RESUMEN
The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.
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Bacterias/inmunología , Bacterias/virología , Bacteriófagos/fisiología , CMP Cíclico/metabolismo , Nucleótidos Cíclicos/metabolismo , Uridina Monofosfato/metabolismo , Secuencia de Aminoácidos , Bacterias/genética , Burkholderia/enzimología , CMP Cíclico/química , Ciclización , Escherichia coli/enzimología , Modelos Moleculares , Mutación/genética , Nucleótidos Cíclicos/química , Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/metabolismo , Pirimidinas/metabolismo , Uridina Monofosfato/químicaRESUMEN
Larotrectinib is a small-molecule kinase inhibitor that targets NTRK fusions that occur in multiple types of cancer. Its FDA approval represents the first instance of a treatment indication being designated "tumor-agnostic" from the outset, being based on actionable genomic insights. To view this Bench to Bedside, open or download the PDF.
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Pirazoles/metabolismo , Pirazoles/farmacología , Pirimidinas/metabolismo , Pirimidinas/farmacología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Humanos , Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/agonistas , Receptor trkB/metabolismoRESUMEN
While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.
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Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Terapia Molecular Dirigida , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Diferenciación Celular , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Células Mieloides/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pirimidinas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It is increasingly appreciated that oncogenic transformation alters cellular metabolism to facilitate cell proliferation, but less is known about the metabolic changes that promote cancer cell aggressiveness. Here, we analyzed metabolic gene expression in cancer cell lines and found that a set of high-grade carcinoma lines expressing mesenchymal markers share a unique 44 gene signature, designated the "mesenchymal metabolic signature" (MMS). A FACS-based shRNA screen identified several MMS genes as essential for the epithelial-mesenchymal transition (EMT), but not for cell proliferation. Dihydropyrimidine dehydrogenase (DPYD), a pyrimidine-degrading enzyme, was highly expressed upon EMT induction and was necessary for cells to acquire mesenchymal characteristics in vitro and for tumorigenic cells to extravasate into the mouse lung. This role of DPYD was mediated through its catalytic activity and enzymatic products, the dihydropyrimidines. Thus, we identify metabolic processes essential for the EMT, a program associated with the acquisition of metastatic and aggressive cancer cell traits.
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Transición Epitelial-Mesenquimal , Pirimidinas/metabolismo , Animales , Carcinoma/metabolismo , Línea Celular Tumoral , Dihidrouracilo Deshidrogenasa (NADP)/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Mesodermo/citología , Mesodermo/metabolismo , Ratones , ARN Interferente Pequeño/metabolismoRESUMEN
The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3-10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.
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Bacteriófagos , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófago T4/metabolismo , Bacteriófagos/fisiología , Sistemas CRISPR-Cas/genética , Endonucleasas/metabolismo , Escherichia coli/metabolismo , Nucleótidos Cíclicos/metabolismo , Oligonucleótidos , Pirimidinas/metabolismoRESUMEN
Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression.
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Genes Mitocondriales , Pirimidinas , Pirimidinas/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Nucleótidos , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Ribonucleótidos/genéticaRESUMEN
N6-(2-deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapyâ¢dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapyâ¢dG preferentially gives rise to G â T transversions and G â A transitions. However, the molecular basis by which Fapyâ¢dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapyâ¢dG, inserts Fapyâ¢dGTP, and extends from Fapyâ¢dG at the primer terminus. When Fapyâ¢dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapyâ¢dGTP is a poor substrate but is incorporated â¼3-times more efficiently opposite dA than dC. Extension from Fapyâ¢dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapyâ¢dG is likely to be the source of the mutagenic effects of the lesion and not Fapyâ¢dGTP. These experiments increase our understanding of the promutagenic effects of Fapyâ¢dG.
