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1.
Cancer Sci ; 112(7): 2855-2869, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33970549

RESUMO

Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.


Assuntos
Proteína HMGA2/genética , Neoplasias Hepáticas/genética , Oxigenases de Função Mista/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Citosina/metabolismo , Metilação de DNA , Dioxigenases/metabolismo , Epigênese Genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGA2/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Oxigenases de Função Mista/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima
2.
Methods Mol Biol ; 2272: 181-194, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009614

RESUMO

Methylation of DNA at cytosine bases is an important DNA modification underlying normal development and disease states. Despite decades of research into the biological function of DNA methylation, most of the observations so far have relied primarily on associative data between observed changes in DNA methylation states and local changes in transcriptional activity or chromatin state processes. This is primarily due to the lack of molecular tools to precisely modify DNA methylation in the genome. Recent advances in genome editing technologies have allowed repurposing the CRISPR-Cas9 system for epigenome editing by fusing the catalytically dead Cas9 (dCas9) to epigenome modifying enzymes. Moreover, methods of recruiting multiple protein domains, including the SunTag system, have increased the efficacy of epigenome editing at target sites. Here, we describe an end-to-end protocol for efficient targeted removal of DNA methylation by recruiting multiple catalytic domain of TET1 enzymes to the target sites with the dCas9-SunTag system, including sgRNA design, molecular cloning, delivery of plasmid into mammalian cells, and targeted DNA methylation analysis.


Assuntos
Sistemas CRISPR-Cas , Desmetilação do DNA , DNA/análise , Edição de Genes , Genoma Humano , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sulfitos/química , Cromatina , Biologia Computacional/métodos , DNA/química , DNA/genética , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/genética , Oxirredução , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética
3.
Methods Mol Biol ; 2272: 195-206, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009615

RESUMO

Aberrant promoter hypermethylation leads to gene silencing and is associated with various pathologies including cancer and organ fibrosis. Active DNA demethylation is mediated by TET enzymes: TET1, TET2, and TET3, which convert 5-methylcytosine to 5-hydroxymethylcytosine. Induction of gene-specific hydroxymethylation via CRISPR/Cas9 gene technology provides an opportunity to reactivate a single target gene silenced in pathological conditions. We utilized a spCas9 variant fused with TET3 catalytic domain to mediate gene-specific hydroxymethylation with subsequent gene reactivation which holds promise for gene therapy. Here, we present guidelines for gene-specific hydroxymethylation targeting with a specific focus on designing sgRNA and functional assessments in vitro.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Sistemas CRISPR-Cas , Metilação de DNA , DNA/análise , Edição de Genes , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Cromatina , Biologia Computacional/métodos , DNA/química , DNA/genética , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/genética , Oxirredução , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Sulfitos/química
4.
Methods Mol Biol ; 2272: 225-237, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009617

RESUMO

The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in "demethylation" of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Metilação de DNA , DNA/análise , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , DNA/química , DNA/genética , Humanos , Oxigenases de Função Mista/genética , Oxirredução , Proteínas Proto-Oncogênicas/genética , Especificidade por Substrato
5.
Methods Mol Biol ; 2272: 251-262, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009619

RESUMO

TET proteins are methylcytosine dioxygenases that interact directly with chromatin to shape the DNA methylation landscape. To increase the understanding of TET protein function in a specific cellular context, it is important to be able to map the interactions between TET proteins and DNA. This ChIP-seq protocol details our procedure to analyze TET2 bound DNA in disuccinimidyl glutarate (DSG) and formaldehyde-crosslinked chromatin but can also be adapted to study other TET enzymes.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Metilação de DNA , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sequência de DNA/métodos , Humanos , Oxigenases de Função Mista/classificação , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/classificação , Proteínas Proto-Oncogênicas/genética
6.
Science ; 372(6538)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33833093

RESUMO

DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.


