RESUMO
Thiol dioxygenases (TDOs) are nonheme Fe(II)dependent enzymes that catalyze the O2-dependent oxidation of thiol substrates to their corresponding sulfinic acids. Six classes of TDOs have thus far been identified and two, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), are found in eukaryotes. All TDOs belong to the cupin superfamily of enzymes, which share a common ßbarrel fold and two cupin motifs: G(X)5HXH(X)3-6E(X)6G and G(X)5-7PXG(X)2H(X)3N. Crystal structures of TDOs revealed that these enzymes contain a relatively rare, neutral 3His ironbinding facial triad. Despite this shared metal-binding site, TDOs vary greatly in their secondary coordination spheres. Sitedirected mutagenesis has been used extensively to explore the impact of changes in secondary sphere residues on substrate specificity and enzymatic efficiency. This chapter summarizes site-directed mutagenesis studies of eukaryotic TDOs, focusing on the tools and practicality of nonstandard amino acid incorporation.
Assuntos
Aminoácidos , Dioxigenases , Mutagênese Sítio-Dirigida , Dioxigenases/química , Dioxigenases/metabolismo , Dioxigenases/genética , Aminoácidos/metabolismo , Aminoácidos/química , Especificidade por Substrato , Cisteína Dioxigenase/química , Cisteína Dioxigenase/metabolismo , Cisteína Dioxigenase/genética , Compostos de Sulfidrila/metabolismo , Compostos de Sulfidrila/química , Humanos , Animais , Modelos MolecularesRESUMO
BACKGROUND: Breast tumorigenesis is a complex and multistep process accompanied by both genetic and epigenetic dysregulation. In contrast to the extensive studies on DNA epigenetic modifications 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in malignant breast tumors, their roles in the early phases of breast tumorigenesis remain ambiguous. RESULTS: DNA 5hmC and 5mC exhibited a consistent and significant decrease from usual ductal hyperplasia to atypical ductal hyperplasia and subsequently to ductal carcinoma in situ (DCIS). However, 5hmC showed a modest increase in invasive ductal breast cancer compared to DCIS. Genomic analyses showed that the changes in 5hmC and 5mC levels occurred around the transcription start sites (TSSs), and the modification levels were strongly correlated with gene expression levels. Meanwhile, it was found that differentially hydroxymethylated regions (DhMRs) and differentially methylated regions (DMRs) were overlapped in the early phases and accompanied by the enrichment of active histone marks. In addition, TET2-related DNA demethylation was found to be involved in breast tumorigenesis, and four transcription factor binding sites (TFs: ESR1, FOXA1, GATA3, FOS) were enriched in TET2-related DhMRs/DMRs. Intriguingly, we also identified a certain number of common DhMRs between tumor samples and cell-free DNA (cfDNA). CONCLUSIONS: Our study reveals that dynamic changes in DNA 5hmC and 5mC play a vital role in propelling breast tumorigenesis. Both TFs and active histone marks are involved in TET2-related DNA demethylation. Concurrent changes in 5hmC signals in primary breast tumors and cfDNA may play a promising role in breast cancer screening.
Assuntos
5-Metilcitosina , Neoplasias da Mama , Proteínas de Ligação a DNA , Dioxigenases , Proteínas Proto-Oncogênicas , Humanos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Feminino , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Carcinogênese/genética , Metilação de DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Desmetilação do DNARESUMO
In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.
Assuntos
5-Metilcitosina , Proteínas de Ligação a DNA , Dioxigenases , Proteínas Proto-Oncogênicas , Dioxigenases/metabolismo , Dioxigenases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análise , 5-Metilcitosina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ensaios Enzimáticos/métodos , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Metilação de DNA , Citosina/análogos & derivados , Citosina/análise , Citosina/metabolismo , Citosina/química , OxirreduçãoRESUMO
Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.
