RESUMEN
The flavivirus NS5 protein contains an N-terminal methyl-transferase (MTase) connected through a flexible linker with a C-terminal RNA-dependent RNA-polymerase (RdRp) domain, that work cooperatively to replicate and methylate the viral genome. In this study we probed the importance of an evolutionary-conserved hydrophobic residue (Val266) located at the start of the ten-residue interdomain linker of Zika virus (ZIKV) NS5. In flavivirus NS5 crystal structures, the start of the linker forms a 310 helix when NS5 adopts a compact conformation, but becomes disordered or extended in open conformations. Using reverse genetics system, we either introduced rigidity in the linker through mutation to a proline or flexibility through a glycine mutation at position 266. ZIKV NS5 Val 266 to Pro mutation was lethal for viral RNA replication while the Gly mutation was severely attenuated. Serial passaging of cell culture supernatant derived from C6/36 mosquito cells transfected with mutant ZIKV RNA showed that the attenuation can be rescued. Next generation deep sequencing revealed four single nucleotide polymorphisms that occur with an allele frequency >98%. The single non-synonymous NS5 mutation Glu419 to Lys is adjacent to RdRp motif G at the tip of the fingers subdomain, while the remaining three are synonymous variants at nucleotide positions 1403, 4403 and 6653 in the genome. Reverse engineering the changes into the ZIKV NS5/Val266Gly background followed by serial passaging revealed that residue 266 is under strong positive selection to revert back to Val. The interaction of the specific conformation of the NS5 linker with Val at position 266 and the RNA binding motif G region may present a potential strategy for allosteric antiviral drug development.
Asunto(s)
Antivirales/química , Metiltransferasas/química , Proteínas no Estructurales Virales/química , Replicación Viral/efectos de los fármacos , Virus Zika/enzimología , Sitio Alostérico , Animales , Línea Celular , Cricetinae , Cristalografía por Rayos X , Diseño de Fármacos , Metiltransferasas/biosíntesis , Modelos Moleculares , Unión Proteica , ARN Polimerasa Dependiente del ARN , Proteínas no Estructurales Virales/biosíntesis , Infección por el Virus ZikaRESUMEN
Alterations in global histone methylation regulate gene expression and participate in cancer onset and progression. The profile of histone methylation marks in pediatric astrocytomas is currently understudied with limited data on their distribution among grades. The global expression patterns of repressive histone marks H3K9me3, H3K27me3, and H4K20me3 and active H3K4me3 and H3K36me3 along with their writers SUV39H1, SETDB1, EZH2, MLL2, and SETD2 were investigated in 46 pediatric astrocytomas and normal brain tissues. Associations between histone marks and modifying enzymes with clinicopathological characteristics and disease-specific survival were studied along with their functional impact in proliferation and migration of pediatric astrocytoma cell lines using selective inhibitors in vitro. Upregulation of histone methyltransferase gene expression and deregulation of histone code were detected in astrocytomas compared to normal brain tissues, with higher levels of SUV39H1, SETDB1, and SETD2 as well as H4K20me3 and H3K4me3 histone marks. Pilocytic astrocytomas exhibited lower MLL2 levels compared to diffusely infiltrating tumors indicating a differential pattern of epigenetic regulator expression between the two types of astrocytic neoplasms. Moreover, higher H3K9me3, H3K36me3, and SETDB1 expression was detected in grade IIΙ/IV compared to grade II astrocytomas. In univariate analysis, elevated H3K9me3 and MLL2 and diminished SUV39H1 expression adversely affected survival. Upon multivariate survival analysis, only SUV39H1 expression was revealed as an independent prognostic factor of adverse significance. Treatment of pediatric astrocytoma cell lines with SUV39H1 inhibitor reduced proliferation and cell migration. Our data implicate H3K9me3 and SUV39H1 in the pathobiology of pediatric astrocytomas, with SUV39H1 yielding prognostic information independent of other clinicopathologic variables.
Asunto(s)
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Perfilación de la Expresión Génica , Código de Histonas/fisiología , N-Metiltransferasa de Histona-Lisina/biosíntesis , Metiltransferasas/biosíntesis , Proteínas Represoras/biosíntesis , Adolescente , Astrocitoma/diagnóstico , Astrocitoma/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Niño , Preescolar , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica/métodos , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Lactante , Masculino , Metilación , Metiltransferasas/genética , Pronóstico , Proteínas Represoras/genéticaRESUMEN
The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.
