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1.
J Agric Food Chem ; 67(31): 8573-8580, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31293156

RESUMO

Glycosylation endows both natural and synthetic small molecules with modulated physicochemical and biological properties. Plant and bacterial glycosyltransferases capable of decorating various privileged scaffolds have been extensively studied, but those from kingdom Fungi still remain underexploited. Here, we use a combination of genome mining and heterologous expression techniques to identify four novel glycosyltransferase-methyltransferase (GT-MT) functional modules from Hypocreales fungi. These GT-MT modules display decent substrate promiscuity and regiospecificity, methylglucosylating a panel of natural products such as flavonoids, stilbenoids, anthraquinones, and benzenediol lactones. Native GT-MT modules can be split up and regrouped into hybrid modules with similar or even improved efficacy as compared with native pairs. Methylglucosylation of kaempferol considerably improves its insecticidal activity against the larvae of oriental armyworm Mythimna separata (Walker). Our work provides a set of efficient biocatalysts for the combinatorial biosynthesis of small molecule glycosides that may have significant importance to the pharmaceutical, agricultural, and food industries.


Assuntos
Proteínas Fúngicas/química , Glicosiltransferases/química , Hypocreales/enzimologia , Metiltransferases/química , Fenóis/química , Animais , Biocatálise , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hypocreales/genética , Inseticidas/química , Inseticidas/farmacologia , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mariposas/efeitos dos fármacos , Fenóis/farmacologia , Especificidade por Substrato
2.
Nat Commun ; 10(1): 2550, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186410

RESUMO

The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (m5C) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of m5C deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for m5C in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation.


Assuntos
5-Metilcitosina/metabolismo , Metilação de DNA , Células Epidérmicas/citologia , Metiltransferases/metabolismo , RNA/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Diferenciação Celular , Linhagem Celular , Citosina/metabolismo , Células Epidérmicas/metabolismo , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Humanos , Metiltransferases/genética , RNA/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo
3.
BMC Plant Biol ; 19(1): 277, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234776

RESUMO

BACKGROUND: The Asia lotus (Nelumbo nucifera Gaertn.) is an ornamental aquatic plant with high economic value. Flower colour is an important ornamental trait, with much of N. nucifera breeding focusing on its yellow flowers. To explore the yellow flower colouration mechanism in N. nucifera, we analysed its pigment constituents and content, as well as gene expression in the flavonoid pathway, in two N. nucifera cultivars. RESULTS: We performed metabolomic and gene expression analyses in two N. nucifera cultivars with yellow and white flowers, Molinqiuse (MLQS) and Yeguangbei (YGB), respectively, at five stages of flower colouration. Based on phenotypic observation and metabolite analyses, the later stages of flower colouration (S3-S5) were determined to be key periods for differences between MLQS and YGB, with dihydroflavonols and flavonols differing significantly between cultivars. Dihydroquercetin, dihydrokaempferol, and isorhamnetin were significantly higher in MLQS than in YGB, whereas kaempferol was significantly higher in YGB. Most of the key homologous structural genes in the flavonoid pathway were significantly more active in MLQS than in YGB at stages S1-S4. CONCLUSION: In this study, we performed the first analyses of primary and secondary N. nucifera metabolites during flower colouration, and found that isorhamnetin and kaempferol shunting resulted in petal colour differences between MLQS and YGB. Based on our data integration analyses of key enzyme expression in the putative flavonoid pathways of the two N. nucifera cultivars, NnFLS gene substrate specificity and differential expression of NnOMTs may be related to petal colour differences between MLQS and YGB. These results will contribute to determining the mechanism of yellow flower colouration in N. nucifera, and will improve yellow petal colour breeding in lotus species.