Assuntos
Oxigenases , Pseudomonas fluorescens , Pseudomonas fluorescens/enzimologia , Oxigenases/metabolismo , Oxigenases/química , Dioxigenases/metabolismo , Dioxigenases/química , Dioxigenases/genética , Ensaios Enzimáticos/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de ElétronsRESUMO
Rieske non-heme iron oxygenases are ubiquitously expressed in prokaryotes. These enzymes catalyze a wide variety of reactions, including cis-dihydroxylation, mono-hydroxylation, sulfoxidation, and demethylation. They contain a Rieske-type [2Fe-2S] cluster and an active site with a mono-nuclear iron bound to a 2-His carboxylate triad. Naphthalene 1,2 dioxygenase, a representative of this family, catalyzes the conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. This transformation requires naphthalene, two electrons, and an oxygen molecule. The first structure of the terminal oxygenase component of a Rieske non-heme iron oxygenase to be determined was naphthalene 1,2 dioxygenase (NDO-O). In this article, we describe in detail the methods used to recombinantly express and purify NDO-O in rich and minimal salts media, the crystallization of NDO-O for structure determination by X-ray crystallography, the challenges faced, and the methods used for the preparation of enzyme ligand complexes. The methods used here resulted in the determination of several NDO-O complexes with aromatic substrates, nitric oxide, oxygen molecule, and products, leading to an initial understanding of the mechanism of enzyme catalysis and the molecular determinants of the regio- and stereo-specificity of this class of enzymes.
Assuntos
Dioxigenases , Dioxigenases/química , Dioxigenases/metabolismo , Dioxigenases/genética , Cristalografia por Raios X/métodos , Naftalenos/química , Naftalenos/metabolismo , Oxigenases/química , Oxigenases/metabolismo , Domínio Catalítico , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Cristalização/métodos , Modelos Moleculares , Complexos MultienzimáticosRESUMO
Extradiol dioxygenases are a class of non-heme iron-dependent enzymes found in eukaryotes and prokaryotes that play a vital role in the aerobic catabolism of aromatic compounds. They are generally divided into three evolutionarily independent superfamilies with different protein folds. Our recent studies have shed light on the catalytic mechanisms and structure-function relationships of two specific extradiol dioxygenases: 3-hydroxyanthranilate-3,4-dioxygenase, a Type III enzyme essential in mammals for producing a precursor for nicotinamide adenine dinucleotide, and L-3,4-dihydroxyphenylalanine dioxygenase, an uncommon form of Type I enzymes involved in natural product biosynthesis. This work details the expression and isolation methods for these extradiol dioxygenases and introduces approaches to achieve homogeneity and high occupancy of the enzyme metal centers. Techniques such as ultraviolet-visible and electron paramagnetic resonance spectroscopies, as well as oxygen electrode measurements, are discussed for probing the interaction of the non-heme iron center with ligands and characterizing enzymatic activities. Moreover, protein crystallization has been demonstrated as a powerful tool to study these enzymes. We highlight in crystallo reactions and single-crystal spectroscopic methods to further elucidate enzymatic functions and protein dynamics.
Assuntos
Cristalino , Cristalino/enzimologia , Cristalino/metabolismo , Animais , Oxigenases/metabolismo , Oxigenases/química , Oxigenases/genética , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Dioxigenases/metabolismo , Dioxigenases/química , Dioxigenases/genéticaRESUMO
Non-heme iron oxygenases constitute a versatile enzyme family that is crucial for incorporating molecular oxygen into diverse biomolecules. Despite their importance, only a limited number of these enzymes have been structurally and functionally characterized. Surprisingly, there remains a significant gap in understanding how these enzymes utilize a typical architecture and reaction mechanism to catalyze a wide range of reactions. Improving our understanding of these catalysts holds promise for advancing both fundamental enzymology and practical applications. This chapter aims to outline methods for heterologous expression, enzyme preparation, in vitro enzyme assays, and crystallization of biphenyl dioxygenase, phthalate dioxygenase and terephthalate dioxygenase. These enzymes catalyze the dihydroxylation of biphenyl, phthalate and terephthalate molecules, serving as a model for functional and structural analysis of other non-heme iron oxygenases.
