RESUMO
The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.
Assuntos
Niacinamida , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Relação Estrutura-Atividade , Compostos de Piridínio/metabolismo , Compostos de Piridínio/química , N-Glicosil Hidrolases/metabolismo , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/química , Uridina/metabolismo , Uridina/análogos & derivados , Uridina/química , Especificidade por Substrato , Humanos , Modelos MolecularesRESUMO
AMPylation is a post-translational modification in which AMP is added to the amino acid side chains of proteins1,2. Here we show that, with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity towards the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family of effectors, and deubiquitinases DupA and DupB in an E1- and E2-independent ubiquitination process3-7. The product of LnaB is further hydrolysed by an ADP-ribosylhydrolase, MavL, to Ub, thereby preventing the accumulation of PRR42-Ub and ADPRR42-Ub and protecting canonical ubiquitination in host cells. LnaB represents a large family of AMPylases that adopt a common structural fold, distinct from those of the previously known AMPylases, and LnaB homologues are found in more than 20 species of bacterial pathogens. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity towards phosphorylated residues and produces unique ADPylation modifications in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family of kinases8,9, which dampens downstream phosphorylation signalling in the host. Structural studies reveal the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study identifies, to our knowledge, an unprecedented molecular regulation mechanism in bacterial pathogenesis and protein phosphorylation.
Assuntos
Monofosfato de Adenosina , Proteínas de Bactérias , Legionella pneumophila , Fosfotirosina , Transdução de Sinais , Humanos , Actinas/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , ADP-Ribosilação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrólise , Legionella pneumophila/enzimologia , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Ligantes , Modelos Moleculares , N-Glicosil Hidrolases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Tirosina/metabolismo , Tirosina/química , Ubiquitina/metabolismo , Ubiquitinação , Enzimas Desubiquitinantes/metabolismo , Dobramento de Proteína , Fosfotirosina/química , Fosfotirosina/metabolismoRESUMO
Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(ß-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(ß-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (Ki = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors.
Assuntos
Leishmania donovani , Leishmaniose Visceral , Humanos , N-Glicosil Hidrolases/metabolismo , Cromatografia de Afinidade , 4-QuinolonasRESUMO
KEY MESSAGE: The gametophytic epigenetic regulators, MEA and DME, extend their synergistic role to the sporophytic development by regulating the meristematic activity via restricting the gene expression in the shoot apex. The gametophyte-to-sporophyte transition facilitates the alternation of generations in a plant life cycle. The epigenetic regulators DEMETER (DME) and MEDEA (MEA) synergistically control central cell proliferation and differentiation, ensuring proper gametophyte-to-sporophyte transition in Arabidopsis. Mutant alleles of DME and MEA are female gametophyte lethal, eluding the recovery of recessive homozygotes to examine their role in the sporophyte. Here, we exploited the paternal transmission of these mutant alleles coupled with CENH3-haploid inducer to generate mea-1;dme-2 sporophytes. Strikingly, the simultaneous loss of function of MEA and DME leads to the emergence of ectopic shoot meristems at the apical pole of the plant body axis. DME and MEA are expressed in the developing shoot apex and regulate the expression of various shoot-promoting factors. Chromatin immunoprecipitation (ChIP), DNA methylation, and gene expression analysis revealed several shoot regulators as potential targets of MEA and DME. RNA interference-mediated transcriptional downregulation of shoot-promoting factors STM, CUC2, and PLT5 rescued the twin-plant phenotype to WT in 9-23% of mea-1-/-;dme-2-/- plants. Our findings reveal a previously unrecognized synergistic role of MEA and DME in restricting the meristematic activity at the shoot apex during sporophytic development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Células Germinativas Vegetais/metabolismo , Impressão Genômica , Metilação de DNA/genética , Regulação da Expressão Gênica de Plantas/genética , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Transativadores/genéticaRESUMO