Assuntos
Flavonoides/metabolismo , Flores/genética , Nelumbo/metabolismo , Pigmentação/genética , Perfilação da Expressão Gênica , Genes de Plantas , Quempferóis/metabolismo , Metaboloma , Metiltransferases/genética , Nelumbo/enzimologia , Nelumbo/genética , Quercetina/análogos & derivados , Quercetina/metabolismo , Especificidade da Espécie
4.
Nat Commun ; 10(1): 2803, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243293

RESUMO

Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to demarcate potentially active enhancers. Here we reveal that putative enhancers marked with H3 lysine 79 (H3K79) di or trimethylation (me2/3) (which we name H3K79me2/3 enhancer elements or KEEs) can be found in multiple cell types. Mixed lineage leukemia gene (MLL) rearrangements (MLL-r) such as MLL-AF4 are a major cause of incurable acute lymphoblastic leukemias (ALL). Using the DOT1L inhibitor EPZ-5676 in MLL-AF4 leukemia cells, we show that H3K79me2/3 is required for maintaining chromatin accessibility, histone acetylation and transcription factor binding specifically at KEEs but not non-KEE enhancers. We go on to show that H3K79me2/3 is essential for maintaining enhancer-promoter interactions at a subset of KEEs. Together, these data implicate H3K79me2/3 as having a functional role at a subset of active enhancers in MLL-AF4 leukemia cells.


Assuntos
Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Metiltransferases/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Estudo de Associação Genômica Ampla , Histonas/genética , Humanos , Metilação , Metiltransferases/genética
5.
J Microbiol Biotechnol ; 29(6): 839-844, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154751

RESUMO

Anthranilate derivatives have been used as flavoring and fragrant agents for a long time. Recently, these compounds are gaining attention due to new biological functions including antinociceptive and analgesic activities. Three anthranilate derivatives, N-methylanthranilate, methyl anthranilate, and methyl N-methylanthranilate were synthesized using metabolically engineered stains of Escherichia coli. NMT encoding N-methyltransferase from Ruta graveolens, AMAT encoding anthraniloyl-coenzyme A (CoA):methanol acyltransferase from Vitis labrusca, and pqsA encoding anthranilate coenzyme A ligase from Pseudomonas aeruginosa were cloned and E. coli strains harboring these genes were used to synthesize the three desired compounds. E. coli mutants (metJ, trpD, tyrR mutants), which provide more anthranilate and/or S-adenosyl methionine, were used to increase the production of the synthesized compounds. MS/MS analysis was used to determine the structure of the products. Approximately, 185.3 µM N-methylanthranilate and 95.2 µM methyl N-methylanthranilate were synthesized. This is the first report about the synthesis of anthranilate derivatives in E. coli.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , ortoaminobenzoatos/metabolismo , Vias Biossintéticas , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/metabolismo , Ruta/enzimologia , Ruta/genética , Vitis/enzimologia , Vitis/genética , ortoaminobenzoatos/química
6.
Nat Commun ; 10(1): 2147, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089132

RESUMO

Cancer-relevant signalling pathways rely on bidirectional nucleocytoplasmic transport events through the nuclear pore complex (NPC). However, mechanisms by which individual NPC components (Nups) participate in the regulation of these pathways remain poorly understood. We discover by integrating large scale proteomics, polysome fractionation and a focused RNAi approach that Nup155 controls mRNA translation of p21 (CDKN1A), a key mediator of the p53 response. The underlying mechanism involves transcriptional regulation of the putative tRNA and rRNA methyltransferase FTSJ1 by Nup155. Furthermore, we observe that Nup155 and FTSJ1 are p53 repression targets and accordingly find a correlation between the p53 status, Nup155 and FTSJ1 expression in murine and human hepatocellular carcinoma. Our data suggest an unanticipated regulatory network linking translational control by and repression of a structural NPC component modulating the p53 pathway through its effectors.