Assuntos
Metilação de DNA , DNA/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Sistemas CRISPR-Cas , DNA (Citosina-5-)-Metiltransferases/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genoma Humano , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transcrição Genética
7.
J Cancer Res Clin Oncol ; 147(7): 1869-1879, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33913031

RESUMO

PURPOSE: Methylation of 5th residue of cytosine in CpG island forms 5-methylcytosine which is stable, heritable epigenetic mark. Methylation levels are broadly governed by methyltransferases and demethylases. An aberration in the demethylation process contributes to the silencing of gene expression. Ten eleven translocation (TET) dioxygenase (1-3) the de novo demethylase is responsible for conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosisne (5-fC) and 5-carboxycytosine (5-caC) during demethylation process. Mutations and abnormal expression of TET proteins contribute to carcinogenesis. Discovery of TET proteins has offered various pathways for the reversal of methylation levels thus, enhancing our knowledge as to how methylation effects cancer progression. METHODS: We searched "PubMed" and "Google scholar" databases and selected studies with the following keywords "TET enzyme", "cancer", "5-hmC", and "DNA demethylation". In this review, we have discussed combinatorial use of vitamin C in inhibiting tumour growth by enhancing the catalytic activity of TET enzymes and consequently, increasing the 5-hmC levels. 5-Hydroxymethylcytosine holds promise as a prognostic biomarker in solid cancers. The contribution of induction and suppression of TET enzymes and 5-hmC carcinogenesis are discussed in haematological and solid cancers. RESULTS: We found that TET enzymes play central role in maintaining the methylation balance. Any anomaly in their expression may dip the balance towards cancer progression. Low levels of TET enzymes and 5-hmC correlate with tumour invasion, progression and metastasis. Also, use of vitamin C enhances TET activity. CONCLUSION: TET enzymes play vital role in shaping the methylation landscape in body. 5-hmC can be used as prognostic marker in solid cancers.


Assuntos
Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Oxigenases de Função Mista/genética , Neoplasias/patologia , Proteínas Proto-Oncogênicas/genética , Humanos , Neoplasias/genética
8.
Int J Mol Sci ; 22(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917711

RESUMO

Quantifying O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation plays an essential role in assessing the potential efficacy of alkylating agents in the chemotherapy of malignant gliomas. MGMT promoter methylation is considered to be a characteristic of subgroups of certain malignancies but has also been described in various peripheral inflammatory diseases. However, MGMT promoter methylation levels have not yet been investigated in non-neoplastic brain diseases. This study demonstrates for the first time that one can indeed detect slightly enhanced MGMT promoter methylation in individual cases of inflammatory demyelinating CNS diseases such as multiple sclerosis and progressive multifocal leucencephalopathy (PML), as well as in other demyelinating diseases such as central pontine and exptrapontine myelinolysis, and diseases with myelin damage such as Wallerian degeneration. In this context, we identified a reduction in the expression of the demethylase TET1 as a possible cause for the enhanced MGMT promoter methylation. Hence, we show for the first time that MGMT hypermethylation occurs in chronic diseases that are not strictly associated to distinct pathogens, oncogenic viruses or neoplasms but that lead to damage of the myelin sheath in various ways. While this gives new insights into epigenetic and pathophysiological processes involved in de- and remyelination, which might offer new therapeutic opportunities for demyelinating diseases in the future, it also reduces the specificity of MGMT hypermethylation as a tumor biomarker.


Assuntos
Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Suscetibilidade a Doenças , Proteínas Supressoras de Tumor/genética , Idoso , Biomarcadores , Biópsia , Doenças do Sistema Nervoso Central/diagnóstico , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo
9.
Int J Mol Sci ; 22(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918911