BACKGROUND: Pseudoenzymes, catalytically deficient variants of active enzymes, have a wide range of regulatory functions. ADP-ribosylhydrolase-like 1 (ADPRHL1), a pseudoenzyme belonging to a small group of ADP-ribosylhydrolase enzymes that lacks the amino acid residues necessary for catalytic activity, may have a significant role in heart development based on accumulating evidence. However, the specific function of ADPRHL1 in this process has not been elucidated. To investigate the role of ADPRHL1 in the heart, we generated the first in vitro human embryonic stem cell model with an ADPRHL1 knockout. METHOD: Using the CRISPR/Cas9 system, we generated ADPRHL1 knockout in the human embryonic stem cell (hESC) H9 line. The cells were differentiated into cardiomyocytes using a chemically defined and xeno-free method. We employed confocal laser microscopy to detect calcium transients and microelectrode array (MEA) to assess the electrophysiological activity of ADPRHL1 deficiency cardiomyocytes. Additionally, we investigated the cellular mechanism of ADPRHL1 by Bulk RNA sequencing and western blot. RESULTS: The results indicate that the absence of ADPRHL1 in cardiomyocytes led to adhered abnormally, as well as perturbations in calcium transients and electrophysiological activity. We also revealed that disruption of focal adhesion formation in these cardiomyocytes was due to an excessive upregulation of the ROCK-myosin II pathway. Notably, inhibition of ROCK and myosin II effectively restores focal adhesions in ADPRHL1-deficient cardiomyocytes and improved electrical conduction and calcium activity. CONCLUSIONS: Our findings demonstrate that ADPRHL1 plays a critical role in maintaining the proper function of cardiomyocytes by regulating the ROCK-myosin II pathway, suggesting that it may serve as a potential drug target for the treatment of ADPRHL1-related diseases.
Assuntos
Cálcio , Miócitos Cardíacos , N-Glicosil Hidrolases , Humanos , Cálcio/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Miócitos Cardíacos/metabolismo , Miosina Tipo II/metabolismo , N-Glicosil Hidrolases/metabolismoRESUMO
2'-Deoxynucleoside 5'-monophosphate N-glycosidase 1 (DNPH1) hydrolyzes the epigenetically modified nucleotide 5-hydroxymethyl 2'-deoxyuridine 5'-monophosphate (hmdUMP) derived from DNA metabolism. Published assays of DNPH1 activity are low throughput, use high concentrations of DNPH1, and have not incorporated or characterized reactivity with the natural substrate. We describe the enzymatic synthesis of hmdUMP from commercially available materials and define its steady-state kinetics with DNPH1 using a sensitive, two-pathway enzyme coupled assay. This continuous absorbance-based assay works in 96-well plate format using nearly 500-fold less DNPH1 than previous methods. With a Z prime value of 0.92, the assay is suitable for high-throughput assays, screening of DNPH1 inhibitors, or characterization of other deoxynucleotide monophosphate hydrolases.
Assuntos
Hidrolases , N-Glicosil Hidrolases , Hidrólise , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Hidrolases/metabolismo , CinéticaRESUMO
Due to the emergence of multidrug resistant pathogens, it is imperative to identify new targets for antibiotic drug discovery. The S-adenosylhomocysteine (SAH) nucleosidase enzyme is a promising target for antimicrobial drug development due to its critical functions in multiple bacterial processes including recycling of toxic byproducts of S-adenosylmethionine (SAM)-mediated reactions and producing the precursor of the universal quorum sensing signal, autoinducer-2 (AI-2). Riboswitches are structured RNA elements typically used by bacteria to precisely monitor and respond to changes in essential bacterial processes, including metabolism. Natural riboswitches fused to a reporter gene can be exploited to detect changes in metabolism or in physiological signaling. We performed a high-throughput screen (HTS) using an SAH-riboswitch controlled ß-galactosidase reporter gene in Escherichia coli to discover small molecules that inhibit SAH recycling. We demonstrate that the assay strategy using SAH riboswitches to detect the effects of SAH nucleosidase inhibitors can quickly identify compounds that penetrate the barriers of Gram-negative bacterial cells and perturb pathways involving SAH.
