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1.
J Inorg Biochem ; 238: 112021, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36395718

RESUMO

Nature exploits transition metal centers to enhance and tune the oxidizing power of natural oxidants such as O2 and H2O2. The design and interrogation of synthetic metallocomplexes with similar reactivity to metalloproteins provides one strategy for gaining insight into the mechanistic underpinnings of oxygen-activating enzymes such as oxidases, oxygenases, and dioxygenases like Ni-quercetinase (Ni-QueD). Ni-QueD catalyzes the oxidative ring opening of the polyphenol quercetin, a natural product with antioxidant properties. Herein, we report the synthesis and characterization of Ni(13-DOB), a Ni(II) species complexed by an N4-macrocycle that has been characterized by single crystal X-ray crystallography. Ni(13-DOB) forms a Ni-superoxide intermediate (Ni(13-DOB)O2•-) upon treatment with H2O2 and Et3N, as verified by resonance Raman spectroscopy. We demonstrate through UV/vis and LCMS that Ni(13-DOB)O2•- is capable of the 1-electron oxidation of flavonols, including both 3-hydroxyflavone (3-HF, the simplest flavonol) and quercetin itself. Incorporation of two O-atoms into the flavonol radical via superoxide from Ni(13-DOB)O2•- precedes oxidative cleavage of the flavonol scaffold in each case, consistent with quercetinase ring cleavage by Ni-QueD in Streptomyces sp. FLA. Conversion of 3-HF into 2-hydroxybenzoylbenzoic acid was accomplished with catalytic turnover of Ni(13-DOB) at ambient temperature, as confirmed by HPLC timecourses and GCMS analysis of isotopic labeling studies. The Ni(13-DOB)-mediated oxidative cleavage of quercetin to the corresponding biomimetic phenolic ester was also verified through 18O-isotopic labeling studies. Through the HPLC characterization of both on- and off-pathway products of flavonol dioxygenation by Ni(13-DOB)O2•-, the stringent reaction pathway control provided by enzyme active sites is highlighted.


Assuntos
Dioxigenases , Níquel , Níquel/química , Superóxidos , Quercetina , Peróxido de Hidrogênio , Dioxigenases/química , Flavonóis/química , Oxigênio/química
2.
Sci Rep ; 12(1): 19875, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36400936

RESUMO

Sparassis latifolia is a valuable edible mushroom, growing on fresh pine wood sawdust substrate. However, the mechanistic bases are poorly understood. The gene expression profiles of S. latifolia were analyzed from submerged cultures with fresh pine wood sawdust substrate for different time (0 h, 1 h, 6 h, 1 day, 5 days, and 10 days, respectively). The total number of differentially expressed genes (DEGs) identified under pine sawdust inducing was 2,659 compared to 0 h (CK). And 1,073, 520, 385, 424, and 257 DEGs were identified at the five time points, respectively. There were 34 genes in common at all inoculated time points, including FAD/NAD(P)-binding domain-containing protein, glucose methanol choline (GMC) oxidoreductase, flavin-containing monooxygenase, and taurine catabolism dioxygenase. Weighted gene co-expression analysis (WGCNA) was then used to compare the molecular characteristics among the groups and identified that the blue module had the highest correlation with the time induced by pine wood sawdust. There were 102 DEGs out of 125 genes in the blue model, which were most enriched in nitronate monooxygenase activity, dioxygenase activity, and oxidation-reduction process GO terms (p < 0.05), and peroxisome in KEGG pathway. This may provide clues into mechanisms that S. latifolia can grow on fresh pine wood sawdust substrate.