Assuntos
Carcinoma Hepatocelular/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Neoplasias Hepáticas/patologia , Metiltransferases/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Metiltransferases/metabolismo , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo
7.
Nat Commun ; 10(1): 2065, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061416

RESUMO

N6-Methyladenosine (m6A) modification has been implicated in the progression of several cancers. We reveal that during epithelial-mesenchymal transition (EMT), one important step for cancer cell metastasis, m6A modification of mRNAs increases in cancer cells. Deletion of methyltransferase-like 3 (METTL3) down-regulates m6A, impairs the migration, invasion and EMT of cancer cells both in vitro and in vivo. m6A-sequencing and functional studies confirm that Snail, a key transcription factor of EMT, is involved in m6A-regulated EMT. m6A in Snail CDS, but not 3'UTR, triggers polysome-mediated translation of Snail mRNA in cancer cells. Loss and gain functional studies confirm that YTHDF1 mediates m6A-increased translation of Snail mRNA. Moreover, the upregulation of METTL3 and YTHDF1 act as adverse prognosis factors for overall survival (OS) rate of liver cancer patients. Our study highlights the critical roles of m6A on regulation of EMT in cancer cells and translation of Snail during this process.


Assuntos
Adenosina/análogos & derivados , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas/genética , RNA/metabolismo , Fatores de Transcrição da Família Snail/genética , Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Fígado/patologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Análise Serial de Tecidos , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
8.
BMC Plant Biol ; 19(1): 208, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31109298

RESUMO

BACKGROUND: Cotton fiber is a single cell that arises from the epidermis of ovule. It is not only a main economic product of cotton, but an ideal material for studying on the growth and development of plant cell. Our previous study indicated that phytosterol content and the ratio of campesterol to sitosterol fluctuated regularly in cotton fiber development. However, what effects of modified phytosterol content and composition on the growth and development of cotton fiber cell is unknown. In this study, we overexpressed the GhSMT2-1, a cotton homologue of sterol C-24 methyltransferase 2 gene in transgenic upland cotton plants to modify phytosterol content and composition in fiber cells and investigated the changes on fiber elongation and secondary cell wall deposition. RESULTS: GhSMT2-1 overexpression led to changes of phytosterol content and the ratio of campesterol to sitosterol in fiber cell. At the rapid elongation stage of fiber cell, total phytosterol and sitosterol contents were increased while campesterol content was decreased in transgenic fibers when compared to control fibers. Accordingly, the ratio of campesterol to sitosterol declined strikingly. Simultaneously, the transgenic fibers were shorter and thicker than control fibers. Exogenous application of sitosterol or campesterol separately inhibited control fiber cell elongation in cotton ovule culture system in vitro. In addition, campesterol treatment partially rescued transgenic fiber elongation. CONCLUSION: These results elucidated that modification of phytosterol content and composition influenced fiber cell elongation and secondary cell wall formation. High sitosterol or low ratio of campesterol to sitosterol suppresses fiber elongation and/or promote secondary cell wall deposition. The roles of sitosterol and campesterol were discussed in fiber cell development. There might be a specific ratio of campesterol to sitosterol in different developmental stage of cotton fibers, in which GhSMT2-1 play an important role. Our study, at a certain degree, provides novel insights into the regulatory mechanisms of fiber cell development.


Assuntos
Gossypium/química , Gossypium/fisiologia , Fitosteróis/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Crescimento Celular , Parede Celular , Fibra de Algodão , Gossypium/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia
9.
Nat Cell Biol ; 21(6): 700-709, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061465

RESUMO

Haematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation, but how these functions are precisely regulated is not fully understood. N6-methyladenosine (m6A) messenger RNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deletion of the m6A methyltransferase Mettl3 from the adult haematopoietic system led to an accumulation of HSCs in the bone marrow and a marked reduction of reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not impact myeloid cell number or function. RNA sequencing revealed 2,073 genes with significant m6A modifications in HSCs. Myc was identified as a direct target of m6A in HSCs. Mettl3-deficient HSCs failed to upregulate MYC expression following stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results reveal a key role of m6A in governing HSC differentiation.


