variação Genética , Genômica , Guanina/análise , Humanos , Saccharomyces cerevisiae/genética , Duplicações Segmentares Genômicas , Deleção de Sequência
15.
Chem Biol Interact ; 327: 109140, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32442416

RESUMO

A liquid chromatograpy-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method was developed for quantitation of the DNA adducts 7-(2'-carboxyethyl)guanine (7-2'-CEG) and N2-(1'-carboxyethyl)guanine (N2-1'-CEG), as their methyl esters, in human leukocyte DNA from smokers and non-smokers. 7-2'-CEG has been previously identified in all human liver samples analyzed and is formed from an unknown carboxyethylating agent while N2-1'-CEG is formed from the advanced glycation endproduct methyl glyoxal. The method was applied for the analysis of these two DNA adducts in leukocyte DNA from 20 smokers and 20 non-smokers, in part to test the hypothesis that 7-2'-CEG could be formed by endogenous nitrosation, as previously observed in rats treated with nitrosodihydrouracil and nitrite. Levels of 7-2'-CEG (mean ± S.D.) were 0.6 ± 0.2 pmol/µmol dG in smokers and 0.5 ± 0.2 pmol/µmol dG in non-smokers, while those of N2-1'-CEG were 4.5 ± 1.9 pmol/µmol dG in smokers and 4.6 ± 2 pmol/µmol dG in non-smokers. These results did not support our hypothesis that endogenous nitrosation of dihydrouracil in smokers leads to higher levels of 7-2'-CEG in leukocyte DNA than in non-smokers. However the study provides the first data on levels of these DNA adducts in human leukocyte DNA, and the LC-NSI-HRMS/MS method developed for their quantitation could be important for future studies of DNA damage by methyl glyoxal.


Assuntos
Adutos de DNA/sangue , Guanina/análogos & derivados , Leucócitos/química , Adolescente , Adulto , Idoso , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , DNA/química , Feminino , Guanina/sangue , Humanos , Masculino , Pessoa de Meia-Idade , não Fumantes , Fumantes , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Adulto Jovem
16.
J Virol ; 94(16)2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32461321

RESUMO

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.


Assuntos
Exorribonucleases/metabolismo , Interferon Tipo I/biossíntese , Interferons/biossíntese , Vírus da Diarreia Epidêmica Suína/fisiologia , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Exorribonucleases/genética , Expressão Gênica , Guanina/metabolismo , Imunidade Inata , Metilação , Mutação , Vírus da Diarreia Epidêmica Suína/enzimologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Suínos , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral
17.
Proc Natl Acad Sci U S A ; 117(21): 11409-11420, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404420

RESUMO

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.


Assuntos
Dano ao DNA , Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Quadruplex G , Células A549 , Acetilação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Expressão Gênica , Genes myc , Genoma Humano , Guanina/química , Guanina/metabolismo , Células HCT116 , Humanos , Oxirredução , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Nat Commun ; 11(1): 2484, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424276

RESUMO

DNA damage contributes to brain aging and neurodegenerative diseases. However, the factors stimulating DNA repair to stave off functional decline remain obscure. We show that HDAC1 modulates OGG1-initated 8-oxoguanine (8-oxoG) repair in the brain. HDAC1-deficient mice display age-associated DNA damage accumulation and cognitive impairment. HDAC1 stimulates OGG1, a DNA glycosylase known to remove 8-oxoG lesions that are associated with transcriptional repression. HDAC1 deficiency causes impaired OGG1 activity, 8-oxoG accumulation at the promoters of genes critical for brain function, and transcriptional repression. Moreover, we observe elevated 8-oxoG along with reduced HDAC1 activity and downregulation of a similar gene set in the 5XFAD mouse model of Alzheimer's disease. Notably, pharmacological activation of HDAC1 alleviates the deleterious effects of 8-oxoG in aged wild-type and 5XFAD mice. Our work uncovers important roles for HDAC1 in 8-oxoG repair and highlights the therapeutic potential of HDAC1 activation to counter functional decline in brain aging and neurodegeneration.


Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Encéfalo/patologia , Dano ao DNA , DNA Glicosilases/metabolismo , Histona Desacetilase 1/metabolismo , Estresse Oxidativo , Acetilação , Envelhecimento/genética , Doença de Alzheimer/complicações , Doença de Alzheimer/fisiopatologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Sequência de Bases , Benzofenonas/farmacologia , Cognição/efeitos dos fármacos , Transtornos Cognitivos/complicações , Transtornos Cognitivos/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Ontologia Genética , Guanina/análogos & derivados , Guanina/metabolismo , Memória/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Regiões Promotoras Genéticas/genética
19.
Mutat Res ; 852: 503160, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265045

RESUMO

Professor Barbara Tudek received the Frits Sobels Award in 2019 from the European Environmental Mutagenesis and Genomics Society (EEMGS). This article presents her outstanding character and most important lines of research. The focus of her studies covered alkylative and oxidative damage to DNA bases, in particular mutagenic and carcinogenic properties of purines with an open imidazole ring and 8-oxo-7,8-dihydroguanine (8-oxoGua). They also included analysis of mutagenic properties and pathways for the repair of DNA adducts of lipid peroxidation (LPO) products arising in large quantities during inflammation. Professor Tudek did all of this in the hope of deciphering the mechanisms of DNA damage removal, in particular by the base excision repair (BER) pathway. Some lines of research aimed at discovering factors that can modulate the activity of DNA damage repair in hope to enhance existing anti-cancer therapies. The group's ongoing research aims at deciphering the resistance mechanisms of cancer cell lines acquired following prolonged exposure to photodynamic therapy (PDT) and the possibility of re-sensitizing cells to PDT in order to increase the application of this minimally invasive therapeutic method.


Assuntos
Carcinogênese/metabolismo , Reparo do DNA , Guanina/análogos & derivados , Neoplasias/história , Fotoquimioterapia/história , Radiossensibilizantes/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA , Guanina/metabolismo , História do Século XX , História do Século XXI , Humanos , Peroxidação de Lipídeos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fotoquimioterapia/métodos
20.
Proc Natl Acad Sci U S A ; 117(18): 9832-9839, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32317383

RESUMO

G-quadruplex, assembled from a square array of guanine (G) molecules, is an important structure with crucial biological roles in vivo but also a versatile template for ordered functional materials. Although the understanding of G-quadruplex structures is the focus of numerous studies, little is known regarding the control of G-quartet stacking modes and the spontaneous orientation of G-quadruplex fibrils. Here, the effects of different metal ions and their concentrations on stacking modes of G-quartets are elucidated. Monovalent cations (typically K+) facilitate the formation of G-quadruplex hydrogels with both heteropolar and homopolar stacking modes, showing weak mechanical strength. In contrast, divalent metal ions (Ca2+, Sr2+, and Ba2+) at given concentrations can control G-quartet stacking modes and increase the mechanical rigidity of the resulting hydrogels through ionic bridge effects between divalent ions and borate. We show that for Ca2+ and Ba2+ at suitable concentrations, the assembly of G-quadruplexes results in the establishment of a mesoscopic chirality of the fibrils with a regular left-handed twist. Finally, we report the discovery of nematic tactoids self-assembled from G-quadruplex fibrils characterized by homeotropic fibril alignment with respect to the interface. We use the Frank-Oseen elastic energy and the Rapini-Papoular anisotropic surface energy to rationalize two different configurations of the tactoids. These results deepen our understanding of G-quadruplex structures and G-quadruplex fibrils, paving the way for their use in self-assembly and biomaterials.


Assuntos
DNA/química , Quadruplex G , Guanina/química , Hidrogéis/química , Anisotropia , Cátions Bivalentes/química , Cátions Monovalentes/química , DNA/ultraestrutura , Metabolismo Energético/efeitos dos fármacos , Líquidos Iônicos/química , Íons/química , Metais/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Propriedades de Superfície
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