RESUMO

Understanding the mechanisms leading to the rise and dissemination of antimicrobial resistance (AMR) is crucially important for the preservation of power of antimicrobials and controlling infectious diseases. Measures to monitor and detect AMR, however, have been significantly delayed and introduced much later after the beginning of industrial production and consumption of antimicrobials. However, monitoring and detection of AMR is largely focused on bacterial pathogens, thus missing multiple key events which take place before the emergence and spread of AMR among the pathogens. In this regard, careful analysis of AMR development towards recently introduced antimicrobials may serve as a valuable example for the better understanding of mechanisms driving AMR evolution. Here, the example of evolution of tet(X), which confers resistance to the next-generation tetracyclines, is summarised and discussed. Initial mechanisms of resistance to these antimicrobials among pathogens were mostly via chromosomal mutations leading to the overexpression of efflux pumps. High-level resistance was achieved only after the acquisition of flavin-dependent monooxygenase-encoding genes from the environmental microbiota. These genes confer resistance to all tetracyclines, including the next-generation tetracyclines, and thus were termed tet(X). ISCR2 and IS26, as well as a variety of conjugative and mobilizable plasmids of different incompatibility groups, played an essential role in the acquisition of tet(X) genes from natural reservoirs and in further dissemination among bacterial commensals and pathogens. This process, which took place within the last decade, demonstrates how rapidly AMR evolution may progress, taking away some drugs of last resort from our arsenal.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Oxigenases de Função Mista/genética , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/classificação , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Transferência Genética Horizontal , Humanos , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Oxigenases de Função Mista/metabolismo , Mutação , Plasmídeos/genética
10.
Gene ; 786: 145623, 2021 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-33798678

RESUMO

Renin, encoded by REN, is an essential enzyme in the renin-angiotensin aldosterone system (RAAS) which is responsible for the maintenance of blood pressure homeostasis. Transcriptional regulation of REN has been linked to enhancer-promoter crosstalk, cAMP response element-binding protein (CREB), the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and a less well-characterized intronic silencer element. We hypothesized that in addition to these, differential DNA methylation is linked to REN expression and influenced by 1,25(OH)2D3. REN expressing cells (HEK293) were used to elucidate the effect of 1,25(OH)2D3 on REN methylation and expression as quantified by methylation-sensitive qPCR and RT-qPCR, respectively. In vitro 1,25(OH)2D3 supplementation (10 nM) induced significant hypomethylation of the REN silencer (P < 0.050), which was linked to a significant reduction in REN expression (P < 0.010) but had no effect on enhancer methylation. In addition, 1,25(OH)2D3 increased VDR (P < 0.05), as well as TET1 (P < 0.05) expression, suggesting an association between 1,25(OH)2D3 and DNA methylation. Thus, it appears that the silencer element, which is controlled by DNA methylation and influenced by 1,25(OH)2D3, plays an essential role in regulating REN expression.


Assuntos
Metilação de DNA/efeitos dos fármacos , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Calcitriol/genética , Renina/genética , Vitamina D/farmacologia , Regulação para Baixo , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico/efeitos dos fármacos
11.
Beijing Da Xue Xue Bao Yi Xue Ban ; 53(2): 420-424, 2021 Feb 22.
Artigo em Chinês | MEDLINE | ID: mdl-33879920