Assuntos
Compostos de Bifenilo , Cristalização , Ácidos Ftálicos , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Compostos de Bifenilo/química , Cinética , Cristalização/métodos , Dioxigenases/química , Dioxigenases/metabolismo , Dioxigenases/genética , Ferro/química , Ferro/metabolismo , Cristalografia por Raios X/métodos , Ensaios Enzimáticos/métodos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , OxigenasesRESUMO
Chronic myelomonocytic leukemia (CMML) is a rare blood cancer of older adults (3 in every 1,000,000 persons) characterized by poor survival and lacking effective mutation-specific therapy. Mutations in the ubiquitin ligase Cbl occur frequently in CMML and share biological and molecular features with a clonal disease occurring in children, juvenile myelomonocytic leukemia (JMML). Here we analyzed the clinical presentations, molecular features and immunophenotype of CMML patients with CBL mutations enrolled in a prospective Phase II clinical trial stratified according to molecular markers. Clinically, CBL mutations were associated with increased bone marrow blasts at diagnosis, leukocytosis and splenomegaly, similar to patients harboring NRAS or KRAS mutations. Interestingly, 64% of patients presented with more than one CBL variant implying a complex subclonal architecture, often with co-occurrence of TET2 mutations. We found CBL mutations in CMML frequently clustered in the RING domain in contrast to JMML, where mutations frequently involve the linker helix region (P<0.0001). According to our comparative alignment of available X-ray structures, mutations in the linker helix region such as Y371E give rise to conformational differences that could be exploited by targeted therapy approaches. Furthermore, we noted an increased percentage of CMML CD34+ stem and progenitor cells expressing CD116 and CD131 in all CBL mutant cases and increased CD116 receptor density compared to healthy controls, similar to CMML overall. In summary, our data demonstrate that CBL mutations are associated with distinct molecular and clinical features in CMML and are potentially targetable with CD116-directed immunotherapy.
Assuntos
Leucemia Mielomonocítica Crônica , Mutação , Proteínas Proto-Oncogênicas c-cbl , Humanos , Proteínas Proto-Oncogênicas c-cbl/genética , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/patologia , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Leucemia Mielomonocítica Juvenil/genética , Idoso de 80 Anos ou mais , Adulto , Dioxigenases/genética , Proteínas de Ligação a DNA/genética , Estudos ProspectivosRESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) is linked to diverse aging-related diseases, including hematologic malignancy and atherosclerotic cardiovascular disease (ASCVD). While CHIP is common among older adults, the underlying factors driving its development are largely unknown. To address this, we performed whole-exome sequencing on 8,374 blood DNA samples collected from 4,187 Atherosclerosis Risk in Communities Study (ARIC) participants over a median follow-up of 21 years. During this period, 735 participants developed incident CHIP. Splicing factor genes (SF3B1, SRSF2, U2AF1, and ZRSR2) and TET2 CHIP grow significantly faster than DNMT3A non-R882 clones. We find that age at baseline and sex significantly influence the incidence of CHIP, while ASCVD and other traditional ASCVD risk factors do not exhibit such associations. Additionally, baseline synonymous passenger mutations are strongly associated with CHIP status and are predictive of new CHIP clone acquisition and clonal growth over extended follow-up, providing valuable insights into clonal dynamics of aging hematopoietic stem and progenitor cells. This study also reveals associations between germline genetic variants and incident CHIP. Our comprehensive longitudinal assessment yields insights into cell-intrinsic and -extrinsic factors contributing to the development and progression of CHIP clones in older adults.
Assuntos
Hematopoiese Clonal , Dioxigenases , Humanos , Hematopoiese Clonal/genética , Masculino , Feminino , Idoso , Estudos Longitudinais , Pessoa de Meia-Idade , Dioxigenases/genética , DNA Metiltransferase 3A , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Aterosclerose/genética , Fatores de Risco , Sequenciamento do Exoma , Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Envelhecimento/genética , Incidência , MutaçãoRESUMO
The isonitrile group is a compact, electron-rich moiety coveted for its commonplace as a building block and bioorthogonal functionality in synthetic chemistry and chemical biology. Hundreds of natural products containing an isonitrile group with intriguing bioactive properties have been isolated from diverse organisms. Our recent discovery of a conserved biosynthetic gene cluster in some Actinobacteria species highlighted a novel enzymatic pathway to isonitrile formation involving a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase. Here, we focus this chapter on recent advances in understanding and probing the biosynthetic machinery for isonitrile synthesis by non-heme iron(II) and α-ketoglutarate-dependent dioxygenases. We will begin by describing how to harness isonitrile enzymatic machinery through heterologous expression, purification, synthetic strategies, and in vitro biochemical/kinetic characterization. We will then describe a generalizable strategy to probe the mechanism for isonitrile formation by combining various spectroscopic methods. The chapter will also cover strategies to study other enzyme homologs by implementing coupled assays using biosynthetic pathway enzymes. We will conclude this chapter by addressing current challenges and future directions in understanding and engineering isonitrile synthesis.