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ADN Polimerasa beta , Replicación del ADN , Formamidas , Furanos , Pirimidinas , Humanos , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , ADN Polimerasa beta/metabolismo , ADN Polimerasa beta/química , Cinética , Modelos Moleculares , Pirimidinas/química , Pirimidinas/metabolismo , Furanos/química , Furanos/metabolismo , Formamidas/metabolismo , MutagénesisRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, in which prognosis is determined by liver fibrosis. A common variant in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13, rs72613567-A) is associated with a reduced risk of fibrosis in NAFLD, but the underlying mechanism(s) remains unclear. We investigated the effects of this variant in the human liver and in Hsd17b13 knockdown in mice by using a state-of-the-art metabolomics approach. We demonstrate that protection against liver fibrosis conferred by the HSD17B13 rs72613567-A variant in humans and by the Hsd17b13 knockdown in mice is associated with decreased pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase. Furthermore, we show that hepatic pyrimidines are depleted in two distinct mouse models of NAFLD and that inhibition of pyrimidine catabolism by gimeracil phenocopies the HSD17B13-induced protection against liver fibrosis. Our data suggest pyrimidine catabolism as a therapeutic target against the development of liver fibrosis in NAFLD.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ratones , Hígado/metabolismo , Cirrosis Hepática/patología , Enfermedad del Hígado Graso no Alcohólico/patología , Pirimidinas/farmacología , Pirimidinas/metabolismoRESUMEN
5-Methylated cytosine is a frequent modification in eukaryotic RNA and DNA influencing mRNA stability and gene expression. Here we show that free 5-methylcytidine (5mC) and 5-methyl-2'-deoxycytidine are generated from nucleic acid turnover in Arabidopsis thaliana, and elucidate how these cytidines are degraded, which is unclear in eukaryotes. First CYTIDINE DEAMINASE produces 5-methyluridine (5mU) and thymidine which are subsequently hydrolyzed by NUCLEOSIDE HYDROLASE 1 (NSH1) to thymine and ribose or deoxyribose. Interestingly, far more thymine is generated from RNA than from DNA turnover, and most 5mU is directly released from RNA without a 5mC intermediate, since 5-methylated uridine (m5U) is an abundant RNA modification (m5U/U â¼1%) in Arabidopsis. We show that m5U is introduced mainly by tRNA-SPECIFIC METHYLTRANSFERASE 2A and 2B. Genetic disruption of 5mU degradation in the NSH1 mutant causes m5U to occur in mRNA and results in reduced seedling growth, which is aggravated by external 5mU supplementation, also leading to more m5U in all RNA species. Given the similarities between pyrimidine catabolism in plants, mammals and other eukaryotes, we hypothesize that the removal of 5mU is an important function of pyrimidine degradation in many organisms, which in plants serves to protect RNA from stochastic m5U modification.
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Arabidopsis , ARN , Animales , Timina , Uridina/metabolismo , Pirimidinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , ADN , Mamíferos/genéticaRESUMEN
Ebola virus (EBOV) disease is characterized by lymphopenia, breach in vascular integrity, cytokine storm, and multiorgan failure. The pathophysiology of organ involvement, however, is incompletely understood. Using [18F]-DPA-714 positron emission tomography (PET) imaging targeting the translocator protein (TSPO), an immune cell marker, we sought to characterize the progression of EBOV-associated organ-level pathophysiology in the EBOV Rhesus macaque model. Dynamic [18F]-DPA-714 PET/computed tomography imaging was performed longitudinally at baseline and at multiple time points after EBOV inoculation, and distribution volumes (Vt) were calculated as a measure of peripheral TSPO binding. Using a mixed-effect linear regression model, spleen and lung Vt decreased, while the bone marrow Vt increased over time after infection. No clear trend was found for liver Vt. Multiple plasma cytokines correlated negatively with lung/spleen Vt and positively with bone marrow Vt. Multiplex immunofluorescence staining in spleen and lung sections confirmed organ-level lymphoid and monocytic loss/apoptosis, thus validating the imaging results. Our findings are consistent with EBOV-induced progressive monocytic and lymphocytic depletion in the spleen, rather than immune activation, as well as depletion of alveolar macrophages in the lungs, with inefficient reactive neutrophilic activation. Increased bone marrow Vt, on the other hand, suggests hematopoietic activation in response to systemic immune cell depletion and leukocytosis and could have prognostic relevance. In vivo PET imaging provided better understanding of organ-level pathophysiology during EBOV infection. A similar approach can be used to delineate the pathophysiology of other systemic infections and to evaluate the effectiveness of newly developed treatment and vaccine strategies.