Assuntos
Adenosina/análogos & derivados , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adenosina/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Hematopoéticas/metabolismo , Metilação , Camundongos , RNA Mensageiro/genética , Análise de Sequência de RNA
10.
Nat Commun ; 10(1): 1898, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015515

RESUMO

N6-methyladenosine (m6A) modification plays important roles in various cellular responses by regulating mRNA biology. However, how m6A modification is involved in innate immunity via affecting the translation of immune transcripts remains to be further investigated. Here we report that RNA methyltransferase Mettl3-mediated mRNA m6A methylation promotes dendritic cell (DC) activation and function. Specific depletion of Mettl3 in DC resulted in impaired phenotypic and functional maturation of DC, with decreased expression of co-stimulatory molecules CD40, CD80 and cytokine IL-12, and reduced ability to stimulate T cell responses both in vitro and in vivo. Mechanistically, Mettl3-mediated m6A of CD40, CD80 and TLR4 signaling adaptor Tirap transcripts enhanced their translation in DC for stimulating T cell activation, and strengthening TLR4/NF-κB signaling-induced cytokine production. Our findings identify a new role for Mettl3-mediated m6A modification in increasing translation of certain immune transcripts for physiological promotion of DC activation and DC-based T cell response.


Assuntos
Adenosina/análogos & derivados , Células Dendríticas/imunologia , Epigênese Genética , Metiltransferases/genética , RNA Mensageiro/genética , Linfócitos T/imunologia , Adenosina/imunologia , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Antígenos/farmacologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Interleucina-12/genética , Interleucina-12/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Metilação , Metiltransferases/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , Peptídeos/imunologia , Peptídeos/farmacologia , Cultura Primária de Células , Biossíntese de Proteínas , RNA Mensageiro/imunologia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia
11.
Nat Commun ; 10(1): 1858, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015415

RESUMO

N6-methyladenosine (m6A) modification is an important mechanism in miRNA processing and maturation, but the role of its aberrant regulation in human diseases remained unclear. Here, we demonstrate that oncogenic primary microRNA-25 (miR-25) in pancreatic duct epithelial cells can be excessively maturated by cigarette smoke condensate (CSC) via enhanced m6A modification that is mediated by NF-κB associated protein (NKAP). This modification is catalyzed by overexpressed methyltransferase-like 3 (METTL3) due to hypomethylation of the METTL3 promoter also caused by CSC. Mature miR-25, miR-25-3p, suppresses PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), resulting in the activation of oncogenic AKT-p70S6K signaling, which provokes malignant phenotypes of pancreatic cancer cells. High levels of miR-25-3p are detected in smokers and in pancreatic cancers tissues that are correlated with poor prognosis of pancreatic cancer patients. These results collectively indicate that cigarette smoke-induced miR-25-3p excessive maturation via m6A modification promotes the development and progression of pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/patologia , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Neoplasias Pancreáticas/patologia , Fumaça/efeitos adversos , Tabaco/toxicidade , Adenosina/análogos & derivados , Adenosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/etiologia , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Proteínas Correpressoras/metabolismo , Metilação de DNA , Progressão da Doença , Células Epiteliais/patologia , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Metiltransferases/genética , MicroRNAs/sangue , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Ductos Pancreáticos/citologia , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/mortalidade , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Prognóstico , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fumar/efeitos adversos , Fumar/sangue , Regulação para Cima
12.
Int J Mol Sci ; 20(7)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934718