Assuntos
Nitrilas , Nitrilas/metabolismo , Nitrilas/química , Ácidos Cetoglutáricos/metabolismo , Oxirredutases/metabolismo , Oxirredutases/genética , Oxirredutases/química , Família Multigênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ferro/metabolismo , Ferro/química , Vias Biossintéticas , Dioxigenases/metabolismo , Dioxigenases/genética , Dioxigenases/química , Cinética , Actinobacteria/enzimologia , Actinobacteria/genética , Actinobacteria/metabolismoRESUMO
This review deals with the functional characteristics and biological roles of enzymes participating in DNA methylation and demethylation as key factors in epigenetic regulation of gene expression. The set of enzymes that carry out such processes in human cells is limited to representatives of two families, namely DNMT (DNA methyltransferases) and TET (DNA dioxygenases). The review presents detailed information known today about each functionally important member of these families and describes the catalytic activity and roles in the mammalian body while also providing examples of dysregulation of the expression and/or activity of these enzymes in conjunction with the development of some human disorders, including cancers, neurodegenerative diseases, and developmental pathologies. By combining the up-to-date information on the dysfunction of various enzymes that control the DNA "methylome" in the human body, we hope not only to draw attention to the importance of the maintenance of a required DNA methylation level (ensuring epigenetic regulation of gene expression and normal functioning of the entire body) but also to help identify new targets for directed control over the activity of the enzymes that implement the balance between processes of DNA methylation and demethylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Dioxigenases , Epigênese Genética , Humanos , Dioxigenases/metabolismo , Dioxigenases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , AnimaisRESUMO
Methylated arsenicals, including highly toxic species, such as methylarsenite [MAs(III)], are pervasive in the environment. Certain microorganisms possess the ability to detoxify MAs(III) by ArsI-catalyzed demethylation. Here, we characterize a bifunctional enzyme encoded by the arsI gene from Acidovorax sp. ST3, which can detoxify MAs(III) through both the demethylation and oxidation pathways. Deletion of the 22 C-terminal amino acids of ArsI increased its demethylation activity while reducing the oxidation activity. Further deletion of 44 C-terminal residues enhanced the MAs(III) demethylation activity. ArsI has four vicinal cysteine pairs, with the first pair being necessary for MAs(III) demethylation, while at least one of the other three pairs contributes to MAs(III) oxidation. Molecular modeling and site-directed mutagenesis indicated that one of the C-terminal vicinal cysteine pairs is involved in modulating the switch between oxidase and demethylase activity. These findings underscore the critical role of the C-terminal region in modulating the enzymatic activities of ArsI, particularly in MAs(III) demethylation. This research reveals the structure-function relationship of the ArsI enzyme and advances our understanding of the MAs(III) metabolism in bacteria.
Assuntos
Dioxigenases , Oxirredução , Dioxigenases/metabolismo , Dioxigenases/genética , Desmetilação , Comamonadaceae/enzimologia , Comamonadaceae/metabolismoRESUMO
The etiology of neurodevelopmental disorders and epilepsy is very heterogeneous and partly still unknown, and the research of causative genes related to these diseases is still in progress. In 2020, pathogenic variants of the TET3 gene were associated with Beck-Fahrner syndrome, which is characterized by neurodevelopmental delay, intellectual and learning disabilities of variable degree, growth abnormalities, hypotonia and seizures. Variants of TET3 have been described having both an autosomal dominant with a milder phenotype and an autosomal recessive pattern. To date, in the literature, only 28 patients are reported with pathogenic variants of the TET3 gene, and only 9 of them have epilepsy. We describe a 31-year-old woman with macrocephaly, mild neurodevelopmental delay and a long history of epilepsy. Trio-based exome sequencing identified a de novo heterozygous TET3 variant, c.2867G>A p.(Arg956Gln), never described before, absent in the general population and predicted to be potentially pathogenetic by bioinformatics tools. This report aims to describe the clinical history of our patient, the pharmacological treatment and clinical response, as well as the biological characteristics of this new variant.