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Fiebre Hemorrágica Ebola , Tomografía de Emisión de Positrones , Receptores de GABA , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Fiebre Hemorrágica Ebola/diagnóstico por imagen , Fiebre Hemorrágica Ebola/patología , Pulmón/patología , Macaca mulatta , Tomografía de Emisión de Positrones/métodos , Pirazoles/metabolismo , Pirimidinas/metabolismo , Receptores de GABA/metabolismo , Bazo/patologíaRESUMEN
An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.
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Biocatálisis , Nitrilos , Oxidorreductasas , Pirazoles , Pirimidinas , Nitrilos/química , Nitrilos/metabolismo , Pirimidinas/química , Pirimidinas/biosíntesis , Pirimidinas/metabolismo , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Pirazoles/química , Pirazoles/metabolismo , Iminas/química , Iminas/metabolismo , Estructura Molecular , Hidrazinas/química , Ingeniería de ProteínasRESUMEN
Etravirine (ETV) is an antiretroviral agent that belongs to the class of non-nucleoside reverse transcriptase inhibitors. This study explores the uptake and distribution of ETV in human aortic endothelial cells (HAECs) using Raman spectroscopy combined with chemometrics. The distinctive chemical structure of ETV facilitates tracking of its uptake by observing the Raman band at 2225 cm-1 in the Raman-silent region. The perinuclear distribution pattern in HAECs depends on drug concentration and incubation time. The uptake of ETV is observed within 5 minutes at a concentration of 10 µM, as evidenced by Raman images. Lower ETV concentrations, reflective of those found in human plasma, are detectable in HAECs by applying chemometric methods to Raman spectra from the perinuclear region. The ETV accumulation process is crucial in advancing our understanding of the drug's impact on biochemical alterations within endothelial cells. Additionally, ETV emerges as a promising Raman reporter for marking subcellular compartments, leveraging the 2225 cm-1 band in the cellular Raman silent region. This research contributes valuable insights into the behavior of ETV at the subcellular level, shedding light on its potential applications and impact on subcellular dynamics.
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Aorta , Células Endoteliales , Nitrilos , Piridazinas , Pirimidinas , Espectrometría Raman , Espectrometría Raman/métodos , Humanos , Nitrilos/química , Nitrilos/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/química , Pirimidinas/química , Pirimidinas/metabolismo , Aorta/metabolismo , Aorta/citología , Piridazinas/química , Piridazinas/metabolismo , Análisis de la Célula Individual/métodos , Células CultivadasRESUMEN
The activation of memory T cells is a very rapid and concerted cellular response that requires coordination between cellular processes in different compartments and on different time scales. In this study, we use ribosome profiling and deep RNA sequencing to define the acute mRNA translation changes in CD8 memory T cells following initial activation events. We find that initial translation enables subsequent events of human and mouse T cell activation and expansion. Briefly, early events in the activation of Ag-experienced CD8 T cells are insensitive to transcriptional blockade with actinomycin D, and instead depend on the translation of pre-existing mRNAs and are blocked by cycloheximide. Ribosome profiling identifies â¼92 mRNAs that are recruited into ribosomes following CD8 T cell stimulation. These mRNAs typically have structured GC and pyrimidine-rich 5' untranslated regions and they encode key regulators of T cell activation and proliferation such as Notch1, Ifngr1, Il2rb, and serine metabolism enzymes Psat1 and Shmt2 (serine hydroxymethyltransferase 2), as well as translation factors eEF1a1 (eukaryotic elongation factor α1) and eEF2 (eukaryotic elongation factor 2). The increased production of receptors of IL-2 and IFN-γ precedes the activation of gene expression and augments cellular signals and T cell activation. Taken together, we identify an early RNA translation program that acts in a feed-forward manner to enable the rapid and dramatic process of CD8 memory T cell expansion and activation.
