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1.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(4): 506-512, 2020 Apr 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895129

RESUMO

OBJECTIVE: To investigate the protective effect of serine hydroxymethyl transferase 2 (SHMT2) against hepatic ischemia-reperfusion injury in mice. METHODS: Sixty C57BL/6 mice were divided equally into sham-operated group, saline adeno-associated virus group (AVV-GFP), and adeno-associated virus silencing group (AAV-SHMT2). The adeno-associated virus and normal saline were injected into the tail vein of the mice 2 weeks before establishment of a 70% ischemia-reperfusion model in the liver. qPCR, Western blotting, immunofluorescence and immunohistochemistry were used to detect the changes of AST/ALT concentration, SHMT2, JNK, NF-κB, caspase-3 and downstream inflammatory factors in the mice, and HE staining was used to observe the pathological damage of the liver tissue in each group; the cell apoptosis in the liver was detected using TUNEL assay. RESULTS: The expression of SHMT2 increased with time after hepatic ischemia-reperfusion and reached the highest level at 24 h (the relative expression was 1.5, P < 0.05). At 24 h after hepatic ischemia-reperfusion, the levels of AST/ALT in AAV-SHMT2 group (588/416 U/L) were significantly higher than those in the control group (416/345 U/L) and the empty vector group (387/321 U/L) (P < 0.05). Compared with those in the control group and the empty vector group, the level of SHMT2 was significantly decreased in AAV-SHMT2 group (with a relative expression of 0.24, P < 0.05), the levels of p-JNK and p-p65 were significantly increased (relative expression of 0.80 and 0.97, respectively, P < 0.05), and the levels TNF-α and IL-1ß were consistently elevated (relative expression levels of 1.6 and 1.2, respectively, P < 0.05). No significant differences were found in these parameters between the empty vector group and the control group (P>0.05). CONCLUSIONS: SHMT2 may alleviate liver cell apoptosis in mice with hepatic ischemia-reperfusion injury by inhibiting the activation of JNK pathway and excessive activation of NF-κB pathway to reduce hepatic damage.


Assuntos
Traumatismo por Reperfusão , Animais , Apoptose , Fígado , Metiltransferases , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B , Serina
2.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(4): 567-572, 2020 Apr 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895140

RESUMO

OBJECTIVE: To investigate the expression of RNA methyltransferase METTL14 in hepatocellular carcinoma (HCC) and its clinical significance. METHODS: Immunohistochemical staining was used to detect the expression of METTL14 in 147 pairs of HCC and adjacent hepatic tissues. According to the scores rated by pathologists, the 147 cases of HCC were divided into high and low METTL14 expression groups. The correlation between the expression of METTL14 and clinicopathological parameters was analyzed, and Kaplan-Meier method was used to analyze the relationship between the expression of METTL14 and the prognosis and survival (including the overall survival and disease-free survival) of the patients with HCC after operation. Univariate analysis and multivariate analysis were carried out to assess the impact of METTL14 expression level on the overall survival and tumor-free survival of the patients after operation using a COX regression model and explore whether METTL14 expression level is an independent prognostic risk factor of the postoperative patients. RESULTS: The expression of METTL14 was significantly lower in HCC tissues than in the adjacent tissues (P < 0.001). METTL14 expression in HCC tissues was significantly correlated with the tumor size (P=0.044) and TNM stage (P=0.046). A low expression of METTL14 in HCC tissues was significantly correlated with a poor prognosis and a significantly shortened overall survival time and disease-free survival time of the patients (P < 0.05), and was an independent risk factor affecting the overall survival and disease-free survival of HCC patients. CONCLUSIONS: METTL14 may be a new prognostic marker for patients with HCC after hepatectomy.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Biomarcadores Tumorais , Intervalo Livre de Doença , Hepatectomia , Humanos , Estimativa de Kaplan-Meier , Metiltransferases , Prognóstico
3.
Int J Mol Sci ; 21(16)2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32824072