Assuntos
Riboswitch , S-Adenosilmetionina/metabolismo , RNA/genética , Bactérias/genética , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismoRESUMO
Tropical legumes transport fixed nitrogen in form of ureides (allantoin and allantoate) over long distances from the nodules to the shoot. Ureides are formed in nodules from purine mononucleotides by a partially unknown reaction network that involves bacteroid-infected and uninfected cells. Here, we demonstrate by metabolic analysis of CRISPR mutant nodules of Phaseolus vulgaris defective in either xanthosine monophosphate phosphatase (XMPP), guanosine deaminase (GSDA), the nucleoside hydrolases 1 and 2 (NSH1, NSH2) or xanthine dehydrogenase (XDH) that nodule ureide biosynthesis involves these enzymes and requires xanthosine and guanosine but not inosine monophosphate catabolism. Interestingly, promoter reporter analyses revealed that XMPP, GSDA and XDH are expressed in infected cells, whereas NSH1, NSH2 and the promoters of the downstream enzymes urate oxidase (UOX) and allantoinase (ALN) are active in uninfected cells. The data suggest a complex cellular organization of ureide biosynthesis with three transitions between infected and uninfected cells.
Assuntos
Nitrogênio , Phaseolus , Alantoína/metabolismo , N-Glicosil Hidrolases/metabolismo , Nitrogênio/metabolismo , Phaseolus/genética , Xantina Desidrogenase/metabolismoRESUMO
S-adenosylhomocysteine nucleosidase (MTAN) is a protein that plays a crucial role in several pathways of bacteria that are essential for its survival and pathogenesis. In addition to the role of MTAN in methyl-transfer reactions, methionine biosynthesis, and polyamine synthesis, MTAN is also involved in bacterial quorum sensing (QS). In QS, chemical signaling autoinducer (AI) secreted by bacteria assists cell to cell communication and is regulated in a cell density-dependent manner. They play a significant role in the formation of bacterial biofilm. MTAN plays a major role in the synthesis of these autoinducers. Signaling molecules secreted by bacteria, i.e., AI-1 are recognized as acylated homoserine lactones (AHL) that function as signaling molecules within bacteria. QS enables bacteria to establish physical interactions leading to biofilm formation. The formation of biofilm is a primary reason for the development of multidrug-resistant properties in pathogenic bacteria like Enterococcus faecalis (E. faecalis). In this regard, inhibition of E. faecalis MTAN (EfMTAN) will block the QS and alter the bacterial biofilm formation. In addition to this, it will also block methionine biosynthesis and many other critical metabolic processes. It should also be noted that inhibition of EfMTAN will not have any effect on human beings as this enzyme is not present in humans. This review provides a comprehensive overview of the structural-functional relationship of MTAN. We have also highlighted the current status, enigmas that warrant further studies, and the prospects for identifying potential inhibitors of EfMTAN for the treatment of E. faecalis infections. In addition to this, we have also reported structural studies of EfMTAN using homology modeling and highlighted the putative binding sites of the protein.
Assuntos
N-Glicosil Hidrolases , Percepção de Quorum , Bactérias/metabolismo , Biofilmes , Homocisteína , Humanos , Metionina , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismoRESUMO
Curtobacterium sp. GD1 was isolated from leaves of conventionally grown soybean in Brazil. It was noteworthy that among all bacteria previously isolated from the same origin, only Curtobacterium sp. GD1 showed a strong chitinase activity. The enzyme was secreted and its production was induced by the presence of colloidal chitin in the medium. The chitinase was partially purified and characterized: molecular weight was approximately 37 kDa and specific activity 90.8 U/mg. Furthermore, Curtobacterium sp. GD1 genome was sequenced and analyzed. Our isolate formed a phylogenetic cluster with four other Curtobacterium spp. strains, with ANIb/ANIm ≥ 98%, representing a new, still non described Curtobacterium species. The circular genome visualization and comparison of genome sequences of strains forming new cluster indicated that most regions within their genomes were highly conserved. The gene associated with chitinase production was identified and the distribution pattern of glycosyl hydrolases genes was assessed. Also, genes associated with catabolism of structural carbohydrates such as oligosaccharides, mixed polysaccharides, plant and animal polysaccharides, as well as genes or gene clusters associated with resistance to antibiotics, toxic compounds and auxin biosynthesis subsystem products were identified. The abundance of putative glycosyl hydrolases in the genome of Curtobacterium sp. GD1 suggests that it has the tools for the hydrolysis of different polysaccharides. Therefore, Curtobacterium sp. GD1 isolated from soybean might be a bioremediator, biocontrol agent, an elicitor of the plant defense responses or simply degrader.
