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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.
Adv Exp Med Biol ; 1143: 75-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338816

RESUMO

As the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), N 6-methyladenosine (m6A) modification has been shown recently to posttranscriptionally regulate expression of thousands of messenger RNA (mRNA) transcripts in each mammalian cell type in a dynamic and reversible manner. This epigenetic mark is deposited by the m6A methyltransferase complex (i.e., the METTL3/METTL14/WTAP complex and other cofactor proteins) and erased by m6A demethylases such as FTO and ALKBH5. Specific recognition of these m6A-modified mRNAs by m6A-binding proteins (i.e., m6A readers) determines the fate of target mRNAs through affecting splicing, nuclear export, RNA stability, and/or translation. During the past few years, m6A modification has been demonstrated to play a critical role in many major normal bioprocesses including self-renewal and differentiation of embryonic stem cells and hematopoietic stem cells, tissue development, circadian rhythm, heat shock or DNA damage response, and sex determination. Thus, it is not surprising that dysregulation of the m6A machinery is also closely associated with pathogenesis and drug response of both solid tumors and hematologic malignancies. In this chapter, we summarize and discuss recent findings regarding the biological functions and underlying mechanisms of m6A modification and the associated machinery in normal hematopoiesis and the initiation, progression, and drug response of acute myeloid leukemia (AML), a major subtype of leukemia usually associated with unfavorable prognosis.


Assuntos
Adenosina , Hematopoese , Leucemia Mieloide Aguda , Metiltransferases , RNA Mensageiro , Adenosina/metabolismo , Animais , Diferenciação Celular , Resistencia a Medicamentos Antineoplásicos/genética , Hematopoese/genética , Humanos , Leucemia Mieloide Aguda/fisiopatologia , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo
3.
Chem Commun (Camb) ; 55(58): 8390-8393, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31257394

RESUMO

Diverse bioactive alkaloids with a tryptophan 2,5-diketopiperazine (DKP) core and an annulated structure forming a methylated pyrroloindoline-DKP assembly have been isolated from various microbial sources. However, little is known about their biosynthesis. In this study, a novel indole C3 methyltransferase from Streptomyces sp. HPH0547 was discovered and characterized. Structural elucidation of the products revealed that this enzyme catalyzed unique pyrroloindoline cyclization in tryptophan-containing cyclodipeptides. This is the first C3 methyltransferase reported to catalyze pyrroloindoline cyclization in cyclic dipeptides, which provides a feasible and simple method to access diverse alkaloids.


Assuntos
Alcaloides/biossíntese , Proteínas de Bactérias/metabolismo , Dipeptídeos/biossíntese , Metiltransferases/metabolismo , Peptídeos Cíclicos/biossíntese , Streptomyces/enzimologia , Ciclização , Dicetopiperazinas/metabolismo , Modelos Químicos , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo
4.
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
5.
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
6.
Food Chem Toxicol ; 131: 110529, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31150784

RESUMO

The health promoting effects of extra virgin olive oil (EVOO) relate to its unique repertoire of phenolic compounds. Here, we used a chemoinformatics approach to computationally identify endogenous ligands and assign putative biomolecular targets to oleacein, one of the most abundant secoiridoids in EVOO. Using a structure-based virtual profiling software tool and reference databases containing more than 9000 binding sites protein cavities, we identified 996 putative oleacein targets involving more than 700 proteins. We subsequently identified the high-level functions of oleacein in terms of biomolecular interactions, signaling pathways, and protein-protein interaction (PPI) networks. Delineation of the oleacein target landscape revealed that the most significant modules affected by oleacein were associated with metabolic processes (e.g., glucose and lipid metabolism) and chromatin-modifying enzymatic activities (i.e., histone post-translational modifications). We experimentally confirmed that, in a low-micromolar physiological range (<20 µmol/l), oleacein was capable of inhibiting the catalytic activities of predicted metabolic and epigenetic targets including nicotinamide N-methyltransferase, ATP-citrate lyase, lysine-specific demethylase 6A, and N-methyltransferase 4. Our computational de-orphanization of oleacein provides new mechanisms through which EVOO biophenols might operate as chemical prototypes capable of modulating the biologic machinery of healthy aging.


Assuntos
Aldeídos/metabolismo , Fenóis/metabolismo , Proteômica/métodos , ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/metabolismo , Aldeídos/química , Domínio Catalítico , Ensaios Enzimáticos , Epigenômica/métodos , Ontologia Genética/estatística & dados numéricos , Histona Desmetilases/química , Histona Desmetilases/metabolismo , Humanos , Informática/métodos , Metiltransferases/química , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Nicotinamida N-Metiltransferase/química , Nicotinamida N-Metiltransferase/metabolismo , Olea/química , Azeite de Oliva/química , Fenóis/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Software
7.
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
8.
J Microbiol Biotechnol ; 29(7): 1137-1143, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31216792