RESUMO

Methoxylated coumarins represent a large proportion of officinal value coumarins while only one enzyme specific to bergaptol O-methylation (BMT) has been identified to date. The multiple types of methoxylated coumarins indicate that at least one unknown enzyme participates in the O-methylation of other hydroxylated coumarins and remains to be identified. Combined transcriptome and metabonomics analysis revealed that an enzyme similar to caffeic acid O-methyltransferase (COMT-S, S is short for similar) was involved in catalyzing all the hydroxylated coumarins in Peucedanum praeruptorum. However, the precise molecular mechanism of its substrate heterozygosis remains unsolved. Pursuing this question, we determined the crystal structure of COMT-S to clarify its substrate preference. The result revealed that Asn132, Asp271, and Asn325 govern the substrate heterozygosis of COMT-S. A single mutation, such as N132A, determines the catalytic selectivity of hydroxyl groups in esculetin and also causes production differences in bergapten. Evolution-based analysis indicated that BMT was only recently derived as a paralogue of caffeic acid O-methyltransferase (COMT) via gene duplication, occurring before the Apiaceae family divergence between 37 and 100 mya. The present study identified the previously unknown O-methylation steps in coumarin biosynthesis. The crystallographic and mutational studies provided a deeper understanding of the substrate preference, which can be used for producing specific O-methylation coumarins. Moreover, the evolutionary relationship between BMT and COMT-S was clarified to facilitate understanding of evolutionary events in the Apiaceae family.


Assuntos
Apiaceae/metabolismo , Vias Biossintéticas , Cumarínicos/metabolismo , Sequência de Aminoácidos , Apiaceae/química , Apiaceae/genética , Cumarínicos/química , Mineração de Dados , Evolução Molecular , Furocumarinas/química , Furocumarinas/metabolismo , Duplicação Gênica , Heterozigoto , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/análise , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , Análise de Sequência de RNA , Especificidade por Substrato , Transcriptoma/genética , Umbeliferonas/química , Umbeliferonas/metabolismo
13.
BMC Cancer ; 19(1): 326, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953473

RESUMO

BACKGROUND: Breast cancer (BC) displays striking genetic, epigenetic and phenotypic diversity. N6-methyladenosine (m6A) in mRNA has emerged as a crucial epitranscriptomic modification that controls cancer self-renewal and cell fate. However, the key enzymes of m6A expression and function in human breast carcinogenesis remain unclear. METHODS: The expression of m6A methylases (METTL3, METTL14 and WTAP) and demethylases (FTO and ALKBH5) were analyzed by using ONCOMINE and The Cancer Genome Atlas databases and in 36 pairs of BC and adjacent non-cancerous tissue. The level of m6A in BC patients was detected by ELISA, and the function of m6A was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and transwell assay. The database of bc-GenExMiner v4.0, Kaplan-Meier Plotter and cBioPortal were queried for correlation, mutation and prognosis analysis of BC. RESULTS: The m6A methylases and demethylases were dysregulated in several major malignant tumors. Specifically, the expression of all m6A methylases was reduced in BC as compared with normal controls, but the demethylase ALKBH5 was induced in ONCOMINE databases and confirmed in clinical patients. METTL14 expression was positively correlated with METTL3 expression, and both showed high expression in normal breast-like and luminal-A and -B BC. Functionally, reducing m6A expression by overexpressing METTL14 and/or knockdown of ALKBH5 could inhibit breast cell viability, colony formation and cell migration. Furthermore, Kaplan-Meier, meta-analysis and univariate Cox assay showed that the expression of m6A members including METTL3, METTL14, WTAP and FTO but not their gene mutation and amplification, was tightly associated with cancer progression and poor survival. CONCLUSIONS: Changes of m6A modulators reduced m6A may promote tumorigenesis and predict poor prognosis in BC.


Assuntos
Adenosina/análogos & derivados , Neoplasias da Mama/patologia , Metiltransferases/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Adenosina/metabolismo , Adulto , Mama/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Carcinogênese/genética , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Progressão da Doença , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metilação , Metiltransferases/genética , Pessoa de Meia-Idade , Mutação , Oxirredutases N-Desmetilantes/genética , Prognóstico , RNA Mensageiro/metabolismo
14.
Molecules ; 24(8)2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999664

RESUMO

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.