RESUMO

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a severe global health crisis. In this paper, we used docking and simulation methods to identify potential targets and the mechanism of action of chloroquine (CQ) and hydroxychloroquine (HCQ) against SARS-CoV-2. Our results showed that both CQ and HCQ influenced the functionality of the envelope (E) protein, necessary in the maturation processes of the virus, due to interactions that modify the flexibility of the protein structure. Furthermore, CQ and HCQ also influenced the proofreading and capping of viral RNA in SARS-CoV-2, performed by nsp10/nsp14 and nsp10/nsp16. In particular, HCQ demonstrated a better energy binding with the examined targets compared to CQ, probably due to the hydrogen bonding of the hydroxyl group of HCQ with polar amino acid residues.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Cloroquina/farmacologia , Exorribonucleases/metabolismo , Hidroxicloroquina/farmacologia , Metiltransferases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Proteínas do Envelope Viral/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos
4.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nat Commun ; 11(1): 3718, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709886

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.


Assuntos
Metiltransferases/química , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Betacoronavirus , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Difração de Raios X
7.
BMC Infect Dis ; 20(1): 544, 2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32711470

RESUMO

BACKGROUND: This study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre. METHODS: A total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with ß-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed. RESULTS: A total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906. CONCLUSION: The present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metiltransferases/genética , beta-Lactamases/genética , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Genes Bacterianos/genética , Humanos , Índia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus
8.
Nat Commun ; 11(1): 3717, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709887

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.


Assuntos
Betacoronavirus/enzimologia , Metiltransferases/química , Proteínas Virais Reguladoras e Acessórias/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Domínio Catalítico , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , Capuzes de RNA , Estabilidade de RNA , RNA Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
9.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: covidwho-595042

RESUMO

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Interferons/antagonistas & inibidores , Interferons/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/metabolismo , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Exorribonucleases/genética , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Metiltransferases/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
10.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32529952

RESUMO

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Interferons/antagonistas & inibidores , Interferons/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/metabolismo , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Exorribonucleases/genética , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Metiltransferases/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
11.
Virol Sin ; 35(3): 321-329, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32500504

RESUMO

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.


Assuntos
Betacoronavirus/metabolismo , Bismuto/farmacologia , Metiltransferases/metabolismo , Nucleosídeo-Trifosfatase/efeitos dos fármacos , RNA Helicases/efeitos dos fármacos , Sais/farmacologia , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Betacoronavirus/enzimologia , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/genética , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Pandemias , Pneumonia Viral/virologia , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes , Síndrome Respiratória Aguda Grave , Proteínas não Estruturais Virais/genética , Replicação Viral
12.
Eur J Med Chem ; 201: 112557, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32563813

RESUMO

The spreading of new viruses is known to provoke global human health threat. The current COVID-19 pandemic caused by the recently emerged coronavirus SARS-CoV-2 is one significant and unfortunate example of what the world will have to face in the future with emerging viruses in absence of appropriate treatment. The discovery of potent and specific antiviral inhibitors and/or vaccines to fight these massive outbreaks is an urgent research priority. Enzymes involved in the capping pathway of viruses and more specifically RNA N7- or 2'O-methyltransferases (MTases) are now admitted as potential targets for antiviral chemotherapy. We designed bisubstrate inhibitors by mimicking the transition state of the 2'-O-methylation of the cap RNA in order to block viral 2'-O MTases. This work resulted in the synthesis of 16 adenine dinucleosides with both adenosines connected by various nitrogen-containing linkers. Unexpectedly, all the bisubstrate compounds were barely active against 2'-O MTases of several flaviviruses or SARS-CoV but surprisingly, seven of them showed efficient and specific inhibition against SARS-CoV N7-MTase (nsp14) in the micromolar to submicromolar range. The most active nsp14 inhibitor identified is as potent as but particularly more specific than the broad-spectrum MTase inhibitor, sinefungin. Molecular docking suggests that the inhibitor binds to a pocket formed by the S-adenosyl methionine (SAM) and cap RNA binding sites, conserved among SARS-CoV nsp14. These dinucleoside SAM analogs will serve as starting points for the development of next inhibitors for SARS-CoV-2 nsp14 N7-MTase.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Nucleosídeos/química , Pneumonia Viral/tratamento farmacológico , Capuzes de RNA/metabolismo , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenina/química , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Exorribonucleases/metabolismo , Humanos , Metilação , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(25): 14251-14258, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513732

RESUMO

Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.


Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Sequências Repetitivas de Ácido Nucleico/fisiologia , Animais , Epigenômica , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Metilação , Metiltransferases/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Repressoras/metabolismo
14.
Bioresour Technol ; 313: 123616, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32563792

RESUMO

Trichoderma harzianum has attracting attention for its potential alternative use in biofuel production, due to a recognized competence for high diversity glycoside hydrolases (GH) enzyme complex, including higher ß-glucosidases and auxiliary proteins, using low-cost carbon sources. This strain constitutively overexpressed the global regulator putative methyltransferase - LAE1, in order to improve the GHs production. The recombinant strain achieved 79-fold increase in lae1 expression and high GHs productivity. The evaluation of the LAE1 impact to induce the GHs used soluble and lignocellulose inexpensive carbon sources in a stirred-tank bioreactor. Using sugarcane bagasse with sucrose, the overexpression of lae1 resulted in significantly increment of gh61b (31x), cel7a (25x), bgl1(20x) and xyn3 (20x) genes expression. Reducing sugar released from pretreated sugarcane bagasse, which hydrolyzed by recombinant crude enzyme cocktail, achieved 41% improvement. Therefore, lae1 overexpression effectively is a promising improving GHs target for biomass degradation by T. harzianum.


Assuntos
Celulases , Saccharum , Trichoderma , Biomassa , Metiltransferases
15.
Proc Natl Acad Sci U S A ; 117(27): 15609-15619, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571953

RESUMO

Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Escherichia coli ΔrrmJ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from ΔrrmJ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the ΔrrmJ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the ΔrrmJ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.


Assuntos
Escherichia coli/fisiologia , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , RNA Ribossômico 23S/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Técnicas de Inativação de Genes , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Uridina/metabolismo
16.
Cells ; 9(5)2020 05 20.
Artigo em Inglês | MEDLINE | ID: covidwho-324261

RESUMO

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Replicação Viral/fisiologia , Animais , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Genoma Viral/genética , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Pandemias , Conformação Proteica em alfa-Hélice , RNA Helicases/química , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo
17.
Nat Commun ; 11(1): 2578, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444598

RESUMO

Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have sprung up in recent years. We find m6A can positively regulate the glycolysis of cancer cells. Specifically, m6A-sequencing and functional studies confirm that pyruvate dehydrogenase kinase 4 (PDK4) is involved in m6A regulated glycolysis and ATP generation. The m6A modified 5'UTR of PDK4 positively regulates its translation elongation and mRNA stability via binding with YTHDF1/eEF-2 complex and IGF2BP3, respectively. Targeted specific demethylation of PDK4 m6A by dm6ACRISPR system can significantly decrease the expression of PDK4 and glycolysis of cancer cells. Further, TATA-binding protein (TBP) can transcriptionally increase the expression of Mettl3 in cervical cancer cells via binding to its promoter. In vivo and clinical data confirm the positive roles of m6A/PDK4 in tumor growth and progression of cervical and liver cancer. Our study reveals that m6A regulates glycolysis of cancer cells through PDK4.