RESUMO

The methylation of cytosine is one of the most fundamental epigenetic modifications in mammalian genomes, and is involved in multiple crucial processes including gene expression, cell differentiation, embryo development and oncogenesis. In the past, DNA methylation was thought to be an irreversible process, which could only be diluted passively through DNA replication. It is now becoming increa-singly obvious that DNA demethylation can be an active process and plays a crucial role in biological processes. Ten eleven translocation (TET) proteins are the key factors modulating DNA demethylation. This family contains three members: TET1, TET2 and TET3. Although three TET proteins have relatively conserved catalytic domains, their roles in organisms are not repeated, and their expression has significant cell/organ specificity. TET1 is mainly expressed in embryonic stem cells, TET2 is mainly expressed in hematopoietic system, and TET3 is widely expressed in cerebellum, cortex and hippocampus. This family catalyzes 5-methylcytosine to 5-hydroxymethylcytosine and other oxidative products, reactivates silenced-gene expression, in turn maintains stem cell pluripotency and regulates lineage specification. With the development of tissue engineering, organ transplantation, autologous tissue transplantation and artificial prosthesis have been widely used in clinical treatment, but these technologies have limitations. Regenerative medicine, which uses stem cells and stem cell related factors for treatment, may provide alternative therapeutic strategies for multiple diseases. Among all kinds of human stem cells, adipose-derived stem cells (ADSCs) are the most prospective stem cell lineage since they have no ethical issues and can be easily obtained with large quantities. To date, ADSCs have been shown to have strong proli-feration capacity, secrete numerous soluble factors and have multipotent differentiation ability. However, the underlying mechanism of the proliferation, secretion, acquired pluripotency, and lineage specific differentiation of ADSCs are still largely unknown. Some studies have explored the role of epigenetic regulation and TET protein in embryonic stem cells, but little is known about its role in ADSCs. By studying the roles of TET proteins and 5-hydroxymethylcytosine in ADSCs, we could provide new theoretical foundation for the clinical application of ADSCs and the stem cell-based therapy. In the future, combined with bioprinting technology, ADSCs may be used in tissue and organ regeneration, plastic surgery reconstruction and other broader fields.


Assuntos
5-Metilcitosina , Epigênese Genética , 5-Metilcitosina/análogos & derivados , Animais , Metilação de DNA , Proteínas de Ligação a DNA/genética , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Estudos Prospectivos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Medicina Regenerativa , Células-Tronco/metabolismo
12.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672312

RESUMO

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host-pathogen interactions.


Assuntos
Proteínas de Bactérias/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Oxigenases de Função Mista/metabolismo , Sideróforos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/isolamento & purificação , Oxigenases de Função Mista/genética , Mutagênese Sítio-Dirigida , Oligopeptídeos/biossíntese , Mapas de Interação de Proteínas , Estabilidade Proteica , Pseudomonas aeruginosa/metabolismo
13.
Appl Environ Microbiol ; 87(11)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33771783

RESUMO

1-Naphthol, a widely used raw material for organic synthesis, is also a well-known organic pollutant. Due to its high toxicity, 1-naphthol is rarely used by microorganisms as the sole carbon source for growth. In this study, catabolism of 1-naphthol by Sphingobium sp. strain B2 was found to be greatly enhanced by additional supplementation with primary carbon sources (e.g., glucose, maltose, and sucrose), and 1-naphthol was even used as the carbon source for growth when strain B2 cells had been preinduced by both 1-naphthol and glucose. A distinct two-component flavin-dependent monooxygenase, NdcA1A2, was found to be responsible for the initial hydroxylation of 1-naphthol to 1,2-dihydroxynaphthalene, a more toxic compound. Transcriptional levels of ndcA1A2 genes were significantly upregulated when strain B2 cells were cultured with both 1-naphthol and glucose compared to cells cultured with only 1-naphthol or glucose. Two transcriptional regulators, the activator NdcS and the inhibitor NdcR, were found to play key roles in the synergistic regulation of the transcription of the 1-naphthol initial catabolism genes ndcA1A2 IMPORTANCE Cometabolism is a widely observed phenomenon, especially in the field of microbial catabolism of highly toxic xenobiotics. However, the mechanisms of cometabolism are ambiguous, and the roles of the obligately coexisting growth substrates remain largely unknown. In this study, we revealed that the roles of the coexisting primary carbon sources (e.g., glucose) in the enhanced catabolism of the toxic compound 1-naphthol in Sphingobium sp. strain B2 were not solely because they were used as growth substrates to support cell growth but, more importantly, because they acted as coinducers to interact with two transcriptional regulators, the activator NdcS and the inhibitor NdcR, to synergistically regulate the transcription of the 1-naphthol initial catabolism genes ndcA1A2 Our findings provide new insights into the cometabolic mechanism of highly toxic compounds in microorganisms.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Oxigenases de Função Mista/genética , Naftóis/metabolismo , Sphingomonadaceae/genética , Proteínas de Bactérias/metabolismo , Oxigenases de Função Mista/metabolismo , Sphingomonadaceae/enzimologia
14.
Carbohydr Polym ; 260: 117814, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33712158

RESUMO

Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan.