Assuntos
Actinobacteria/fisiologia , Quitina/química , Quitinases/genética , Glycine max/microbiologia , Sequenciamento Completo do Genoma/métodos , Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Hidrólise , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Filogenia , Folhas de Planta/química , Folhas de Planta/microbiologia , Glycine max/metabolismoRESUMO
Adaptive evolution to cellular stress is a process implicated in a wide range of biological and clinical phenomena. Two major routes of adaptation have been identified: non-genetic changes, which allow expression of different phenotypes in novel environments, and genetic variation achieved by selection of fitter phenotypes. While these processes are broadly accepted, their temporal and epistatic features in the context of cellular evolution and emerging drug resistance are contentious. In this manuscript, we generated hypomorphic alleles of the essential nuclear pore complex (NPC) gene NUP58. By dissecting early and long-term mechanisms of adaptation in independent clones, we observed that early physiological adaptation correlated with transcriptome rewiring and upregulation of genes known to interact with the NPC; long-term adaptation and fitness recovery instead occurred via focal amplification of NUP58 and restoration of mutant protein expression. These data support the concept that early phenotypic plasticity allows later acquisition of genetic adaptations to a specific impairment. We propose this approach as a genetic model to mimic targeted drug therapy in human cells and to dissect mechanisms of adaptation.
Assuntos
Adaptação Fisiológica/genética , Alelos , Receptor Quinase 1 Acoplada a Proteína G/genética , Aptidão Genética , N-Glicosil Hidrolases/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Células HEK293 , Haploidia , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Células Mieloides/metabolismo , Células Mieloides/patologia , N-Glicosil Hidrolases/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transdução de Sinais , Transcriptoma , Proteína Vermelha FluorescenteRESUMO
The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Células Germinativas Vegetais/enzimologia , Meristema/enzimologia , N-Glicosil Hidrolases/metabolismo , Transativadores/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Diferenciação Celular , Proliferação de Células , Células Germinativas Vegetais/citologia , Meristema/genética , Meristema/crescimento & desenvolvimento , N-Glicosil Hidrolases/genética , Transativadores/genéticaRESUMO
Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.
Assuntos
Helicobacter pylori/metabolismo , Nucleosídeos/metabolismo , Vitamina K 2/metabolismo , Domínio Catalítico , Cristalografia por Raios X/métodos , Desoxiadenosinas , Helicobacter pylori/química , Helicobacter pylori/enzimologia , Modelos Moleculares , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Nucleosídeos/química , Purina-Núcleosídeo Fosforilase/química , Especificidade por Substrato , Tionucleosídeos , Vitamina K 2/análogos & derivadosRESUMO
In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.
Assuntos
Arabidopsis/imunologia , Cloroplastos/imunologia , Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Epiderme Vegetal/imunologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Auxilinas/genética , Auxilinas/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Colletotrichum/imunologia , Colletotrichum/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Magnaporthe/imunologia , Magnaporthe/patogenicidade , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Epiderme Vegetal/microbiologia , Folhas de Planta/citologia , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Pseudomonas syringae/imunologia , Pseudomonas syringae/patogenicidadeRESUMO
Patients with hepatocellular carcinoma (HCC) suffer from few treatment options and poor survival rates. Here we report that endonuclease VIII-like protein 3 (NEIL3) is overexpressed in HCC and correlates with poor survival. All six HCC cell lines investigated were dependent on NEIL3 catalytic activity for survival and prevention of senescence, while NEIL3 was dispensable for nontransformed cells. NEIL3-depleted HCC cell lines accumulated oxidative DNA lesions specifically at telomeres, resulting in telomere dysfunctional foci and 53BP1 foci formation. Following oxidative DNA damage during mitosis, NEIL3 relocated to telomeres and recruited apurinic endonuclease 1 (APE1), indicating activation of base excision repair. META-FISH revealed that NEIL3, but not NEIL1 or NEIL2, is required to initiate APE1 