Asunto(s)
Activación de Linfocitos , Metiltransferasas/fisiología , Biosíntesis de Proteínas , Ribosomas/metabolismo , Linfocitos T/enzimología , Animales , Técnicas de Inactivación de Genes , Guanosina/metabolismo , Activación de Linfocitos/genética , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Ratones , Caperuzas de ARN/química , Caperuzas de ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , Proteínas de Unión al ARN/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Regulación hacia ArribaRESUMEN
Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determine that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A-binding-dependent manner. Importantly, we find that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.
Asunto(s)
Adenosina/análogos & derivados , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , Transcripción Genética , Estomatitis Vesicular/genética , Vesiculovirus/patogenicidad , Células A549 , Adenosina/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Antivirales/farmacología , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Interferón beta/farmacología , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcripción Genética/efectos de los fármacos , Células Vero , Estomatitis Vesicular/metabolismo , Estomatitis Vesicular/virología , Vesiculovirus/crecimiento & desarrollo , Replicación ViralRESUMEN
Protein α-N-methylation is an evolutionarily conserved type of post-translational modification; however, little is known about the regulatory mechanisms for this modification. Methylation at the N6 position of adenosine in mRNAs is dynamic and modulates their stability, splicing, and translational efficiency. Here, we found that the expression of N-terminal methyltransferase 1 (NTMT1) protein is altered by depletion of those genes encoding the reader/writer/eraser proteins of N6-methyladenosine (m6A). We also observed that MRG15 is N-terminally methylated by NTMT1, and this methylation could also be modulated by reader/writer/eraser proteins of m6A. Together, these results revealed a novel m6A-based epitranscriptomic mechanism in regulating protein N-terminal methylation.
Asunto(s)
Adenosina/análogos & derivados , Epigénesis Genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Transcriptoma , Adenosina/metabolismo , Células HEK293 , Humanos , Metilación , Metiltransferasas/biosíntesis , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Lung cancer is a common type of cancer that causes a very large public health burden worldwide. Achieving a better understanding of the molecular mechanism underlying the progression of lung cancer is of benefit for the diagnosis, prognosis, and treatment of lung cancer. Here, we first identified dramatically decreased expression of miR-338-5p in lung cancer tissues and cells using quantitative polymerase chain reaction (qPCR) analysis. We then revealed that miR-338-5p inhibited the cell growth and migration of lung cancer cells using cell counting kit 8 (CCK8), EdU, and Transwell analysis. Furthermore, we demonstrated that miR-338-5p inhibited METTL3 expression by qPCR, western blot analysis, and luciferase reporter assay, while upregulation of METTL3 alleviated the role of miR-338-5p in lung cancer cells. We also showed that METTL3 promoted c-Myc expression by increasing the m6A modification of c-Myc, and overexpression of c-Myc restored the inhibition of cell growth and migration of lung cancer cells induced by METTL3 silencing. Ultimately, this research illustrated that modification of the miR-338-5p/METTL3/c-Myc pathway affected cellular progression in lung cancer cells. Collectively, our study revealed the underlying mechanism of miR-338-5p in lung cancer, providing a novel regulatory pathway in lung cancer. There is potential for this pathway to serve as a diagnostic, prognostic, and therapeutic biomarker for lung cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Metiltransferasas/biosíntesis , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , ARN Neoplásico/metabolismo , Transducción de Señal , Células A549 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Metiltransferasas/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas c-myc/genética , ARN Neoplásico/genéticaRESUMEN
Putative methyltranferase LaeA and LaeA-like proteins, which are conserved in many filamentous fungi, regulate the sporogenesis and biosynthesis of secondary metabolites. In this study, we reported the biological function of a LaeA-like methyltransferase, Penicillium oxalicum Mtr23B, which contains a methyltransf_23 domain and an S-adenosylmethionine binding domain, in controlling spore pigment formation and in the expression of secondary metabolic gene cluster and glycoside hydrolase genes. Additionally, we compared Mtr23B and LaeA, and determined their similarities and differences in terms of their roles in regulating the above biological processes. mtr23B had the highest transcriptional level among the 12 members of the methyltransf_23 family in P. oxalicum. The colony color of Δmtr23B (deletion of mtr23B) was lighter than that of ΔlaeA, although Δmtr23B produced ~ 19.2-fold more conidia than ΔlaeA. The transcriptional levels of abrA, abrB/yA, albA/wA, arpA, arpB, and aygA, which are involved in the dihydroxynaphtalene-melanin pathway, decreased in Δmtr23B. However, Mtr23B had a little effect on brush-like structures and conidium formation, and had a different function from LaeA. Mtr23B extensively regulated glycoside hydrolase gene expression. The absence of Mtr23B remarkably repressed prominent cellulase- and amylase-encoding genes in the whole culture period, while the effect of LaeA mainly occurred in the later phases of prolonged batch cultures. Similar to LaeA, Mtr23B was involved in the expression of 10 physically linked regions containing secondary metabolic gene clusters; the highest regulatory activities of Mtr23B and LaeA were observed in BrlA-dependent cascades. Although LaeA interacted with VeA, Mtr23B did not interact with VeA directly. We assumed that Mtr23B regulates cellulase and amylase gene transcription by interacting with the CCAAT-binding transcription factor HAP5 and chromatin remodeling complex.