Assuntos
Celulases/metabolismo , Celulose/metabolismo , Ativação Enzimática/efeitos da radiação , Proteínas Fúngicas/metabolismo , Luz , Oxigenases de Função Mista/metabolismo , Sordariales/enzimologia , Biomassa , Catálise , Celulose/química , Proteínas Fúngicas/química , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Glucanos/química , Glucanos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/classificação , Oxigenases de Função Mista/genética , Oxirredução , Filogenia , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espalhamento a Baixo Ângulo , Estereoisomerismo , Especificidade por Substrato , Difração de Raios X , Xilanos/química , Xilanos/metabolismo
15.
Neurosci Lett ; 750: 135712, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33647394

RESUMO

Molecule's mechanism of action interacting with CasL 1 (MICAL1) in spinal cord injury (SCI) is unclear. This study aimed to detect the function of MICAL1 in SCI. Western blot was used to analyze the change of MICAL1 in vivo. Immunofluorescence staining was used to detect the location of MICAL1 expression. Oligodendrocyte cells were treated with H2O2 to induce oxidative injury. Subsequently, siRNA transfection was performed to decrease MICAL1 expression in oligodendrocyte cells. Then, the effects of MICAL1 on oxidative stress, apoptosis, and autophagy were assessed. We found that silencing of MICAL1 could significantly reduce the levels of the nuclear factor erythroid 2-related factor 2 (Nrf2), increase the expression of pro-apoptotic factors (Bax and C-caspase 3), decrease the levels of anti-apoptotic factor (Bcl-2) and pro-autophagy factors (Beclin1 and LC3B). Therefore, MICAL1 is a potential target gene for SCI clinical therapy.


Assuntos
Apoptose , Autofagia , Proteínas dos Microfilamentos/metabolismo , Oxigenases de Função Mista/metabolismo , Oligodendroglia/metabolismo , Estresse Oxidativo , Traumatismos da Medula Espinal/metabolismo , Animais , Proteína Beclina-1/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Feminino , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Oxigenases de Função Mista/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
16.
Plant Sci ; 306: 110857, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33775356

RESUMO

Dwarfism is an important agronomic trait in pumpkin that can increase yield. In this study, the dwarf Cucurbita pepo L. line X10 exhibited significantly longitudinally shorter cell length in the stem than did the normal-vine line JIN234. The dwarf stature of X10 was recovered with exogenous gibberellin (GA3) application, suggesting that X10 might be sensitive to GA biosynthesis. Genetic analysis revealed that this dwarf trait is controlled by a single completely dominant locus: CpDw (Cucurbita pepo L. Dwarf). Using 1,300 F2 individuals derived from a cross between X10 and JIN234, we mapped the CpDw locus to a region of approximately 24.6 kb on chromosome 10 that contain 5 annotated genes. The high expression level of Cp4.1LG10g05910.1 and high GA2ox enzyme activity in X10 revealed that the GA 2-oxidase gene Cp4.1LG10g05910.1 is a candidate gene for CpDw. Alignment of the Cp4.1LG10g05910.1 gene revealed two nonsynonymous single nucleotide polymorphism (SNP) mutations in the two exons, as well as several SNPs and InDels in the important functional elements of promoter between parental lines. Further allelic diversity analysis of the Cucurbita spp. germplasm resources indicated that Cp4.1LG10g05910.1 may be involved in vine growth during the early developmental stage in C. pepo but not in C. maxima or C. moschata. This study provides an important theoretical basis for the genetic regulation of vine length and crop breeding in pumpkin.