Assuntos
Dioxigenases , Pinus , Polyporales , Transcriptoma , Polyporales/genética , Pinus/genética , Dioxigenases/genética
3.
Sci Rep ; 12(1): 19223, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357504

RESUMO

Polychlorinated Biphenyls (PCBs) are persistence in the contaminated sites as a result of lacking PCBs-degrading microorganisms. Cultivation-independent technique called single-strand-conformation polymorphism (SSCP) based on 16SrRNA genes was chosen to characterize the diversity of bacterial communities in PCBs polluted soil samples. The bacterial communities showed an increasing diversity from the genetic profiles using SSCP technique. 51 single products were identified from the profiles using PCR reamplification and cloning. DNA sequencing of the 51 products, it showed similarities to Acidobacteria, Actinobacteria, Betaproteobateria, Gammaproteobacteria and Alphaproteobacteria, the range of similarities were 92.3 to 100%. Pure 23 isolates were identified from PCBs contaminated sites. The identified isolates belonged to genus Bacillus, Brevibacillus, Burkholderia, Pandoraea, Pseudomonas, and Rhodococcus. The new strains have the capability to use PCBs as a source of sole carbon and harbor 2,3-dihydroxybiphenyl dioxygenase (DHBDO) which could be used as molecular marker for detection PCBs-degrading bacteria in the PCBs contaminated sites. This finding may enhance the PCBs bioremediation by monitoring and characterization of the PCBs degraders using DHBDO in PCBs contaminated sites.


Assuntos
Dioxigenases , Bifenilos Policlorados , Poluentes do Solo , Solo , Microbiologia do Solo , Biodegradação Ambiental , Bactérias/genética , Biotecnologia , Dioxigenases/genética
4.
World J Gastroenterol ; 28(40): 5845-5864, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36353202

RESUMO

BACKGROUND: Inflammatory bowel disease (IBD) constitutes a substantial risk factor for colorectal cancer. Connexin 43 (Cx43) is a protein that forms gap junction (GJ) complexes involved in intercellular communication, and its expression is altered under pathological conditions, such as IBD and cancer. Recent studies have implicated epigenetic processes modulating DNA methylation in the pathogenesis of diverse inflammatory and malignant diseases. The ten-eleven translocation-2 (TET-2) enzyme catalyzes the demethylation, hence, regulating the activity of various cancer-promoting and tumor-suppressor genes. AIM: To investigate Cx43 and TET-2 expression levels and presence of 5-hydroxymethylcytosine (5-hmC) marks under inflammatory conditions both in vitro and in vivo. METHODS: TET-2 expression was evaluated in parental HT-29 cells and in HT-29 cells expressing low or high levels of Cx43, a putative tumor-suppressor gene whose expression varies in IBD and colorectal cancer, and which has been implicated in the inflammatory process and in tumor onset. The dextran sulfate sodium-induced colitis model was reproduced in BALB/c mice to evaluate the expression of TET-2 and Cx43 under inflammatory conditions in vivo. In addition, archived colon tissue sections from normal, IBD (ulcerative colitis), and sporadic colon adenocarcinoma patients were obtained and evaluated for the expression of TET-2 and Cx43. Expression levels were reported at the transcriptional level by quantitative real-time polymerase chain reaction, and at the translational level by Western blotting and immunofluorescence. RESULTS: Under inflammatory conditions, Cx43 and TET-2 expression levels increased compared to non-inflammatory conditions. TET-2 upregulation was more pronounced in Cx43-deficient cells. Moreover, colon tissue sections from normal, ulcerative colitis, and sporadic colon adenocarcinoma patients corroborated that Cx43 expression increased in IBD and decreased in adenocarcinoma, compared to tissues from non-IBD subjects. However, TET-2 expression and 5-hmC mark levels decreased in samples from patients with ulcerative colitis or cancer. Cx43 and TET-2 expression levels were also investigated in an experimental colitis mouse model. Interestingly, mice exposed to carbenoxolone (CBX), a GJ inhibitor, had upregulated TET-2 levels. Collectively, these results show that TET-2 levels and activity increased under inflammatory conditions, in cells downregulating gap junctional protein Cx43, and in colon tissues from mice exposed to CBX. CONCLUSION: These results suggest that TET-2 expression levels, as well as Cx43 expression levels, are modulated in models of intestinal inflammation. We hypothesize that TET-2 may demethylate genes involved in inflammation and tumorigenesis, such as Cx43, potentially contributing to intestinal inflammation and associated carcinogenesis.