Asunto(s)
Proteínas Fúngicas/genética , Glicósido Hidrolasas/genética , Metiltransferasas/genética , Penicillium/genética , Regulación Fúngica de la Expresión Génica/genética , Metiltransferasas/biosíntesis , Penicillium/metabolismo , Reproducción Asexuada/genética , S-Adenosilmetionina/metabolismo , Metabolismo Secundario/genética , Esporas Fúngicas/genéticaRESUMEN
Methyltransferase-like protein 3 (METTL3) regulates multiple cell functions and diseases by modulating N6-methyladenosine (m6A) modifications. However, it is still unclear whether METTL3 involves in the pathogenesis of diabetic retinopathy (DR). In the present study, we found that high-glucose inhibited RPE cell proliferation, promoted cell apoptosis and pyroptosis in a time-dependent manner. In addition, both METTL3 mRNA and miR-25-3p were low-expressed in the peripheral venous blood samples of diabetes mellitus (DM) patients compared to normal volunteers, and high-glucose inhibited METTL3 and miR-25-3p expressions in RPE cells. As expected, upregulation of METTL3 and miR-25-3p alleviated the cytotoxic effects of high-glucose on RPE cells, and knock-down of METTL3 and miR-25-3p had opposite effects. Additionally, METTL3 overexpression increased miR-25-3p levels in RPE cells in a microprocessor protein DGCR8-dependent manner, and miR-25-3p ablation abrogated the effects of overexpressed METTL3 on cell functions in high-glucose treated RPE cells. Furthermore, PTEN could be negatively regulated by miR-25-3p, and overexpression of METTL3 increased phosphorylated Akt (p-Akt) levels by targeting miR-25-3p/PTEN axis. Consistently, upregulation of PTEN abrogated the protective effects of METTL3 overexpression on RPE cells treated with high-glucose. Collectively, METTL3 rescued cell viability in high-glucose treated RPE cells by targeting miR-25-3p/PTEN/Akt signaling cascade.
Asunto(s)
Retinopatía Diabética/genética , Regulación de la Expresión Génica , Metiltransferasas/genética , MicroARNs/genética , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas c-akt/genética , Epitelio Pigmentado de la Retina/metabolismo , Apoptosis , Proliferación Celular , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Glucosa/farmacología , Humanos , Metiltransferasas/biosíntesis , MicroARNs/biosíntesis , Fosfohidrolasa PTEN/biosíntesis , Subunidades de Proteína , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Piroptosis , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Proteínas Supresoras de TumorRESUMEN
Human WBSCR22 is involved in cancer proliferation, invasion and metastasis; however, its function in glioma remains unexplored. In our research, we aimed to investigate the role of WBSCR22 in the development of glioma and its possible molecular mechanisms. Using bioinformatic analysis of public datasets, we determined that WBSCR22 overexpression in glioma specimens was correlated with an unfavorable patient prognosis. Our results revealed that WBSCR22 was highly expressed in glioma cell lines. The loss of WBSCR22 inhibited the growth, invasion and migration of glioma cells, while WBSCR22 overexpression produced the opposite effects. Moreover, we found that WBSCR22 downregulation reduced the phosphorylation of Akt and GSK3ß and decreased the levels of ß-catenin and CyclinD1 in glioma cells. The opposite effects were observed when WBSCR22 was overexpressed. Additionally, we verified with a dual-luciferase reporter assay that WBSCR22 was a direct target of miR-146b-5p. Furthermore, overexpression of miR-146b-5p suppressed WBSCR22 mRNA and protein expression. Notably, the restoration of WBSCR22 expression remarkably reversed the effects of miR-146b-5p overexpression on cell survival, apoptosis and the cell cycle in glioma cells. Collectively, our findings revealed a tumor-promoting role for WBSCR22 in glioma cells, thus providing molecular evidence for WBSCR22 as a novel therapeutic target in glioma.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Glioma/metabolismo , Glioma/mortalidad , Metiltransferasas/biosíntesis , Anciano , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Supervivencia Celular/fisiología , Femenino , Glioma/diagnóstico , Glioma/genética , Humanos , Masculino , Metiltransferasas/genética , Persona de Mediana Edad , Pronóstico , Tasa de Supervivencia/tendenciasRESUMEN