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Glicina Hidroximetiltransferasa , Interleucina-2 , Regiones no Traducidas 5' , Animales , Linfocitos T CD8-positivos , Cicloheximida/metabolismo , Dactinomicina/metabolismo , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/metabolismo , Humanos , Memoria Inmunológica , Interleucina-2/metabolismo , Activación de Linfocitos , Células T de Memoria , Ratones , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Factores de Elongación de Péptidos/genética , Pirimidinas/metabolismo , ARN Mensajero/genética , Serina/genéticaRESUMEN
Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.
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Melanoma/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Pirimidinas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Melanoma/patología , Melanoma Experimental , Proteínas Oncogénicas/genética , Factores de Transcripción , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Background: Amino acid metabolism, including ATP production, nucleotide synthesis, and redox homeostatic processes, are associated with proliferation and differentiation of tumor cells. This study aimed to identify novel prognostic biomarkers and potential therapeutic targets of amino acid metabolism-related genes for stomach adenocarcinoma (STAD). Methods: RNA sequencing transcriptome data in the TCGA-STAD (training set) and GTEx datasets (validation set) were used. The LIMMA R program enabled the differentially expressed amino acid metabolism-related genes (AAMRGs) to be found. A prognostic risk score model based on clinical phenotypic features was built using LASSO regression and step multi-Cox analyses. Gene set enrichment analysis (GSEA) was used to find potential molecular pathways associated with STAD. Hierarchical cluster analysis was used to evaluate pyrimidine metabolism. Cultured STAD cells assessed the proliferation of STAD and upregulation of GPX3 expression by CCK8 and flow cytometry. Transwell and wound healing assays assessed the impact of GPX3 on invasion and migration of STAD cells. Western blot and qRT-PCR were used to measure changes in pyrimidine metabolism-related markers and active molecules involved in the AMPK/mTOR signaling pathway. Results: Three AAMRGs, DNMT1, F2R, and GPX3, could independently predict the course of STAD. Pyrimidine metabolism appeared to be significantly associated with these by GSEA and clustering analyses. Pyrimidine metabolism was negatively correlated with GPX3. Functional studies using an overexpressed GPX3 plasmid showed an enhanced migration and invasion of STAD cells as well as the expression of genes associated with pyrimidine metabolism and the AMPK/mTOR signaling pathway. By using a CAD siRNA, it was found that that GPX3 affected 5-fluorouracil resistance during de novo synthesis of pyrimidine through the CAD-UMPS signaling axis. Conclusions: GPX3 which regulates the level of pyrimidine metabolism through the AMPK/mTOR pathway was found to be closely associated with STAD. Our findings demonstrate GPX3 is a reliable biomarker for the prognosis of amino acid metabolism and a probable target for STAD therapy.
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Adenocarcinoma , Glutatión Peroxidasa , Estrés Oxidativo , Pirimidinas , Neoplasias Gástricas , Humanos , Adenocarcinoma/metabolismo , Aminoácidos , Proteínas Quinasas Activadas por AMP , Glutatión Peroxidasa/metabolismo , Pronóstico , Neoplasias Gástricas/metabolismo , Serina-Treonina Quinasas TOR , Pirimidinas/metabolismoRESUMEN
Bruton's tyrosine kinase (BTK) is pivotal in B-cell signaling and a target for potential anti-cancer and immunological disorder therapies. Improved selective reversible BTK inhibitors are in demand due to the absence of direct BTK engagement measurement tools. Promisingly, PET imaging can non-invasively evaluate BTK expression. In this study, radiolabeled BIO-2008846 ([11C]BIO-2008846-A), a BTK inhibitor, was used for PET imaging in NHPs to track brain biodistribution. Radiolabeling BIO-2008846 with carbon-11, alongside four PET scans on two NHPs each, showed a homogeneous distribution of [11C]BIO-2008846-A in NHP brains. Brain uptake ranged from 1.8% ID at baseline to a maximum of 3.2% post-pretreatment. The study found no significant decrease in regional VT values post-dose, implying minimal specific binding of [11C]BIO-2008846-A compared to free and non-specific components in the brain. Radiometabolite analysis revealed polar metabolites with 10% unchanged radioligand after 30 min. The research highlighted strong brain uptake despite minor distribution variability, confirming passive diffusion kinetics dominated by free and non-specific binding.