Assuntos
Adenocarcinoma , Colite Ulcerativa , Colite , Neoplasias do Colo , Dioxigenases , Doenças Inflamatórias Intestinais , Animais , Camundongos , Adenocarcinoma/patologia , Carcinogênese/patologia , Colite/induzido quimicamente , Colite Ulcerativa/patologia , Colo/patologia , Neoplasias do Colo/patologia , Conexina 43/genética , Conexina 43/metabolismo , Sulfato de Dextrana/toxicidade , Dioxigenases/metabolismo , Modelos Animais de Doenças , Inflamação/patologia , Doenças Inflamatórias Intestinais/patologia
5.
Int J Mol Sci ; 23(22)2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36430449

RESUMO

Plant senescence is a complex process that is controlled by developmental regulation and genetic programs. A senescence-related gene CpSRG1, which belongs to the 2OG-Fe(II) dioxygenase superfamily, was characterized from wintersweet, and the phylogenetic relationship of CpSRG1 with homologs from other species was investigated. The expression analysis by qRT-PCR (quantitative real-time PCR) indicated that CpSRG1 is abundant in flower organs, especially in petals and stamens, and the highest expression of CpSRG1 was detected in stage 6 (withering period). The expression patterns of the CpSRG1 gene were further confirmed in CpSRG1pro::GUS (ß-glucuronidase) plants, and the activity of the CpSRG1 promoter was enhanced by exogenous Eth (ethylene), SA (salicylic acid), and GA3 (gibberellin). Heterologous overexpression of CpSRG1 in Arabidopsis promoted growth and flowering, and delayed senescence. Moreover, the survival rates were significantly higher and the root lengths were significantly longer in the transgenic lines than in the wild-type plants, both under low nitrogen stress and GA3 treatment. This indicated that the CpSRG1 gene may promote the synthesis of assimilates in plants through the GA pathway, thereby improving growth and flowering, and delaying senescence in transgenic Arabidopsis. Our study has laid a satisfactory foundation for further analysis of senescence-related genes in wintersweet and wood plants. It also enriched our knowledge of the 2OG-Fe(II) dioxygenase superfamily, which plays a variety of important roles in plants.


Assuntos
Arabidopsis , Calycanthaceae , Dioxigenases , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Filogenia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Calycanthaceae/genética , Dioxigenases/genética , Compostos Ferrosos/metabolismo
6.
Cell Rep ; 41(6): 111612, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36351399

RESUMO

DNA methylation has emerged as a critical modulator of neuronal plasticity and cognitive function. Notwithstanding, the role of enzymes that demethylate DNA remain to be fully explored. Here, we report that loss of ten-eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), in adult neurons enhances cognitive function. In the adult mouse hippocampus, we detected an enrichment of Tet2 in neurons. Viral-mediated neuronal overexpression and RNA interference of Tet2 altered dendritic complexity and synaptic-plasticity-related gene expression in vitro. Overexpression of neuronal Tet2 in adult hippocampus, and loss of Tet2 in adult glutamatergic neurons, resulted in differential hydroxymethylation associated with genes involved in synaptic transmission. Functionally, overexpression of neuronal Tet2 impaired hippocampal-dependent memory, while loss of neuronal Tet2 enhanced memory. Ultimately, these data identify neuronal Tet2 as a molecular target to boost cognitive function.


Assuntos
Dioxigenases , Proteínas Proto-Oncogênicas , Animais , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a DNA/metabolismo , 5-Metilcitosina/metabolismo , Dioxigenases/genética , Metilação de DNA/genética , Cognição , Neurônios/metabolismo , Hipocampo/metabolismo
7.
Molecules ; 27(22)2022 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-36431912