Assuntos
Epilepsia , Megalencefalia , Mutação de Sentido Incorreto , Humanos , Feminino , Adulto , Epilepsia/genética , Megalencefalia/genética , Dioxigenases/genética , Proteínas de Ligação a DNA/genética , Sequenciamento do Exoma , FenótipoRESUMO
The inversion of substrate size specificity is an evolutionary roadblock for proteins. The Duf4243 dioxygenases GedK and BTG13 are known to catalyze the aromatic cleavage of bulky tricyclic hydroquinone. In this study, we discover a Duf4243 dioxygenase PaD that favors small monocyclic hydroquinones from the penicillic-acid biosynthetic pathway. Sequence alignments between PaD and GedK and BTG13 suggest PaD has three additional motifs, namely motifs 1-3, distributed at different positions in the protein sequence. X-ray crystal structures of PaD with the substrate at high resolution show motifs 1-3 determine three loops (loops 1-3). Most intriguing, loops 1-3 stack together at the top of the pocket, creating a lid-like tertiary structure with a narrow channel and a clearly constricted opening. This drastically changes the substrate specificity by determining the entry and binding of much smaller substrates. Further genome mining suggests Duf4243 dioxygenases with motifs 1-3 belong to an evolutionary branch that is extensively involved in the biosynthesis of natural products and has the ability to degrade diverse monocyclic hydroquinone pollutants. This study showcases how natural enzymes alter the substrate specificity fundamentally by incorporating new small motifs, with a fixed overall scaffold-architecture. It will also offer a theoretical foundation for the engineering of substrate specificity in enzymes and act as a guide for the identification of aromatic dioxygenases with distinct substrate specificities.
Assuntos
Motivos de Aminoácidos , Dioxigenases , Especificidade por Substrato , Dioxigenases/metabolismo , Dioxigenases/genética , Dioxigenases/química , Cristalografia por Raios X , Hidroquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Sequência de Aminoácidos , Modelos Moleculares , Alinhamento de SequênciaRESUMO
Clonal haematopoiesis of indeterminate potential (CHIP) has been associated with many adverse health outcomes. However, further research is required to understand the critical genes and pathways relevant to CHIP subtypes, evaluate how CHIP clones evolve with time, and further advance functional characterisation and therapeutic studies. Large epidemiological studies are well placed to address these questions but often collect saliva rather than blood from participants. Paired saliva- and blood-derived DNA samples from 94 study participants were sequenced using a targeted CHIP-gene panel. The ten genes most frequently identified to carry CHIP-associated variants were analysed. Fourteen unique variants associated with CHIP, ten in DNMT3A, two in TP53 and two in TET2, were identified with a variant allele fraction (VAF) between 0.02 and 0.2 and variant depth ≥ 5 reads. Eleven of these CHIP-associated variants were detected in both the blood- and saliva-derived DNA sample. Three variants were detected in blood with a VAF > 0.02 but fell below this threshold in the paired saliva sample (VAF 0.008-0.013). Saliva-derived DNA is suitable for detecting CHIP-associated variants. Saliva can offer a cost-effective biospecimen that could both advance CHIP research and facilitate clinical translation into settings such as risk prediction, precision prevention, and treatment monitoring.