and polymerase beta (POLB)-dependent base excision repair at oxidized telomeres. Repeated exposure of NEIL3-depleted cells to oxidizing damage induced chromatin bridges and damaged telomeres. These results demonstrate a novel function for NEIL3 in repair of oxidative DNA damage at telomeres in mitosis, which is important to prevent senescence of HCC cells. Furthermore, these data suggest that NEIL3 could be a target for therapeutic intervention for HCC. SIGNIFICANCE: This study describes compartmentalization of base excision repair during mitosis that is dependent on NEIL3, APE1, and POLB to repair oxidative damage accumulating at telomeres in hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mitose/genética , N-Glicosil Hidrolases/metabolismo , Telômero/metabolismo , Humanos , Oxirredução , TransfecçãoRESUMO
Acquired treatment resistance is an important cause of death in prostate cancer, and this study aimed to explore the mechanisms of chemotherapy resistance in prostate cancer. We employed castration-resistant prostate cancer (CRPC), neuroendocrine prostate cancer (NEPC), and chemotherapy-resistant prostate cancer datasets to screen for potential target genes. The Cancer Genome Atlas (TCGA) was used to detect the correlation between the target genes and prognosis and clinical characteristics. Nei endonuclease VIII-like 3 (NEIL3) knockdown cell lines were constructed with RNA interference. Prostate cancer cells were treated with enzalutamide for the androgen deprivation therapy (ADT) model, and with docetaxel and cisplatin for the chemotherapy model. Apoptosis and the cell cycle were examined using flow cytometry. RNA sequencing and western blotting were performed in the knockdown Duke University 145 (DU145) cell line to explore the possible mechanisms. The TCGA dataset demonstrated that high NEIL3 was associated with a high T stage and Gleason score, and indicated a possibility of lymph node metastasis, but a good prognosis. The cell therapy models showed that the loss of NEIL3 could promote the chemotherapy resistance (but not ADT resistance) of prostate cancer (PCa). Flow cytometry revealed that the loss of NEIL3 in PCa could inhibit cell apoptosis and cell cycle arrest under cisplatin treatment. RNA sequencing showed that the knockdown of NEIL3 changes the expression of neuroendocrine-related genes. Further western blotting revealed that the loss of NEIL3 could significantly promote the phosphorylation of ATR serine/threonine kinase (ATR) and ATM serine/threonine kinase (ATM) under chemotherapy, thus initiating downstream pathways related to DNA repair. In summary, the loss of NEIL3 promotes chemotherapy resistance in prostate cancer, and NEIL3 may serve as a diagnostic marker for chemotherapy-resistant patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos , N-Glicosil Hidrolases/deficiência , Neoplasias da Próstata/tratamento farmacológico , Antagonistas de Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Invasividade Neoplásica , Sistemas Neurossecretores/efeitos dos fármacos , Sistemas Neurossecretores/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Fase S/efeitos dos fármacosRESUMO
Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.
Assuntos
Antineoplásicos/farmacologia , N-Glicosil Hidrolases/antagonistas & inibidores , N-Glicosil Hidrolases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Apoptose , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA de Neoplasias/metabolismo , Desoxicitidina Monofosfato/análogos & derivados , Desoxicitidina Monofosfato/metabolismo , Desoxicitidina Monofosfato/farmacologia , Nucleotídeos de Desoxiuracil/metabolismo , Resistencia a Medicamentos Antineoplásicos , Genes BRCA1 , Humanos , Hidrólise , N-Glicosil Hidrolases/genética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/genética , Mutações Sintéticas Letais , Timidina/análogos & derivados , Timidina/antagonistas & inibidores , Timidina/metabolismo , Timidina/farmacologia , Uracila-DNA Glicosidase/metabolismoRESUMO