RESUMO

Carotenoids are isoprenoid pigments, and sources of vitamin A in humans. The first metabolic pathway for their synthesis is mediated by the enzymes ß,ß-carotene-15,15'-dioxygenase (BCO1) and ß,ß-carotene-9',10'-dioxygenase (BCO2), which cleave carotenoids into smaller compounds, called apocarotenoids. The objective of this study is to gain insight into the interaction of BCO1 and BCO2 with carotenoids, adding structural diversity and importance in the agro-food and/or health sectors. Homology modeling of BCO1 and BCO2, and the molecular dynamics of complexes with all carotenoids were performed. Interaction energy and structures were analyzed. For both enzymes, the general structure is conserved with a seven beta-sheet structure, and the ß-carotene is positioned at an optimal distance from the catalytic center. Fe2+ forms in an octahedral coordination sphere with four perfectly conserved histidine residues. BCO1 finds stability in a structure in which the ß-carotene is positioned ready for enzymatic catalysis at the 15-15' bond, and BCO2 in positioning the bond to be cleaved (C9-C10) close to the active site. In BCO1 the carotenoids interact with only seven residues with aromatic rings, while the interaction of BCO2 is much more varied in terms of the type of interaction, with more residues of different chemical natures.


Assuntos
Dioxigenases , beta-Caroteno 15,15'-Mono-Oxigenase , Humanos , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismo , beta Caroteno/metabolismo , Simulação por Computador , Dioxigenases/metabolismo , Carotenoides/metabolismo
8.
Sci Rep ; 12(1): 19452, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376482

RESUMO

Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD's quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD's ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z' values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU.


Assuntos
Alcaptonúria , Dioxigenases , Humanos , Alcaptonúria/genética , Homogentisato 1,2-Dioxigenase/genética , Dioxigenases/genética , Polimorfismo de Nucleotídeo Único , Ensaios de Triagem em Larga Escala , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Homogentísico
9.
Life Sci Alliance ; 5(12)2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-36260750

RESUMO

The immunosuppressive function "licensed" by IFN-γ is a vital attribute of mesenchymal stem cells (MSCs) widely used in the treatment of inflammatory diseases. However, the mechanism and impact of metabolic reprogramming on MSC immunomodulatory plasticity remain unclear. Here, we explored the mechanism by which glucose metabolism affects the immunomodulatory reprogramming of MSCs "licensed" by IFN-γ. Our data showed that glucose metabolism regulates the immunosuppressive function of human umbilical cord MSCs (hUC-MSCs) challenged by IFN-γ through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Furthermore, ATP facilitated the cross talk between glucose metabolism and the JAK-STAT system, which stimulates the phosphorylation of JAK2 and STATs, as well as the expression of indoleamine 2, 3-dioxygenase and programmed cell death-1 ligand. Moreover, ATP synergistically enhanced the therapeutic efficacy of IFN-γ-primed hUC-MSCs against acute pneumonia in mice. These results indicate a novel cross talk between the immunosuppressive function, glucose metabolism, and mitochondrial oxidation and provide a novel targeting strategy to enhance the therapeutic efficacies of hUC-MSCs.


Assuntos
Dioxigenases , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Ligantes , Células-Tronco Mesenquimais/metabolismo , Interferon gama/metabolismo , Terapia de Imunossupressão , Janus Quinases/metabolismo , Dioxigenases/metabolismo , Glucose/metabolismo , Trifosfato de Adenosina/metabolismo
10.
Angew Chem Int Ed Engl ; 61(48): e202210938, 2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36196912

RESUMO

Brevione E (1) is a fungal hexacyclic meroditerpenoid with unique oxepane and cycloheptenone moieties. In this study, we identified the biosynthetic gene cluster of 1 and elucidated its biosynthetic pathway via heterologous expression of the biosynthetic genes and in vitro enzymatic reactions. Surprisingly, reexamination of the structure of 1 revealed a substituted tetrahydrofuran ring instead of the previously proposed oxepane system. Moreover, we determined that cycloheptenone synthesis involves skeletal rearrangement catalyzed by the α-ketoglutarate-dependent dioxygenase BrvJ. BrvJ is highly homologous to SetK, which engages in the biosynthesis of another fungal metabolite, setosusin, and accepts the same substrate as BrvJ but performs only simple hydroxylation. Finally, we identified the key amino acid residues critical for product selectivity of BrvJ and SetK, providing insight into how the biosynthesis pathways of 1 and setosusin diverge and how fungi diversify natural products.