Assuntos
Hematopoiese Clonal , DNA Metiltransferase 3A , Proteínas de Ligação a DNA , Saliva , Humanos , Saliva/metabolismo , Hematopoiese Clonal/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Masculino , DNA/genética , Dioxigenases/genética , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Adulto , Pessoa de Meia-Idade , Idoso , AlelosRESUMO
BACKGROUND: Cardiac fibrosis is the hallmark of all forms of chronic heart disease. Activation and proliferation of cardiac fibroblasts are the prime mediators of cardiac fibrosis. Existing studies show that ROS and inflammatory cytokines produced during fibrosis not only signal proliferative stimuli but also contribute to DNA damage. Therefore, as a prerequisite to maintain sustained proliferation in fibroblasts, activation of distinct DNA repair mechanism is essential. RESULT: In this study, we report that TET3, a DNA demethylating enzyme, which has been shown to be reduced in cardiac fibrosis and to exert antifibrotic effects does so not only through its demethylating activity but also through maintaining genomic integrity by facilitating error-free homologous recombination (HR) repair of DNA damage. Using both in vitro and in vivo models of cardiac fibrosis as well as data from human heart tissue, we demonstrate that the loss of TET3 in cardiac fibroblasts leads to spontaneous DNA damage and in the presence of TGF-ß to a shift from HR to the fast but more error-prone non-homologous end joining repair pathway. This shift contributes to increased fibroblast proliferation in a fibrotic environment. In vitro experiments showed TET3's recruitment to H2O2-induced DNA double-strand breaks (DSBs) in mouse cardiac fibroblasts, promoting HR repair. Overexpressing TET3 counteracted TGF-ß-induced fibroblast proliferation and restored HR repair efficiency. Extending these findings to human cardiac fibrosis, we confirmed TET3 expression loss in fibrotic hearts and identified a negative correlation between TET3 levels, fibrosis markers, and DNA repair pathway alteration. CONCLUSION: Collectively, our findings demonstrate TET3's pivotal role in modulating DDR and fibroblast proliferation in cardiac fibrosis and further highlight TET3 as a potential therapeutic target.
Assuntos
Dioxigenases , Fibroblastos , Fibrose , Animais , Fibrose/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Camundongos , Humanos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Dano ao DNA/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Miocárdio/patologia , Miocárdio/metabolismo , Masculino , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
BACKGROUND: Cardiac surgery triggers sterile innate immune responses leading to postoperative complications. Clonal hematopoiesis (CH) is associated with short-term inflammation-mediated outcomes after cardiac surgery. The impact of CH on long-term postoperative outcomes remains unknown. METHODS AND RESULTS: In this cohort study, patients undergoing elective cardiac surgery were included from January 2017 to September 2019. Patients were screened for CH using a predefined gene panel of 19 genes. Recorded clinical events were all-cause death, major adverse cardiac and cerebral events including cardiovascular death, myocardial infarction or nonscheduled coronary revascularization, stroke, and hospitalization for acute heart failure. The primary study outcome was time to a composite criterion including all-cause mortality and major adverse cardiac and cerebral events. Among 314 genotyped patients (median age: 67 years; interquartile range 59-74 years), 139 (44%) presented with CH, based on a variant allelic frequency ≥1%. Carriers of CH had a higher proportion of patients with a history of atrial fibrillation (26% for CH versus 17% for non-CH carriers, P=0.022). The most frequently mutated genes were DNMT3A, TET2, and ASXL1. After a median follow-up of 1203 [813-1435] days, the primary outcome occurred in 50 patients. After multivariable adjustment, CH was independently associated with a higher risk for the primary outcome (hazard ratio, 1.88 [95% CI, 1.05-3.41], P=0.035). Most adverse events occurred in patients carrying TET2 variants. CONCLUSIONS: In patients undergoing cardiac surgery, CH is frequent and associated with a 2-fold increased long-term risk for major adverse clinical outcomes. CH is a novel risk factor for long-term postcardiac surgery complications and might be useful to personalize management decisions. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03376165.