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Agammaglobulinemia Tirosina Quinasa , Encéfalo , Radioisótopos de Carbono , Tomografía de Emisión de Positrones , Inhibidores de Proteínas Quinasas , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Tomografía de Emisión de Positrones/métodos , Animales , Inhibidores de Proteínas Quinasas/farmacología , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagen , Distribución Tisular , Radiofármacos/química , Radiofármacos/farmacocinética , Masculino , Macaca mulatta , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , HumanosRESUMEN
Dihydropyrimidine dehydrogenase (DPD) is a flavin dependent enzyme that catalyzes the reduction of the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons from NADPH. DPD has two active sites that are separated by â¼60 Å. At one site NADPH binds adjacent to an FAD cofactor and at the other pyrimidine binds proximal to an FMN. Four Fe4S4 centers span the distance between these active sites. It has recently been established that the enzyme undergoes reductive activation prior to reducing the pyrimidine. In this initial process NADPH is oxidized at the FAD site and electrons are transmitted to the FMN via the Fe4S4 centers to yield the active state with a cofactor set of FADâ¢4(Fe4S4)â¢FMNH2. The catalytic chemistry of DPD can be studied in transient-state by observation of either NADPH consumption or charge transfer absorption associated with complexation of NADPH adjacent to the FAD. Here we have utilized both sets of absorption transitions to find evidence for specific additional aspects of the DPD mechanism. Competition for binding with NADP+ indicates that the two charge transfer species observed in activation/single turnover reactions arise from NADPH populating the FAD site before and after reductive activation. An additional charge transfer species is observed to accumulate at longer times when high NADPH concentrations are mixed with the enzymeâ¢pyrimidine complex and this data can be modelled based on asymmetry in the homodimer. It was also shown that, like pyrimidines, dihydropyrimidines induce rapid reductive activation indicating that the reduced pyrimidine formed in turnover can stimulate the reinstatement of the active state of the enzyme. Investigation of the reverse reaction revealed that dihydropyrimidines alone can reductively activate the enzyme, albeit inefficiently. In the presence of dihydropyrimidine and NADP+ DPD will form NADPH but apparently without measurable reductive activation. Pyrimidines that have 5-substituent halogens were utilized to probe both reductive activation and turnover. The linearity of the Hammett plot based on the rate of hydride transfer to the pyrimidine establishes that, at least to the radius of an iodo-group, the 5-substituent volume does not have influence on the observed kinetics of pyrimidine reduction.
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Dihidrouracilo Deshidrogenasa (NADP) , Pirimidinas , Animales , Oxidación-Reducción , Dihidrouracilo Deshidrogenasa (NADP)/química , NADP/metabolismo , Espectrofotometría , Pirimidinas/metabolismo , Cinética , Flavina-Adenina Dinucleótido/química , Mamíferos/metabolismoRESUMEN
Among eukaryotes, the major spliceosomal pathway is highly conserved. While long introns may contain additional regulatory sequences, the ones in short introns seem to be nearly exclusively related to splicing. Although these regulatory sequences involved in splicing are well-characterized, little is known about their evolution. At the 3' end of introns, the splice signal nearly universally contains the dimer AG, which consists of purines, and the polypyrimidine tract upstream of this 3' splice signal is characterized by over-representation of pyrimidines. If the over-representation of pyrimidines in the polypyrimidine tract is also due to avoidance of a premature splicing signal, we hypothesize that AG should be the most under-represented dimer. Through the use of DNA-strand asymmetry patterns, we confirm this prediction in fruit flies of the genus Drosophila and by comparing the asymmetry patterns to a presumably neutrally evolving region, we quantify the selection strength acting on each motif. Moreover, our inference and simulation method revealed that the best explanation for the base composition evolution of the polypyrimidine tract is the joint action of purifying selection against a spurious 3' splice signal and the selection for pyrimidines. Patterns of asymmetry in other eukaryotes indicate that avoidance of premature splicing similarly affects the nucleotide composition in their polypyrimidine tracts.