Rhamnogalacturonan-I biosynthesis occurs in the lumen of the Golgi apparatus, a compartment where UDP-Rhamnose and UDP-Galacturonic Acid are the main substrates for synthesis of the backbone polymer of pectin. Recent studies showed that UDP-Rha is transported from the cytosol into the Golgi apparatus by a family of six UDP-rhamnose/UDP-galactose transporters (URGT1-6). In this study, analysis of adherent and soluble mucilage (SM) of Arabidopsis thaliana seeds revealed distinct roles of URGT2, URGT4, and URGT6 in mucilage biosynthesis. Characterization of SM polymer size showed shorter chains in the urgt2 urgt4 and urgt2 urgt4 urgt6 mutants, suggesting that URGT2 and URGT4 are mainly involved in Rhamnogalacturonan-I (RG-I) elongation. Meanwhile, mutants in urgt6 exhibited changes only in adherent mucilage (AM). Surprisingly, the estimated number of RG-I polymer chains present in urgt2 urgt4 and urgt2 urgt4 urgt6 mutants was higher than in wild-type. Interestingly, the increased number of shorter RG-I chains was accompanied by an increased amount of xylan. In the urgt mutants, expression analysis of other genes involved in mucilage biosynthesis showed some compensation. Studies of mutants of transcription factors regulating mucilage formation indicated that URGT2, URGT4, and URGT6 are likely part of a gene network controlled by these regulators and involved in RG-I synthesis. These results suggest that URGT2, URGT4, and URGT6 play different roles in the biosynthesis of mucilage, and the lack of all three affects the production of shorter RG-I polymers and longer xylan domains.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Pectinas/metabolismo , Ramnogalacturonanos/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Transporte de Monossacarídeos/genética , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismoRESUMO
Plant sucrose-phosphate synthase (SPS) contains a glycosyltransferase domain, which specifically catalyzes reactions with the nucleotide sugar uridine diphosphate glucose (UDP-G) as a donor substrate. Unlike plant SPS, bacterial SPS is predicted to bind other nucleotide sugars, such as adenosine diphosphate glucose (ADP-G). This study aimed to identify the UDP-G binding site of sugarcane (Saccharum officinarum) SPS (SoSPS1) and to improve its affinity for ADP-G by site-directed mutagenesis. To achieve targeted mutagenesis, amino acid distribution and comparative modeling studies were performed, followed by site-directed mutagenesis of SoSPS1 in the putative UDP-G binding motif. The N-terminal deletion of SoSPS1 (∆N-SoSPS1) was used for enzymatic analysis. The results showed that mutations in the R-X4-K, E-X7-E, and H-X5-V motifs significantly affect UDP-G and ADP-G binding. Mutations at R496 and K501 severely attenuate the affinity for UDP-G. Additionally, alanine substitutions at E591 and V570 decreased the UDP-G affinity but remarkably increased its ADP-G affinity. The R-X4-K motif plays a crucial role in the UDP-G binding site and catalytic activity of plant SPS; thus, its alteration to other amino acids was not viable. The E-X7-E and H-X5-V motifs may bind to the nucleotide glucose substrate, indicating that these motifs are involved in substrate specificity. These results agree with substrate docking simulations at the mutated residue positions, supporting the experimental results. These results demonstrate that mutation of E591 and V570 severely attenuated the UDP-G affinity, while retaining its activity against ADP-G, offering strategic insights into increasing sucrose synthesis and plant growth.
Assuntos
Adenosina Difosfato Glucose/química , Glucosiltransferases/química , Saccharum/enzimologia , Saccharum/genética , Uridina Difosfato Glucose/química , Adenosina Difosfato Glucose/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Escherichia coli/metabolismo , Expressão Gênica , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , N-Glicosil Hidrolases/metabolismo , Proteínas Recombinantes , Saccharum/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Uridina Difosfato Glucose/metabolismoRESUMO
Rhizoctonia solani causes damaging yield losses on most major food crops. R. solani isolates belonging to anastomosis group 8 (AG8) are soil-borne, root-infecting pathogens with a broad host range. AG8 isolates can cause disease on wheat, canola and legumes, however Arabidopsis thaliana is heretofore thought to possess non-host resistance as A. thaliana ecotypes, including the reference strain Col-0, are resistant to AG8 infection. Using a mitochondria-targeted redox sensor (mt-roGFP2) and cell death staining, we demonstrate that both AG8 and a host isolate (AG2-1) of R. solani are able to infect A. thaliana roots. Above ground tissue of A. thaliana was found to be resistant to AG8 but not AG2. Genetic analysis revealed that ethylene, jasmonate and PENETRATION2-mediated defense pathways work together to provide resistance to AG8 in the leaves which subsequently enable tolerance of root infections. Overall, we demonstrate a significant difference in defense capabilities of above and below ground tissue in providing resistance to R. solani AG8 in Arabidopsis.