Macarpine is a minor benzophenanthridine alkaloid with interesting biological activities, which is produced in only a few species of the Papaveraceae family, including Eschscholzia californica. Our present study was focused on the enhancement of macarpine production in E. californica suspension cultures using three elicitation models: salicylic acid (SA) (4; 6; 8 mg/L) elicitation, and simultaneous or sequential combinations of SA and L-tyrosine (1 mmol/L). Sanguinarine production was assessed along with macarpine formation in elicited suspension cultures. Alkaloid production was evaluated after 24, 48 and 72 h of elicitation. Among the tested elicitation models, the SA (4 mg/L), supported by L-tyrosine, stimulated sanguinarine and macarpine production the most efficiently. While sequential treatment led to a peak accumulation of sanguinarine at 24 h and macarpine at 48 h, simultaneous treatment resulted in maximum sanguinarine accumulation at 48 h and macarpine at 72 h. The effect of SA elicitation and precursor supplementation was evaluated also based on the gene expression of 4'-OMT, CYP719A2, and CYP719A3. The gene expression of investigated enzymes was increased at all used elicitation models and their changes correlated with sanguinarine but not macarpine accumulation.
Asunto(s)
Benzofenantridinas/biosíntesis , Eschscholzia/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/farmacología , Ácido Salicílico/farmacología , Tirosina/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Relación Dosis-Respuesta a Droga , Eschscholzia/genética , Eschscholzia/crecimiento & desarrollo , Eschscholzia/metabolismo , Regulación de la Expresión Génica de las Plantas , Hidroponía/métodos , Isoquinolinas , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Proteínas de Plantas/agonistas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tirosina/metabolismoRESUMEN
Pyocyanin produced by Pseudomonas aeruginosa is a key virulence factor that often causes heavy damages to airway and lung in patients. Conversion of phenazine-1-carboxylic acid to pyocyanin involves an extrametabolic pathway that contains two enzymes encoded, respectively, by phzM and phzS. In this study, with construction of the rpoS-deficient mutant, we first found that although phenazine production increased, pyocyanin produced in the mutant YTΔrpoS was fourfold much higher than that in the wild-type strain YT. To investigate this issue, we constructed phzM-lacZ fusion on a vector and on the chromosome. By quantifying ß-galactosidase activities, we confirmed that expression of the phzM was up-regulated when the rpoS gene was inactivated. However, no changes occurred in the expression of phzS and phzH when the rpoS was knocked out. Taken together, overproduction of the SAM-dependent methyltransferase (PhzM) might contribute to the increased pyocyanin in the absence of RpoS in P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Metiltransferasas/biosíntesis , Oxigenasas de Función Mixta/biosíntesis , Pseudomonas aeruginosa/metabolismo , Piocianina/biosíntesis , Factor sigma/genética , Humanos , Metiltransferasas/genética , Oxigenasas de Función Mixta/genética , Fenazinas/metabolismo , Pseudomonas aeruginosa/genética , Factores de Virulencia/metabolismoRESUMEN
Pancreatic cancer is a leading cause of cancer-related death worldwide. Cisplatin is an essential drug treating patients with BRCA1/2 or PALB2 mutations. Whether other genetic determinants of cisplatin sensitivity exist and their underlying mechanisms remain unclear. Immunohistochemistry was used to determine METTL14 expression in pancreatic cancer tissues and non-tumoural tissues. Cell proliferation was detected with CCK-8 assays. Apoptosis was analysed via Western blotting and flow cytometry, and autophagy was analysed via Western blotting and immunofluorescence. In this work, we found higher METTL14 expression in pancreatic cancer tissues than in non-tumoural tissues, and METTL14 expression was associated with pathological characteristics. Downregulation of METTL14 with siRNA sensitized pancreatic cancer cells to cisplatin. Specifically, apoptosis and autophagy were significantly enhanced in METT14 knockdown cells compared with control cells after treatment with cisplatin. Mechanistically, the AMPKα, ERK1/2 and mTOR signalling pathways were disturbed by downregulation of METTL14. We further found that METTL14 knockdown-mediated autophagy was dependent on mTOR signalling and that mTOR activation decreased autophagy to the level observed in the control group. Collectively, our results indicate that METTL14 is upregulated in pancreatic cancer, downregulation of METTL14 sensitizes pancreatic cancer cells to cisplatin by enhancing apoptosis, and autophagy is improved via an mTOR signalling-dependent pathway.