RESUMO

Hepatitis E virus (HEV) accounts for 20 million infections in humans worldwide. In most cases, the infections are self-limiting while HEV genotype 1 infection cases may lead to lethal infections in pregnant women (~ 20% fatality). The lack of small animal models has hampered detailed analysis of virus-host interactions and HEV-induced pathology. Here, by employing a recently developed culture-adapted HEV, we demonstrated that methyltransferase, a nonstructural protein, strongly inhibits melanoma differentiation-associated gene 5 (MDA5)- mediated activation of type I interferon responses. Compared to uninfected controls, HEVinfected cells display significantly lower levels of IFN-ß promoter activation when assessed by luciferase assay and RT-PCR. HEV genome-wide screening showed that HEV-encoded methyltransferase (MeT) strongly inhibits MDA5-mediated transcriptional activation of IFN-ß and NF-κB in a dose-responsive manner whether or not it is expressed in the presence/ absence of a tag fused to it. Taken together, current studies clearly demonstrated that HEV MeT is a novel antagonist of MDA5-mediated induction of IFN-ß signaling.


Assuntos
Vírus da Hepatite E/fisiologia , Hepatite E/metabolismo , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Metiltransferases/metabolismo , Proteínas Virais/metabolismo , Células A549 , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/genética , Interferon beta/genética , Interferon beta/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais , Transcrição Genética
9.
Mol Biol (Mosk) ; 53(3): 436-445, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184609

RESUMO

N^(6)-methyladenosine (m^(6)A) has been identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, and plays important biological roles in the regulation of cellular metabolic processes. However, its role in myogenic differentiation is unclear. Here, we altered the m^(6)A RNA methylation level by overexpression of METTL3, and explored the effect of m^(6)A RNA methylation on myogenic differentiation of murine myoblasts in vitro. The m6A RNA methylation level is regulated by exogenous methylation inhibitor cycloleucine (Cyc) and methyl donor betaine (Bet). Therefore, chemical reagents of Cyc and Bet were used to test the regulatory effect of m^(6)A RNA methylation on myogenic differentiation. Results showed that METTL3 and Bet positively regulated the m^(6)A RNA methylation levels, and Cyc negatively regulated m^(6)A RNA methylation levels. In addition, m^(6)A methylation positively regulated myogenic differentiation in murine myoblasts. These findings provide insight in the mechanisms underlying the effect of m^(6)A RNA methylation on myogenesis.


Assuntos
Diferenciação Celular , Metilação , Metiltransferases/metabolismo , Desenvolvimento Muscular , Mioblastos/citologia , Mioblastos/metabolismo , Animais , Camundongos
10.
Plant Mol Biol ; 101(3): 235-255, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31254267

RESUMO

KEY MESSAGE: The core set of biosynthetic genes potentially involved in developmental lignification was identified in the model C4 grass Setaria viridis. Lignin has been recognized as a major recalcitrant factor negatively affecting the processing of plant biomass into bioproducts. However, the efficient manipulation of lignin deposition in order to generate optimized crops for the biorefinery requires a fundamental knowledge of several aspects of lignin metabolism, including regulation, biosynthesis and polymerization. The current availability of an annotated genome for the model grass Setaria viridis allows the genome-wide characterization of genes involved in the metabolic pathway leading to the production of monolignols, the main building blocks of lignin. Here we performed a comprehensive study of monolignol biosynthetic genes as an initial step into the characterization of lignin metabolism in S. viridis. A total of 56 genes encoding bona fide enzymes catalyzing the consecutive ten steps of the monolignol biosynthetic pathway were identified in the S. viridis genome. A combination of comparative phylogenetic studies, high-throughput expression analysis and quantitative RT-PCR analysis was further employed to identify the family members potentially involved in developmental lignification. Accordingly, 14 genes clustered with genes from closely related species with a known function in lignification and showed an expression pattern that correlates with lignin deposition. These genes were considered the "core lignin toolbox" responsible for the constitutive, developmental lignification in S. viridis. These results provide the basis for further understanding lignin deposition in C4 grasses and will ultimately allow the validation of biotechnological strategies to produce crops with enhanced processing properties.


Assuntos
Lignina/metabolismo , Poaceae/metabolismo , Biomassa , Vias Biossintéticas , Coenzima A Ligases/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Funções Verossimilhança , Metiltransferases/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Filogenia , Plantas Geneticamente Modificadas/metabolismo , Transcinamato 4-Mono-Oxigenase/metabolismo
11.
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
12.
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
13.
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
14.
Phytochemistry ; 164: 50-59, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078779