Assuntos
Lycopersicon esculentum , Melatonina , Metiltransferases , Proteínas de Plantas , Plantas Geneticamente Modificadas , Estresse Salino , Tolerância ao Sal , Frutas/enzimologia , Frutas/genética , Peróxido de Hidrogênio/metabolismo , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/genética , Melatonina/biossíntese , Melatonina/genética , Metiltransferases/biossíntese , Metiltransferases/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética
15.
J Dairy Sci ; 102(6): 5706-5712, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30954263

RESUMO

Antimicrobial peptides are a common defense against bacterial infections in many species and a significant part of the innate immune response of the bovine mammary gland. The objective of this study was to investigate the influence of epigenetic factors on vitamin D and toll-like receptor-mediated induction of ß-defensins in mammary epithelial cells. Primary bovine mammary epithelial cells were treated with lipopolysaccharide (LPS, 0 or 100 ng/mL), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 0 or 10 nM], and 5-aza-2'-deoxycytidine (5-Aza, inhibitor of DNA methyltransferase, 0 or 5 µM) or trichostatin A (TSA, inhibitor of histone deacetylase, 0 or 80 nM) in a factorial arrangement. Effects of treatments on ß-defensin gene expression along with genes for cytokines and enzymes known to be induced by LPS or 1,25(OH)2D3 were evaluated by quantitative PCR. The LPS treatment induced expression of ß-defensin (DEFB)3, DEFB5, DEFB7, DEFB10, enteric ß-defensin (EBD), lingual antimicrobial peptide (LAP), and tracheal antimicrobial peptide (TAP); whereas, the 1,25(OH)2D3 treatment increased DEFB5 and DEFB7 expression and decreased LAP. The 5-Aza treatment increased expression of DEFB3, DEFB5, DEFB10, EBD, LAP, and TAP in the presence and absence of LPS. The TSA treatment increased expression of DEFB3, DEFB4, DEFB5, DEFB7, and DEFB10 in the absence of LPS but decreased LPS-induced expression of and LAP and TAP. Together these results indicate that ß-defensin expression in bovine mammary epithelial cells is likely influenced by DNA methylation and histone acetylation. Investigation of environmental and nutritional factors that influence epigenetic control of ß-defensins in the mammary gland may be beneficial for improving resistance to intramammary infections.


Assuntos
Bovinos/metabolismo , Células Epiteliais/metabolismo , Histona Desacetilases/metabolismo , Lipopolissacarídeos/metabolismo , Glândulas Mamárias Animais/metabolismo , Metiltransferases/metabolismo , Vitamina D/análogos & derivados , beta-Defensinas/genética , Animais , Bovinos/genética , Metilação de DNA , Feminino , Histona Desacetilases/genética , Glândulas Mamárias Animais/citologia , Metiltransferases/genética , Vitamina D/metabolismo , beta-Defensinas/metabolismo
16.
Artigo em Inglês | MEDLINE | ID: mdl-30880278

RESUMO

Methyl farnesoate (MF), a sesquiterpenoid synthesized in the mandibular organ, regulates many physiological processes in crustaceans including growth and reproduction. In the present study, farnesoic acid O-methyltransferase (FAMeT), the key enzyme responsible for final step conversion of farnesoic acid (FA) to methyl farnesoate (MF), was cloned and characterized from the nervous tissues of Penaeus indicus. Multiple sequence alignment, prediction of conserved domain regions, phosphorylation sites identification and phylogenetic analysis indicated that putative FAMeT fragment from P. indicus (PiFAMeT), shares a high degree of sequence identity to FAMeT proteins isolated from other crustaceans species. Quantitative real-time PCR analysis revealed ubiquitous expression of PiFAMeT in all the tissues examined, with comparative higher mRNA levels in nervous tissue and ovary. Additionally, the levels of PiFAMeT also showed gradual increase of expression correlating with the advancement in ovarian maturation. Further to support their role in promoting ovarian development, serotonin treatment (5HT, 50 µg/g body weight) was given to eyestalk intact and unilaterally eyestalk ablated females which resulted in significant increase in PiFAMeT transcript levels at day 7 and day 14. The relatively higher levels of PiFAMeT, reflecting higher levels of MF, suggest a role during secondary vitellogenesis thereby regulating ovarian development in P. indicus. Further research is required to understand the synergistic interaction of MF pathways with serotonergic and other regulatory pathways in regulating ovarian maturation in penaeid shrimps.