Assuntos
Cucurbita/crescimento & desenvolvimento , Cucurbita/genética , Cucurbita/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/genética , Mapeamento Cromossômico , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fenótipo , Caules de Planta/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas
17.
BMC Cardiovasc Disord ; 21(1): 51, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33526034

RESUMO

BACKGROUND: Atherosclerosis (AS) is a chronic inflammatory disorder. The aim of our study was to explore the role of circular RNA (circRNA) transmembrane 7 superfamily member 3 (circTM7SF3) in AS progression. METHODS: Experiments were conducted using oxidized low-density lipoprotein (ox-LDL)-induced THP-1-derived macrophages and differentiated human monocyte-derived macrophages (hMDMs). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circTM7SF3, its linear form TM7SF3, microRNA-206 (miR-206) and aspartyl (asparaginyl) ß-hydroxylase (ASPH) messenger RNA (mRNA). Cell viability and apoptosis were examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Cell inflammation was analyzed by measuring the production of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) using enzyme-linked immunosorbent assay (ELISA) kits. Cell oxidative stress was assessed through analyzing the levels of oxidative stress markers using their corresponding commercial kits. Dual-luciferase reporter assay and RNA-pull down assay were used to confirm the interaction between miR-206 and circTM7SF3 or ASPH. The protein level of ASPH was examined by Western blot assay. RESULTS: CircTM7SF3 level was markedly increased in the serum samples of AS patients and ox-LDL-induced THP-1-derived macrophages compared with their matching counterparts. ox-LDL induced-damage in THP-1 cells was partly attenuated by the interference of circTM7SF3. MiR-206 was a downstream molecular target of circTM7SF3. Si-circTM7SF3-mediated effects in ox-LDL-induced THP-1-derived macrophages were partly ameliorated by the addition of anti-miR-206. MiR-206 directly interacted with ASPH mRNA. CircTM7SF3 silencing reduced the expression of ASPH partly through up-regulating miR-206 in THP-1-derived macrophages. ASPH overexpression partly counteracted the effects induced by miR-206 overexpression in ox-LDL-induced THP-1-derived macrophages. CONCLUSION: CircTM7SF3 contributed to ox-LDL-induced injury in AS cell model through up-regulating the expression of ASPH via targeting miR-206.


Assuntos
Apoptose/efeitos dos fármacos , Aterosclerose/enzimologia , Proteínas de Ligação ao Cálcio/metabolismo , Mediadores da Inflamação/metabolismo , Lipoproteínas LDL/toxicidade , Macrófagos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Musculares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Circular/metabolismo , Idoso , Aterosclerose/genética , Aterosclerose/patologia , Proteínas de Ligação ao Cálcio/genética , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Proteínas de Membrana/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Proteínas Musculares/genética , RNA Circular/genética , Transdução de Sinais , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo
18.
Artigo em Chinês | MEDLINE | ID: mdl-33540974

RESUMO

Objective:To investigate the expression and clinical significance of TET gene and 5hmC in chronic sinusitis. Methods:Acquiring 20 cases of nasal polyps from chronic sinusitis with polyps(CW), 20 cases of uncinate process tissues from chronic sinusitis without polyps(CS), 10 cases of middle turbinate tissues from patients with nasal septum deviated as normal group(N).The expression of TET gene and 5hmC in chronic sinusitis was measured by immunofluorescence, Western-Blot and Quantitative real-time PCR. Results:Both TET1/2/3 gene and 5 hmC were highly expressed in the CS group, but were lower in the CW group and the N group.There were statistically significant differences between the CS group and the CW group(P<0.05), and between the CS group and the N group(P<0.05).The expressions of TET1 and TET3 in the N group were higher than those in the CW group, and the difference was statistically significant(P<0.05). Conclusion:DNA demethylation plays an important role in the formation of nasal polyps.High expression of TET1, TET2, TET3 and 5hmC May reduce the risk of nasal polyps.Increased DNA demethylation in chronic sinusitis may reduce the risk of nasal polyp formation.When the degree of DNA methylation in chronic sinusitis is high and the degree of DNA demethylation is low, the disease may develop to CRSwNP, and when the degree of DNA methylation is low and the degree of DNA demethylation is high, the disease may develop to CRSsNP.