Asunto(s)
Cisplatino/farmacología , Metiltransferasas/biosíntesis , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/fisiología , Regulación hacia Abajo , Humanos , Metiltransferasas/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Transducción de Señal , Transfección , Regulación hacia ArribaRESUMEN
Our study is based on the establishment of a cohort of human obese omental adipose tissue and the culture of adipocytes in vitro. To observe the effect of high level of free fatty acid (FFA) on the expression of DNA methyltransferases (DNMTs) and the anti-inflammatory factor Kruppel-like factor 4 (KLF4) in adipocytes and evaluate the role of methyltransferases in FFA inhibiting KLF4 expression. A total of 20 normal patients and 20 obese patients were selected for further test. qRT-PCR and western blot were used to detect the mRNA and protein expression levels of DNMT1/DNMT3a/DNMT3b and KLF4 in human adipose tissue and 3T3-L1 adipocytes which stimulated with saturated fatty acid, palmitic acid (PA). Bisulfite sequencing PCR (BSP) detected methylation status of KLF4 gene in human adipose tissue. It was found that the mRNA and protein expression levels of DNMT1 and DNMT3a in the omental tissue of obese individuals were higher than those in normal group, but the expression of KLF4 was decreased. The positive methylation rate of KLF4 promoter region in obese individuals were significantly higher than those in normal individuals, especially at CpG_33 and CpG_34 sites. Meanwhile compared with non-methylated group at CpG_33 and CpG_34 sites of KLF4 promoter region, the DNMT3a mRNA expression in methylated group were significantly increased. A total of 200 µM PA significantly promoted DNMT1, DNMT3a, and DNMT3b and inhibited KLF4 protein expression levels in 3T3-L1 adipocytes. Our findings suggest that under obesity status, the lower expression level of KLF4 of visceral adipose tissue may correlate with palmitic acid promoted DNMTs expression in adipocytes.
Asunto(s)
Tejido Adiposo/metabolismo , Regulación Enzimológica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/biosíntesis , Metiltransferasas/biosíntesis , Obesidad/metabolismo , Ácido Palmítico/farmacología , Células 3T3 , Tejido Adiposo/efectos de los fármacos , Adulto , Animales , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/genética , Ratones , Persona de Mediana Edad , Obesidad/genética , Adulto JovenRESUMEN
Bacteria have evolved a defense system to resist external stressors, such as heat, pH, and salt, so as to facilitate survival in changing or harsh environments. However, the specific mechanisms by which bacteria respond to such environmental changes are not completely elucidated. Here, we used halotolerant bacteria as a model to understand the mechanism conferring high tolerance to NaCl. We screened for genes related to halotolerance in Halomonas socia, which can provide guidance for practical application. Phospholipid fatty acid analysis showed that H. socia cultured under high osmotic pressure produced a high portion of cyclopropane fatty acid derivatives, encoded by the cyclopropane-fatty acid-acyl phospholipid synthase gene (cfa). Therefore, H. socia cfa was cloned and introduced into Escherichia coli for expression. The cfa-overexpressing E. coli strain showed better growth, compared with the control strain under normal cultivation condition as well as under osmotic pressure (> 3% salinity). Moreover, the cfa-overexpressing E. coli strain showed 1.58-, 1.78-, 3.3-, and 2.19-fold higher growth than the control strain in the presence of the inhibitors furfural, 4-hydroxybenzaldehyde, vanillin, and acetate from lignocellulosic biomass pretreatment, respectively. From a practical application perspective, cfa was co-expressed in E. coli with the polyhydroxyalkanoate (PHA) synthetic operon of Ralstonia eutropha using synthetic and biosugar media, resulting in a 1.5-fold higher in PHA production than that of the control strain. Overall, this study demonstrates the potential of the cfa gene to boost cell growth and production even in heterologous strains under stress conditions.