Assuntos
Produtos Biológicos , Dioxigenases , Dioxigenases/genética , Dioxigenases/metabolismo , Família Multigênica , Vias Biossintéticas , Hidroxilação , Produtos Biológicos/metabolismo
12.
Dalton Trans ; 51(44): 17064-17080, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36314263

RESUMO

This paper demonstrates the metal ion effects on the quercetin 2,4-dioxygenase (2,4-QD)-like reactivity. For this purpose, a series of five metal(II)-acetato complexes [MII(L)(OAc)] {M = Mn (1OAc), Co (2OAc), Ni (3OAc), Cu (4OAc), Zn (5OAc); OAc = acetate} supported with a newly designed N3O-donor carboxylato ligand L- {L- = 2-((benzyl((6'-methyl-[2,2'-bipyridin]-6-yl)methyl)amino)methyl)benzoate} has been synthesised as models for the active sites of MII-substituted 2,4-QDs. The enzyme-substrate (ES) model complexes [MII(L)(fla)] {M = Mn (1fla), Co (2fla), Ni (3fla), Cu (4fla), Zn (5fla); flaH = flavonol} have been synthesised by reacting flaH with their corresponding acetate-bound complexes in basic conditions. Detailed physicochemical properties of all the compounds are reported. Furthermore, single-crystal X-ray diffractions have been done to determine the structures of the compounds 2OAc·2H2O, 3OAc, 4OAc·CH2Cl2·2H2O, 5OAc·2H2O and 2fla·MeOH. The enzymatic reactivities of complexes 1OAc-5OAc towards the dioxygenation of flavonol have been explored in detail. All the complexes effectively catalyse the oxygenative degradation of flavonol in N,N-dimethylformamide (DMF) medium at 70 °C under multiple-turnover conditions and produce enzyme-type products. Kinetic investigations were performed to see the metal ions' effects on reactivity. The reaction rates vary with the metal ions, showing the order Co > Ni > Zn > Mn > Cu. The studies reveal that the reactivities of the [MII(L)(OAc)] complexes are governed primarily by three factors viz the ES adduct formation constant (Kf), the redox potential (Epa) of the bound fla-/fla˙ couple, and the degree of delocalisation of the fla˙ radical with the metal electrons, which are drastically influenced by the M2+ ions. In the mechanistic interpretation, a single-electron transfer (SET) from the bound-flavonolate to dioxygen has been proposed to generate the catalytically important "M(II)-fla˙" radical and superoxide ion, which react further to bring about the dioxygenation reaction. The identification of the metal(II)-bound flavonoxy radical intermediate for the case of cobalt using EPR spectroscopy and the detection of superoxide ion by NBT2+ test and EPR spin-trapping experiment (DMPO test) are remarkable in envisaging the reaction pathway.


Assuntos
Complexos de Coordenação , Dioxigenases , Dioxigenases/química , Quercetina , Complexos de Coordenação/química , Superóxidos , Modelos Moleculares , Metais , Catálise , Flavonóis/química , Zinco/química , Acetatos
13.
Cancer Discov ; 12(10): 2234-2236, 2022 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-36196576

RESUMO

Transgenic knockin mice expressing a common loss-of-function mutation in human TET2 exhibit aging-related accelerated myeloid leukemia development and skewing of myelopoiesis toward the production of proinflammatory MHC-IIhi monocytes that may contribute to disease. See related article by Yeaton et al., p. 2392 (2).


Assuntos
Dioxigenases , Leucemia , Neoplasias , Animais , Carcinogênese , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Humanos , Inflamação/genética , Leucemia/genética , Camundongos , Monócitos , Mutação , Proteínas Proto-Oncogênicas/genética
14.
Nat Commun ; 13(1): 6230, 2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36266342