Assuntos
Adenosina/análogos & derivados , Glicólise/fisiologia , Quinase Piruvato Desidrogenase (Transferência de Acetil)/metabolismo , Neoplasias do Colo do Útero/genética , Regiões 5' não Traduzidas , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Glicólise/genética , Células HeLa , Humanos , Estimativa de Kaplan-Meier , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Quinase Piruvato Desidrogenase (Transferência de Acetil)/genética , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Neoplasias do Colo do Útero/mortalidade , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Drugs ; 80(10): 941-946, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32451923

RESUMO

G-Quadruplexes (G4s) are non-canonical secondary structures formed within guanine-rich regions of DNA or RNA. G4 sequences/structures have been detected in human and in viral genomes, including Coronaviruses Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and SARS-CoV-2. Here, we outline the existing evidence indicating that G4 ligands and inhibitors of SARS-CoV-2 helicase may exert some antiviral activity reducing viral replication and can represent a potential therapeutic approach to tackle the COVID-19 pandemic due to SARS-CoV-2 infection. We also discuss how repositioning of FDA-approved drugs against helicase activity of other viruses, could represent a rapid strategy to limit deaths associated with COVID-19 pandemic.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Quadruplex G , Genoma Viral/genética , Pneumonia Viral/tratamento farmacológico , RNA Helicases/antagonistas & inibidores , Reposicionamento de Medicamentos , Humanos , Metiltransferases/antagonistas & inibidores , Pandemias , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
19.
Nucleic Acids Res ; 48(11): 6157-6169, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32392304

RESUMO

The TRM10 family of methyltransferases is responsible for the N1-methylation of purines at position 9 of tRNAs in Archaea and Eukarya. The human genome encodes three TRM10-type enzymes, of which only the mitochondrial TRMT10C was previously characterized in detail, whereas the functional significance of the two presumably nuclear enzymes TRMT10A and TRMT10B remained unexplained. Here we show that TRMT10A is m1G9-specific and methylates a subset of nuclear-encoded tRNAs, whilst TRMT10B is the first m1A9-specific tRNA methyltransferase found in eukaryotes and is responsible for the modification of a single nuclear-encoded tRNA. Furthermore, we show that the lack of G9 methylation causes a decrease in the steady-state levels of the initiator tRNAiMet-CAT and an alteration in its further post-transcriptional modification. Our work finally clarifies the function of TRMT10A and TRMT10B in vivo and provides evidence that the loss of TRMT10A affects the pool of cytosolic tRNAs required for protein synthesis.


Assuntos
Metiltransferases/metabolismo , tRNA Metiltransferases/metabolismo , Sequência de Bases , Linhagem Celular , Humanos , Metilação , Metiltransferases/deficiência , Biossíntese de Proteínas , Purinas/metabolismo , RNA de Transferência/metabolismo
20.
Nucleic Acids Res ; 48(11): 6251-6264, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32406913

RESUMO

m6A is a prevalent internal modification in mRNAs and has been linked to the diverse effects on mRNA fate. To explore the landscape and evolution of human m6A, we generated 27 m6A methylomes across major adult tissues. These data reveal dynamic m6A methylation across tissue types, uncover both broadly or tissue-specifically methylated sites, and identify an unexpected enrichment of m6A methylation at non-canonical cleavage sites. A comparison of fetal and adult m6A methylomes reveals that m6A preferentially occupies CDS regions in fetal tissues. Moreover, the m6A sub-motifs vary between fetal and adult tissues or across tissue types. From the evolutionary perspective, we uncover that the selection pressure on m6A sites varies and depends on their genic locations. Unexpectedly, we found that ∼40% of the 3'UTR m6A sites are under negative selection, which is higher than the evolutionary constraint on miRNA binding sites, and much higher than that on A-to-I RNA modification. Moreover, the recently gained m6A sites in human populations are clearly under positive selection and associated with traits or diseases. Our work provides a resource of human m6A profile for future studies of m6A functions, and suggests a role of m6A modification in human evolutionary adaptation and disease susceptibility.


Assuntos
Adenosina/análogos & derivados , Metilação de DNA , Evolução Molecular , Regiões 3' não Traduzidas , Adenosina/metabolismo , Adulto , Suscetibilidade a Doenças , Epigenoma , Feto/metabolismo , Genética Populacional , Células HEK293 , Humanos , Metiltransferases/deficiência , Metiltransferases/genética , Especificidade de Órgãos
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