Asunto(s)
Proteínas Bacterianas , Escherichia coli , Expresión Génica , Metiltransferasas , Microorganismos Modificados Genéticamente , Presión Osmótica/efectos de los fármacos , Cloruro de Sodio/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Cupriavidus necator/enzimología , Cupriavidus necator/genética , Escherichia coli/enzimología , Escherichia coli/genética , Halomonas/enzimología , Halomonas/genética , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Microorganismos Modificados Genéticamente/enzimología , Microorganismos Modificados Genéticamente/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA. It has been reported that there is a stimulus-dependent regulation of m6A in the mammalian central nervous system in response to sensory experience, learning, and injury. The mRNA m6A methylation pattern in rat cortex after traumatic brain injury (TBI) has not been investigated. RESULTS: In this study, we conducted a genome-wide profiling of mRNA m6A methylation in rat cortex via methylated RNA immunoprecipitation sequencing (MeRIP-Seq). After TBI, the expressions of METTL14 and FTO were significantly down-regulated in rat cerebral cortex. Using MeRIP-Seq, we identified a total of 2165 significantly changed peaks, of which 1062 were significantly up-regulated and 1103 peaks were significantly down-regulated. These m6A peaks were located across 1850 genes. The analysis of both m6A peaks and mRNA expression revealed that there were 175 mRNA significantly altered methylation and expression levels after TBI. Moreover, it was found that functional FTO is necessary to repair neurological damage caused by TBI but has no effect on the spatial learning and memory abilities of TBI rats by using FTO inhibitor FB23-2. CONCLUSION: This study explored the m6A methylation pattern of mRNA after TBI in rat cortex and identified FTO as possible intervention targets in the epigenetic modification of TBI.
Asunto(s)
Adenosina/análogos & derivados , Lesiones Traumáticas del Encéfalo/genética , Corteza Cerebral/metabolismo , Epigénesis Genética , Procesamiento Postranscripcional del ARN , Adenosina/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/biosíntesis , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Animales , Regulación hacia Abajo , Regulación de la Expresión Génica , Ontología de Genes , Inmunoprecipitación , Masculino , Metilación , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVES: To characterize methyltransferases involved in the biosynthesis of benzylisoquinoline alkaloids in Stephania intermedia. RESULTS: Three N-methyltransferases, SiCNMT1, SiCNMT2, SiCNMT3, and O-methyltransferase SiSOMT were identified in Stephania intermedia. Then, four methyltransferase genes were cloned into the pGEX-6P-1 vector. The recombinant vectors were transformed into Escherichia coli BL21(DE3) for expression and were functionally tested. SiCNMT1, SiCNMT2, and SiCNMT3 could methylate (R)-coclaurine to produce (R)-N-methylcoclaurine. SiCNMT2 further methylated the product of (R)-N-methylcoclaurine to produce (R)-magnocurarine. Similarly, (R)-norcoclaurine was continuously catalyzed to yield (R)-N-methylnorcoclaurine and (R)-N, N-dimethylnorcoclaurine by SiCNMT2. Furthermore, SiSOMT was shown to catalyze the conversion of (S)-scoulerine to (S)-tetrahydropalmatine. CONCLUSIONS: The key methyltransferases, which were in the last step biosynthesis of (R)-magnocurarine, (R)-N, N-dimethylnorcoclaurine and (S)-tetrahydropalmatine were revealed and their activities were verified in vitro. Four novel methyltransferases will be promising candidates for methylation of benzylisoquinoline alkaloids.
Asunto(s)
Bencilisoquinolinas/química , Metiltransferasas/química , Proteínas de Plantas/química , Stephania/enzimología , Bencilisoquinolinas/metabolismo , Clonación Molecular , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Stephania/genéticaRESUMEN
Epstein-Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers.