RESUMO

TET (Ten-Eleven Translocation) dioxygenases effect DNA demethylation through successive oxidation of the methyl group of 5-methylcytosine (5mC) in DNA. In humans and in mouse models, TET loss-of-function has been linked to DNA damage, genome instability and oncogenesis. Here we show that acute deletion of all three Tet genes, after brief exposure of triple-floxed, Cre-ERT2-expressing mouse embryonic stem cells (mESC) to 4-hydroxytamoxifen, results in chromosome mis-segregation and aneuploidy; moreover, embryos lacking all three TET proteins showed striking variation in blastomere numbers and nuclear morphology at the 8-cell stage. Transcriptional profiling revealed that mRNA encoding a KH-domain protein, Khdc3 (Filia), was downregulated in triple TET-deficient mESC, concomitantly with increased methylation of CpG dinucleotides in the vicinity of the Khdc3 gene. Restoring KHDC3 levels in triple Tet-deficient mESC prevented aneuploidy. Thus, TET proteins regulate Khdc3 gene expression, and TET deficiency results in mitotic infidelity and genome instability in mESC at least partly through decreased expression of KHDC3.


Assuntos
Aneuploidia , Proteínas de Ligação a DNA , Dioxigenases , Células-Tronco Embrionárias Murinas , Animais , Camundongos , 5-Metilcitosina/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo
16.
Reprod Biol ; 22(4): 100701, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36242939

RESUMO

This study intends to investigate the effects of DNA methyltransferase 3B (DNMT3B) and ten-eleven translocation 3 (TET3) on transforming growth factor-ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) in ovarian cancer (OV) cells. According to the specific experiments, the cells were treated with TGF-ß1 for 48 h, and then the expressions of EMT-related proteins (E-cadherin, Vimentin and Snail), TET3 and DNMT3B were quantified by Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Methprimer, methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP) and chromatin immunoprecipitation (ChIP) were used to determine the regulation of DNMT3B on TET3 promoter. The impacts of DNMT3B and TET3 upon the EMT-related proteins and OV cell migration and invasion abilities were evaluated through the rescue experiments and the loss- and gain-of-function experiments. In line with the results, TGF-ß1 down-regulated TET3 and E-cadherin levels, up-regulated Vimentin and Snail levels, promoted migratory and invasive abilities, and increased methylation level of TET3 promoter in OV cells, which however were reversed by shDNMT3B. The binding of DNMT3B to TET3 promoter facilitated the methylation of TET3 promoter. Overexpressed TET3 inhibited the migratory and invasive abilities and EMT of OV cells, whereas shTET3 did the opposite. ShTET3 also offset the regulatory effects of shDNMT3B on EMT, migration and invasion of OV cells. To conclude, DNMT3B mitigates the suppression of TET3 on TGF-ß1-induced EMT in OV cells by methylating the promoter region of TET3.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Dioxigenases , Neoplasias Ovarianas , Feminino , Humanos , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Dioxigenases/genética , Dioxigenases/metabolismo , Transição Epitelial-Mesenquimal/genética , Neoplasias Ovarianas/genética , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta1/farmacologia , Vimentina/genética , Vimentina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo
17.
Leukemia ; 36(12): 2883-2893, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36271153

RESUMO

Recurrent mutations in RNA splicing proteins and epigenetic regulators contribute to the development of myelodysplastic syndrome (MDS) and related myeloid neoplasms. In chronic myelomonocytic leukemia (CMML), SRSF2 mutations occur in ~50% of patients and TET2 mutations in ~60%. Clonal analysis indicates that either mutation can arise as the founder lesion. Based on human cancer genetics we crossed an inducible Srsf2P95H/+ mutant model with Tet2fl/fl mice to mutate both concomitantly in hematopoietic stem cells. At 20-24 weeks post mutation induction, we observed subtle differences in the Srsf2/Tet2 mutants compared to either single mutant. Under conditions of native hematopoiesis with aging, we see a distinct myeloid bias and monocytosis in the Srsf2/Tet2 mutants. A subset of the compound Srsf2/Tet2 mutants display an increased granulocytic and distinctive monocytic proliferation (myelomonocytic hyperplasia), with increased immature promonocytes and monoblasts and binucleate promonocytes. Exome analysis of progressed disease demonstrated mutations in genes and pathways similar to those reported in human CMML. Upon transplantation, recipients developed leukocytosis, monocytosis, and splenomegaly. We reproduce Srsf2/Tet2 co-operativity in vivo, yielding a disease with core characteristics of CMML, unlike single Srsf2 or Tet2 mutation. This model represents a significant step toward building high fidelity and genetically tractable models of CMML.