Assuntos
Proteínas de Artrópodes , Regulação Enzimológica da Expressão Gênica/fisiologia , Metiltransferases , Ovário/enzimologia , Penaeidae , Vitelogênese/fisiologia , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Clonagem Molecular , Feminino , Metiltransferases/biossíntese , Metiltransferases/genética , Ovário/citologia , Penaeidae/enzimologia , Penaeidae/genética
17.
Enzyme Microb Technol ; 125: 1-5, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30885319

RESUMO

O-Methylation of N-acetylserotonin (NAS) has been identified as the bottleneck in melatonin biosynthesis pathway. In the present paper, caffeic acid O-methyltransferase from Arabidopsis thaliana (AtCOMT) was engineered by rational design to improve its catalytic efficiency in conversion of NAS to melatonin. Based on the notable difference in the terminal structure of caffeic acid and NAS, mutants were designed to strengthen the interactions between the substrate binding pocket of the enzyme and the terminal structure of the unnatural substrate NAS. The final triple mutant (C296F-Q310L-V314T) showed 9.5-fold activity improvement in O-methylation of NAS. Molecular dynamics simulations and binding free energy analysis attributed the increased activity to the higher affinity between the substrate terminal structure and AtCOMT, resulting from the introduction of NH⋯π interaction by Phe296 substitution, hydrophobic interaction by Thr314 substitution and elimination of electrostatic repulsion by substitution of Gln310 with Leu310. This work provides hints for O-methyltransferase engineering and meanwhile lays foundation for biotechnological production of melatonin.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Engenharia de Proteínas , Serotonina/análogos & derivados , Substituição de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Sítios de Ligação , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Catálise , Melatonina/biossíntese , Melatonina/metabolismo , Metiltransferases/genética , Simulação de Dinâmica Molecular , Estrutura Molecular , Serotonina/química , Serotonina/metabolismo , Relação Estrutura-Atividade , Termodinâmica
18.
Food Chem ; 288: 368-376, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902306

RESUMO

Exogenous Ca2+ affects the phenolic metabolism and physiological indices of germinated wheat under ultraviolet-B (UV-B) radiation, but the mechanism is unclear. The current study applied exogenous Ca2+ and Ca2+ channel blocker LaCl3 to the germinated wheat under UV-B radiation to unravel the role of Ca2+. The results indicated that total phenolic content (TPC) of the 4-day germinated wheat under UV-B radiation with CaCl2 (UV-B+Ca) treatment significantly increased by 10.3% as compared to the UV-B treatment. Gene expression levels of p-coumarate 3-hydroxylase, cinnamic acid 4-hydroxylase and caffeic acid O-methyltransferase were positively correlated with the content of ferulic and p-coumaric acids, respectively. Exogenous Ca2+ could significantly alleviate the membrane lipid peroxidation, activate the antioxidant enzymes and regulate the phytohormone level under UV-B radiation. This study suggested that exogenous Ca2+ participated in the phenolic metabolism and physiological regulation in germinated wheat under UV-B radiation.


Assuntos
Germinação/efeitos da radiação , Fenóis/metabolismo , Triticum/crescimento & desenvolvimento , Raios Ultravioleta , Antioxidantes/metabolismo , Cloreto de Cálcio/química , Cloreto de Cálcio/farmacologia , Cromatografia Líquida de Alta Pressão , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Germinação/efeitos dos fármacos , Malondialdeído/análise , Metiltransferases/genética , Metiltransferases/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Fenóis/análise , Reguladores de Crescimento de Planta/análise , Triticum/química , Triticum/metabolismo
19.
Mol Med Rep ; 19(4): 2999-3008, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816500