Asunto(s)
Transformación Celular Viral , Infecciones por Virus de Epstein-Barr/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Metiltransferasas/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neoplasias/metabolismo , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Animales , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Células HEK293 , Humanos , Masculino , Metiltransferasas/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Neoplasias/virologíaRESUMEN
Melatonin (Mel) serves as an important signalling molecule in various aspects of stress tolerance in plants. However, the function of Mel in pesticide metabolism remains unknown. Here, selecting the widely used fungicide carbendazim (MBC) as the model, we found that exogenous Mel had the ability to alleviate pesticide phytotoxicity and residues in tomato as well as in some other vegetables. Additionally, overexpression of the Mel biosynthetic gene caffeic acid O-methyltransferase 1 (COMT1) significantly enhanced the capacity of the tomato to reduce MBC phytotoxicity and residue. This outcome was mainly because of the Mel-induced antioxidant capability, as well as the key detoxification process. Indeed, levels of reactive oxygen species (ROS) and lipid peroxides significantly decreased after applying exogenous Mel or overexpressing COMT1, which resulted from direct ROS scavenging, and increased Mel levels significantly enhanced antioxidant enzymatic activity. More importantly, Mel activated the ascorbate-glutathione cycle to participate in glutathione S-transferase-mediated pesticide detoxification. A grafting experiment showed that rootstocks from COMT1 transgenic plants increased the Mel accumulation of wild-type scions, resulting in MBC metabolism in the scions. To our knowledge, this is the first report providing evidence of Mel-induced pesticide metabolism, which provides a novel approach for minimizing pesticide residues in crops by exploiting plant self-detoxification mechanisms.
Asunto(s)
Bencimidazoles/metabolismo , Carbamatos/metabolismo , Fungicidas Industriales/metabolismo , Inactivación Metabólica/fisiología , Melatonina/metabolismo , Metiltransferasas/metabolismo , Solanum lycopersicum/metabolismo , Bencimidazoles/toxicidad , Carbamatos/toxicidad , Fungicidas Industriales/toxicidad , Glutatión Transferasa/metabolismo , Peróxidos Lipídicos/metabolismo , Melatonina/biosíntesis , Metiltransferasas/biosíntesis , Plantas Modificadas Genéticamente/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Melatonin can increase plant resistance to stress, and exogenous melatonin has been reported to promote stress resistance in plants. In this study, a melatonin biosynthesis-related SlCOMT1 gene was cloned from tomato (Solanum lycopersicum Mill. cv. Ailsa Craig), which is highly expressed in fruits compared with other organs. The protein was found to locate in the cytoplasm. Melatonin content in SlCOMT1 overexpression transgenic tomato plants was significantly higher than that in wild-type plants. Under 800 mM NaCl stress, the transcript level of SlCOMT1 in tomato leaf was positively related to the melatonin contents. Furthermore, compared with that in wild-type plants, levels of superoxide and hydrogen peroxide were lower while the content of proline was higher in SlCOMT1 transgenic tomatoes. Therefore, SlCOMT1 was closely associated with melatonin biosynthesis confers the significant salt tolerance, providing a clue to cope with the growing global problem of salination in agricultural production.
Asunto(s)
Melatonina , Metiltransferasas , Proteínas de Plantas , Plantas Modificadas Genéticamente , Estrés Salino , Tolerancia a la Sal , Solanum lycopersicum , Frutas/enzimología , Frutas/genética , Peróxido de Hidrógeno/metabolismo , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Melatonina/biosíntesis , Melatonina/genética , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genéticaRESUMEN
The transition from vegetative to reproductive growth is a key developmental event in a plant's life cycle. The process is mediated by a combination of phytohormones, including melatonin (MT) and strigolactone (SL). Here, the Arabidopsis mutants, d14-1 and max4-1, which are compromised with respect to either SL synthesis or signaling, were shown to flower earlier than wild types. The tissue MT content in both mutants was higher than in wild types, as a result of the up-regulation of various genes encoding enzymes involved in MT synthesis. The abundance in the mutants of transcripts derived from each of the genes SPLs, AP1, and SOC1 was reduced with exogenously supplied MT, while FLC was induced. Plants exposed to a high concentration of MT did not flower earlier than wild types. The tissue MT content of a mutant unable to synthesize caffeic acid O-methyltransferase was less than that of wild type and flowered earlier than did wild types. The suggestion is that the flowering time of Arabidopsis is altered if the tissue content of MT is either higher than ~ 8 ng/g F.W, or lower than ~ 0.9 ng/g. Within this range, SL acts to determine flowering time by its regulation of SPL genes. The application of exogenous SL reduces tissue MT content. The flowering time of the flc-3 mutant was unaffected by exogenously supplying either MT or/and SL. It is proposed that MT acts downstream of SL to activate FLC, inducing a delay to flowering if its concentration lies outside a certain range.