Assuntos
Procedimentos Cirúrgicos Cardíacos , Hematopoiese Clonal , Complicações Pós-Operatórias , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Hematopoiese Clonal/genética , Dioxigenases/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Mutação , Complicações Pós-Operatórias/genética , Complicações Pós-Operatórias/epidemiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Medição de Risco/métodos , Fatores de Risco , Fatores de TempoRESUMO
In this study, a novel strain Burkholderia stabilis TF-2 capable of assimilatory and co-metabolic degradation of chlorobenzenes was obtained. The interaction between chlorobenzene (CB) and target enzymes, as well as the metabolic pathways in TF-2, were elucidated using multi-omics and molecular docking techniques. Results of degradation experiments indicated that TF-2 assimilated CB at a rate of 0.22-0.66 mg·gcell-1·h-1 in concentrations of 20-200 mg L-1. Additionally, TF-2 also used sodium succinate and sodium citrate as substrates to co-metabolize CB, with degradation rates of 0.26-2.00 and 0.31-1.72 mol·gcell-1·h-1, respectively. Whole-genome sequencing revealed over 18 novel genes associated with aromatic hydrocarbon degradation in TF-2. Transcriptomic analysis showed that CB induced the high expression of 119 genes involved in CB metabolism and late mineralization. The significant up-regulation of the bedC1 (encoding a ring-hydroxylated dioxygenase), CatA (chlorocatechol 1,2-dioxygenase), pcaJ (3-oxoadipate CoA-transferase alpha subunit) and fadA (acetyl-CoA acyltransferase) genes facilitated CB metabolism. Based on these findings, a metabolic pathway for CB was constructed, with the key step involving ortho cleavage of the aromatic ring under the action of the catA gene. Furthermore, molecular docking revealed that CB bound to bedC1 with -4.5 kcal mol-1 through hydrophobic bonds, π-stacking, and a halogen bond. These results provide strong support for development of efficient strains to enhance the removal of chlorinated organic compounds.
Assuntos
Biodegradação Ambiental , Burkholderia , Clorobenzenos , Simulação de Acoplamento Molecular , Clorobenzenos/metabolismo , Burkholderia/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Dioxigenases/metabolismo , Dioxigenases/genéticaRESUMO
Matrix stiffness is a crucial factor in the tumor microenvironment, impacting tumor progression and development. TET2 is vital for epigenetic regulation in melanoma and is significantly reduced in advanced melanomas compared with nevi and thin melanomas. However, it is unclear how TET2 mediates the effect of matrix stiffness on melanoma cells. This study utilized A2058 cell lines and prepared different stiffness collagen hydrogels to evaluate TET2 overexpression (TET2OE) and mutant (TET2M) melanoma cells' activity, proliferation, and invasion. A2058 melanoma cells' viability and invasion decreased with increased matrix stiffness, with TET2OE cells experiencing a more significant impact than TET2M cells. Methylation analysis revealed that TET2 determines gene methylation levels, influencing cell-ECM interactions. Transcriptome analysis confirmed that TET2 promotes matrix stiffness's effect on melanoma cell fate. This research provides promising directions and opportunities for melanoma treatment.
Assuntos
Proliferação de Células , Metilação de DNA , Proteínas de Ligação a DNA , Dioxigenases , Matriz Extracelular , Melanoma , Proteínas Proto-Oncogênicas , Humanos , Melanoma/genética , Melanoma/patologia , Melanoma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Linhagem Celular Tumoral , Metilação de DNA/genética , Matriz Extracelular/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proliferação de Células/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Cultura de Células/métodos , Microambiente Tumoral/genética , Invasividade Neoplásica/genética , Hidrogéis/química , Sobrevivência Celular/genéticaRESUMO
Tamoxifen, a selective estrogen receptor modulator (SERM), exhibits dual agonist or antagonist effects contingent upon its binding to either G-protein-coupled estrogen receptor (GPER) or estrogen nuclear receptor (ESR). Estrogen signaling plays a pivotal role in initiating epigenetic alterations and regulating estrogen-responsive genes in breast cancer. Employing three distinct breast cancer cell lines-MCF-7 (ESR+; GPER+), MDA-MB-231 (ESR-; GPER-), and SkBr3 (ESR-; GPER+)-this study subjected them to treatment with two tamoxifen derivatives: 4-hydroxytamoxifen (4-HT) and endoxifen (Endox). Through 2D high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS), varying levels of 5-methylcytosine (5-mC) were found, with MCF-7 displaying the highest levels. Furthermore, TET3 mRNA expression levels varied among the cell lines, with MCF-7 exhibiting the lowest expression. Notably, treatment with 4-HT induced significant changes in TET3 expression across all cell lines, with the most pronounced increase seen in MCF-7 and the least in MDA-MB-231. These findings underscore the influence of tamoxifen derivatives on DNA methylation patterns, particularly through modulating TET3 expression, which appears to be contingent on the presence of estrogen receptors. This study highlights the potential of targeting epigenetic modifications for personalized anti-cancer therapy, offering a novel avenue to improve treatment outcomes.