Assuntos
Dioxigenases , Leucemia Mielomonocítica Crônica , Leucemia Mielomonocítica Juvenil , Síndromes Mielodisplásicas , Humanos , Camundongos , Animais , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/patologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Hematopoese/genética , Mutação , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética
19.
J Clin Invest ; 132(19)2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36189793

RESUMO

The TET family of dioxygenases promote DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Hypothalamic agouti-related peptide-expressing (AGRP-expressing) neurons play an essential role in driving feeding, while also modulating nonfeeding behaviors. Besides AGRP, these neurons produce neuropeptide Y (NPY) and the neurotransmitter GABA, which act in concert to stimulate food intake and decrease energy expenditure. Notably, AGRP, NPY, and GABA can also elicit anxiolytic effects. Here, we report that in adult mouse AGRP neurons, CRISPR-mediated genetic ablation of Tet3, not previously known to be involved in central control of appetite and metabolism, induced hyperphagia, obesity, and diabetes, in addition to a reduction of stress-like behaviors. TET3 deficiency activated AGRP neurons, simultaneously upregulated the expression of Agrp, Npy, and the vesicular GABA transporter Slc32a1, and impeded leptin signaling. In particular, we uncovered a dynamic association of TET3 with the Agrp promoter in response to leptin signaling, which induced 5hmC modification that was associated with a chromatin-modifying complex leading to transcription inhibition, and this regulation occurred in both the mouse models and human cells. Our results unmasked TET3 as a critical central regulator of appetite and energy metabolism and revealed its unexpected dual role in the control of feeding and other complex behaviors through AGRP neurons.


Assuntos
Ansiolíticos , Dioxigenases , 5-Metilcitosina/metabolismo , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , Ansiolíticos/farmacologia , Cromatina/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Humanos , Hipotálamo/metabolismo , Leptina/metabolismo , Camundongos , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia
20.
Microb Cell Fact ; 21(1): 215, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36243861

RESUMO

BACKGROUND: Flavonoids are necessary for plant growth and resistance to adversity and stress. They are also an essential nutrient for human diet and health. Among the metabolites produced in Cannabis sativa (C. sativa), phytocannabinoids have undergone extensive research on their structures, biosynthesis, and biological activities. Besides the phytocannabinoids, C. sativa is also rich in terpenes, alkaloids, and flavonoids, although little research has been conducted in this area. RESULTS: In this study, we identified 11 classes of key enzyme-encoding genes, including 56 members involved in the flavonoid biosynthesis in C. sativa, from their physical characteristics to their expression patterns. We screened the potentially step-by-step enzymes catalyzing the precursor phenylalanine to the end flavonoids using a conjoin analysis of gene expression with metabolomics from different tissues and chemovars. Flavonol synthase (FLS), belonging to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily, catalyzes the dihydroflavonols to flavonols. In vitro recombinant protein activity analysis revealed that CsFLS2 and CsFLS3 had a dual function in converting naringenin (Nar) to dihydrokaempferol (DHK), as well as dihydroflavonols to flavonols with different substrate preferences. Meanwhile, we found that CsFLS2 produced apigenin (Api) in addition to DHK and kaempferol when Nar was used as the substrate, indicating that CsFLS2 has an evolutionary relationship with Cannabis flavone synthase I. CONCLUSIONS: Our study identified key enzyme-encoding genes involved in the biosynthesis of flavonoids in C. sativa and highlighted the key CsFLS genes that generate flavonols and their diversified functions in C. sativa flavonoid production. This study paves the way for reconstructing the entire pathway for C. sativa's flavonols and cannflavins production in heterologous systems or plant culture, and provides a theoretical foundation for discovering new cannabis-specific flavonoids.


Assuntos
Cannabis , Dioxigenases , Apigenina , Cannabis/genética , Cannabis/metabolismo , Dioxigenases/genética , Flavonoides , Flavonóis , Humanos , Quempferóis , Ácidos Cetoglutáricos/metabolismo , Fenilalanina , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Terpenos
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