RESUMO

NOP2/Sun domain family member 2 (NSUN2) is upregulated in numerous types of tumors and may be implicated in multiple biological processes, including cell proliferation, migration and human tumorigenesis. However, little is known about how NSUN2 serves a role in these processes. In the present study, expression profiles of long noncoding RNAs (lncRNAs) and mRNAs were developed in NSUN2­deficient HepG2 cells by RNA­sequencing analysis. A total of 757 lncRNAs were differentially expressed, 392 of which were upregulated, and 365 were downregulated compared with wild­type HepG2 cells. Moreover, 212 lncRNAs were co­expressed with 368 target mRNAs. It was also observed that 253 pairs of lncRNAs and mRNAs exhibited negative correlations and that 290 pairs had positive correlations. Bioinformatics analysis indicated that these lncRNAs regulated by NSUN2 were associated with 'signal transduction', 'extracellular exosome' and 'calcium ion binding', and were enriched in 'pathways in cancer', 'PI3K­Akt signaling pathway' and 'ECM­receptor interaction pathway'. These results illustrate the landscape and co­expression network of lncRNAs regulated by NSUN2 and provide invaluable information for studying the molecular function of NSUN2.


Assuntos
Metiltransferases/genética , RNA Longo não Codificante , Transcriptoma , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interferência de RNA , RNA Mensageiro
20.
PLoS Genet ; 15(3): e1007984, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30893314

RESUMO

Inorganic arsenic (iAs) is a carcinogen, and exposure to iAs via food and water is a global public health problem. iAs-contaminated drinking water alone affects >100 million people worldwide, including ~50 million in Bangladesh. Once absorbed into the blood stream, most iAs is converted to mono-methylated (MMA) and then di-methylated (DMA) forms, facilitating excretion in urine. Arsenic metabolism efficiency varies among individuals, in part due to genetic variation near AS3MT (arsenite methyltransferase; 10q24.32). To identify additional arsenic metabolism loci, we measured protein-coding variants across the human exome for 1,660 Bangladeshi individuals participating in the Health Effects of Arsenic Longitudinal Study (HEALS). Among the 19,992 coding variants analyzed exome-wide, the minor allele (A) of rs61735836 (p.Val101Met) in exon 3 of FTCD (formiminotransferase cyclodeaminase) was associated with increased urinary iAs% (P = 8x10-13), increased MMA% (P = 2x10-16) and decreased DMA% (P = 6x10-23). Among 2,401 individuals with arsenic-induced skin lesions (an indicator of arsenic toxicity and cancer risk) and 2,472 controls, carrying the low-efficiency A allele (frequency = 7%) was associated with increased skin lesion risk (odds ratio = 1.35; P = 1x10-5). rs61735836 is in weak linkage disequilibrium with all nearby variants. The high-efficiency/major allele (G/Valine) is human-specific and eliminates a start codon at the first 5´-proximal Kozak sequence in FTCD, suggesting selection against an alternative translation start site. FTCD is critical for catabolism of histidine, a process that generates one-carbon units that can enter the one-carbon/folate cycle, which provides methyl groups for arsenic metabolism. In our study population, FTCD and AS3MT SNPs together explain ~10% of the variation in DMA% and support a causal effect of arsenic metabolism efficiency on arsenic toxicity (i.e., skin lesions). In summary, this work identifies a coding variant in FTCD associated with arsenic metabolism efficiency, providing new evidence supporting the established link between one-carbon/folate metabolism and arsenic toxicity.


Assuntos
Amônia-Liases/genética , Arsênico/toxicidade , Glutamato Formimidoiltransferase/genética , Metiltransferases/genética , Adulto , Alelos , Amônia-Liases/fisiologia , Arsênico/metabolismo , Intoxicação por Arsênico , Bangladesh , Exposição Ambiental , Feminino , Ácido Fólico/metabolismo , Frequência do Gene/genética , Glutamato Formimidoiltransferase/fisiologia , Humanos , Masculino , Metilação , Metiltransferases/metabolismo , Mutação de Sentido Incorreto , Razão de Chances , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Dermatopatias/induzido quimicamente , Dermatopatias/genética , Poluentes Químicos da Água
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