Asunto(s)
Pirimidinas , Empalme del ARN , Secuencia de Bases , Composición de Base , Mutación , Intrones , Pirimidinas/metabolismoRESUMEN
OBJECTIVE: Neuroprotection is a precise target for the treatment of neurodegenerative diseases, ischemic stroke, and traumatic brain injury. Pyrimidine and its derivatives have been proven to use antiviral, anticancer, antioxidant, and antimicrobial activity prompting us to study the neuroprotection and anti-inflammatory activity of the triazole-pyrimidine hybrid on human microglia and neuronal cell model. METHODS: A series of novel triazole-pyrimidine-based compounds were designed, synthesized and characterized by mass spectra, 1HNMR, 13CNMR, and a single X-Ray diffraction analysis. Further, the neuroprotective, anti-neuroinflammatory activity was evaluated by cell viability assay (MTT), Elisa, qRT-PCR, western blotting, and molecular docking. RESULTS: The molecular results revealed that triazole-pyrimidine hybrid compounds have promising neuroprotective and anti-inflammatory properties. Among the 14 synthesized compounds, ZA3-ZA5, ZB2-ZB6, and intermediate S5 showed significant anti-neuroinflammatory properties through inhibition of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in LPS-stimulated human microglia cells. From 14 compounds, six (ZA2 to ZA6 and intermediate S5) exhibited promising neuroprotective activity by reduced expression of the endoplasmic reticulum (ER) chaperone, BIP, and apoptosis marker cleaved caspase-3 in human neuronal cells. Also, a molecular docking study showed that lead compounds have favorable interaction with active residues of ATF4 and NF-kB proteins. CONCLUSION: The possible mechanism of action was observed through the inhibition of ER stress, apoptosis, and the NF-kB inflammatory pathway. Thus, our study strongly indicates that the novel scaffolds of triazole-pyrimidine-based compounds can potentially be developed as neuroprotective and anti-neuroinflammatory agents.
Asunto(s)
Neuroprotección , Fármacos Neuroprotectores , Humanos , FN-kappa B/metabolismo , Triazoles/farmacología , Triazoles/metabolismo , Simulación del Acoplamiento Molecular , Antiinflamatorios/farmacología , Microglía/patología , Pirimidinas/farmacología , Pirimidinas/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/metabolismo , Lipopolisacáridos/farmacologíaRESUMEN
CCR6+CXCR3+CCR4-CD4+ memory T cells, termed Th1*, are important for long-term immunity to Mycobacterium tuberculosis and the pathogenesis of autoimmune diseases. Th1* cells express a unique set of lineage-specific transcription factors characteristic of both Th1 and Th17 cells and display distinct gene expression profiles compared with other CD4+ T cell subsets. To examine molecules and signaling pathways important for the effector function of Th1* cells, we performed loss-of-function screening of genes selectively enriched in the Th1* subset. The genetic screen yielded candidates whose depletion significantly impaired TCR-induced IFN-γ production. These included genes previously linked to IFN-γ or M. tuberculosis susceptibility and novel candidates, such as ISOC1, encoding a metabolic enzyme of unknown function in mammalian cells. ISOC1-depleted T cells, which produced less IFN-γ and IL-17, displayed defects in oxidative phosphorylation and glycolysis and impairment of pyrimidine metabolic pathway. Supplementation with extracellular pyrimidines rescued both bioenergetics and IFN-γ production in ISOC1-deficient T cells, indicating that pyrimidine metabolism is a key driver of effector functions in CD4+ T cells and Th1* cells. Results provide new insights into the immune-stimulatory function of ISOC1 as well as the particular metabolic requirements of human memory T cells, providing a novel resource for understanding long-term T cell-driven responses.