RESUMO

Methyl (E)-cinnamate is a specialized metabolite that occurs in a variety of land plants. In flowering plants, it is synthesized by cinnamic acid methyltransferase (CAMT) that belongs to the SABATH family. While rarely reported in bryophytes, methyl (E)-cinnamate is produced by some liverworts of the Conocephalum conicum complex, including C. salebrosum. In axenically grown thalli of C. salebrosum, methyl (E)-cinnamate was detected as the dominant compound. To characterize its biosynthesis, six full-length SABATH genes, which were designated CsSABATH1-6, were cloned from C. salebrosum. These six genes showed different levels of expression in the thalli of C. salebrosum. Next, CsSABATH1-6 were expressed in Escherichia coli to produce recombinant proteins, which were tested for methyltransferase activity with cinnamic acid and a few related compounds as substrates. Among the six SABATH proteins, CsSABATH6 exhibited the highest level of activity with cinnamic acid. It was renamed CsCAMT. The apparent Km value of CsCAMT using (E)-cinnamic acid as substrate was determined to be 50.5 µM. In contrast, CsSABATH4 was demonstrated to function as salicylic acid methyltransferase and was renamed CsSAMT. Interestingly, the CsCAMT gene from a sabinene-dominant chemotype of C. salebrosum is identical to that of the methyl (E)-cinnamate-dominant chemotype. Structure models for CsCAMT, CsSAMT and one flowering plant CAMT (ObCCMT1) in complex with (E)-cinnamic acid and salicylic acid were built, which provided structural explanations to substrate specificity of these three enzymes. In phylogenetic analysis, CsCAMT and ObCCMT1 were in different clades, implying that methyl (E)-cinnamate biosynthesis in bryophytes and flowering plants originated through convergent evolution.


Assuntos
Cinamatos/metabolismo , Hepatófitas/metabolismo , Metiltransferases/metabolismo , Cinamatos/química , Relação Dose-Resposta a Droga , Hepatófitas/química , Metiltransferases/química , Estrutura Molecular , Relação Estrutura-Atividade
15.
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
16.
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
17.
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
18.
Molecules ; 24(8)2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31027295

RESUMO

In cells, thymidylate synthases provide the only de novo source of 2'-deoxythymidine-5'-monophosphate (dTMP), required for DNA synthesis. The activity of these enzymes is pivotal for cell survival and proliferation. Two main families of thymidylate synthases have been identified in bacteria, folate-dependent thymidylate synthase (TS) and flavin-dependent TS (FDTS). TS and FDTS are highly divergent enzymes, characterized by exclusive catalytic mechanisms, involving different sets of cofactors. TS and FDTS mechanisms of action have been recently revised, providing new perspectives for the development of antibacterial drugs targeting these enzymes. Nonetheless, some catalytic details still remain elusive. For bacterial TSs, half-site reactivity is still an open debate and the recent evidences are somehow controversial. Furthermore, different behaviors have been identified among bacterial TSs, compromising the definition of common mechanisms. Moreover, the redox reaction responsible for the regeneration of reduced flavin in FDTSs is not completely clarified. This review describes the recent advances in the structural and functional characterization of bacterial TSs and FDTSs and the current understanding of their mechanisms of action. Furthermore, the recent progresses in the development of inhibitors targeting TS and FDTS in human pathogenic bacteria are summarized.


Assuntos
Metiltransferases/metabolismo , Timidilato Sintase/metabolismo , Sequência de Aminoácidos , Flavinas/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína
19.
J Sci Food Agric ; 99(11): 5176-5186, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31021402

RESUMO

BACKGROUND: The literature on the role of calcium ion (Ca2+ ) in relation to phenolic compounds metabolism and related enzymes activities remains controversial. It is still unclear whether Ca2+ affects phenolic compounds content of barley sprouts. This study investigated the role and function of Ca2+ in phenolic compound metabolism of barley (Hordeum vulgare L.) sprouts under sodium chloride (NaCl) stress. RESULTS: Calcium chloride (CaCl2 ) significantly improved total calcium and calmodulin (CaM) contents as well as Ca2+ concentration, and enhanced phenolic compound accumulation by regulating the gene, protein expression and the activities of enzymes related to phenolics biosynthesis. Specifically, CaCl2 significantly increased the activities of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL) and ferulic acid 5-hydroxylase (F5H) by up-regulating the corresponding protein expression. The activity of p-coumaric acid 3-hydroxylase (C3H) decreased during germination while caffeic acid O-methyltransferase (COMT) increased initially and then decreased, which was consistent with the changes in gene and protein expression under CaCl2 treatment. Conversely, lanthanum(III) chloride (LaCl3 ), ethylene glycol tetraacetic acid (EGTA) and 2-aminoethoxydiphenyl borate (2-APB) induced opposite effects. Decreased calcium and CaM contents and Ca2+ concentration were observed, and fluctuation change of relevant gene and protein expressions and PAL, C4H, 4CL, C3H, COMT and F5H activitives were also detected. CONCLUSION: Calcium ion played an important role for mediating NaCl stress-induced phenolics accumulation in barley sprouts. It required both Ca2+ influx and release from apoplast and intracellular stores, respectively. © 2019 Society of Chemical Industry.


Assuntos
Hordeum/metabolismo , Fenóis/metabolismo , Sementes/crescimento & desenvolvimento , Cloreto de Sódio/metabolismo , Cloreto de Cálcio/metabolismo , Coenzima A Ligases/metabolismo , Germinação , Hordeum/enzimologia , Hordeum/crescimento & desenvolvimento , Metiltransferases/metabolismo , Sementes/enzimologia , Sementes/metabolismo
20.
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
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