Assuntos
Pólipos Nasais , Rinite , Sinusite , Doença Crônica , Metilação de DNA , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Pólipos Nasais/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Rinite/genética , Sinusite/genética , Conchas Nasais/metabolismo
19.
Mol Med Rep ; 23(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33495813

RESUMO

Renal interstitial fibrosis is one of the common causes, and a major pathological basis for the development of various types of chronic progressive renal to end­stage renal diseases. Therefore, it is important to clarify the underlying mechanisms of disease progression in order to develop effective strategies for the treatment and prevention of these pathologies. The aim of the present study was to investigate the association between microRNA (miR)­212 expression and the development of renal interstitial fibrosis, as well as analyzing the role of miR­212 in the disease. The expression of miR­212 was significantly increased in the peripheral blood of patients with renal interstitial fibrosis and in the kidney tissues of unilateral ureteral obstruction (UUO) mice. Angiotensin (Ang) II, TGF­ß1 and hypoxia were found to increase the expression of miR­212 and α smooth muscle actin (α­SMA) in NRK49F cells. Ang II stimulation induced the expression of miR­212 and α­SMA in NRK49F cells, while transfection of miR­212 mimics further upregulated the expression of α­SMA. miR­212 was also revealed to target hypoxia­inducible factor 1α inhibitor (HIF1AN) and to upregulate the expression of hypoxia­inducible factor 1α, α­SMA, connective tissue growth factor, collagen α­1(I) chain and collagen α­1(III) chain, whereas HIF1AN overexpression reversed the regulatory effects of miR­212. In UUO mice, miR­212 overexpression promoted the progression of renal interstitial fibrosis, whereas inhibiting miR­212 resulted in the opposite effect. These results indicated that high expression of miR­212 was closely associated with the occurrence of renal interstitial fibrosis, and that miR­212 may promote its development by targeting HIF1AN.


Assuntos
Nefropatias/metabolismo , MicroRNAs/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Repressoras/metabolismo , Adulto , Idoso , Animais , Linhagem Celular , Feminino , Fibrose , Humanos , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos , MicroRNAs/genética , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Proteínas Repressoras/genética , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
20.
Plant Cell Rep ; 40(3): 479-489, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33386962

RESUMO

KEY MESSAGE: Microarray and genetic analyses reveal that ZTL induces the expression of genes related to auxin synthesis, thereby promoting hypocotyl elongation. ZTL is a blue-light receptor that possesses a light-oxygen-voltage-sensing (LOV) domain, an F-box motif, and a kelch repeat domain. ZTL promotes hypocotyl elongation under high temperature (28 °C) in Arabidopsis thaliana; however, the mechanism of this regulation is unknown. Here, we divided seedlings into hypocotyls and upper aerial parts, and performed microarray analyses. In hypocotyl, 1062 genes were down-regulated in ztl mutants (ztl-3 and ztl-105) compared with wild type; some of these genes encoded enzymes involved in cell wall modification, consistent with reduced hypocotyl elongation. In upper aerial parts, 1038 genes were down-regulated in the ztl mutants compared with wild type; these included genes involved in auxin synthesis and auxin response. Furthermore, the expression of the PHYTOCHROME INTERACTING FACTOR 4 (PIF4) gene, which encodes a transcription factor known to positively regulate YUCCA genes (YUCs), was also decreased in the ztl mutants. Genetic analysis revealed that overexpression of PIF4 and YUC8 could restore the suppressed hypocotyl length in the ztl mutants. Our results suggest that ZTL induces expression of YUC8 via PIF4 in upper aerial parts and promotes hypocotyl elongation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Oxigenases de Função Mista/genética , Arabidopsis/crescimento & desenvolvimento , Parede Celular/genética , Regulação da Expressão Gênica de Plantas , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Mutação , Fitocromo B/genética , Componentes Aéreos da Planta/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Plântula/genética , Plântula/crescimento & desenvolvimento
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