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1.
PLoS One ; 15(7): e0235433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726316

RESUMO

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.


Assuntos
Ventrículos do Coração/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , N-Glicosil Hidrolases/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Catálise , Domínio Catalítico/genética , Coração/fisiopatologia , Ventrículos do Coração/patologia , Humanos , Camundongos , Camundongos Knockout , Morfolinos/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Organogênese/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
2.
Toxicology ; 441: 152531, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32593706

RESUMO

Gene-regulatory networks reconstruction has become a very popular approach in applied biology to infer and dissect functional interactions of Transcription Factors (TFs) driving a defined phenotypic state, termed as Master Regulators (MRs). In the present work, cutting-edge bioinformatic methods were applied to re-analyze experimental data on leukemia cells (human myelogenous leukemia cell line THP-1 and acute myeloid leukemia MOLM-13 cells) treated for 6 h with two different Ribosome-Inactivating Proteins (RIPs), namely Shiga toxin type 1 (400 ng/mL) produced by Escherichia coli strains and the plant toxin stenodactylin (60 ng/mL), purified from the caudex of Adenia stenodactyla Harms. This analysis allowed us to identify the common early transcriptional response to 28S rRNA damage based on gene-regulatory network inference and Master Regulator Analysis (MRA). Both toxins induce a common response at 6 h which involves inflammatory mediators triggered by AP-1 family transcriptional factors and ATF3 in leukemia cells. We describe for the first time the involvement of MAFF, KLF2 and KLF6 in regulating RIP-induced apoptotic cell death, while receptor-mediated downstream signaling through ANXA1 and TLR4 is suggested for both toxins.


Assuntos
Redes Reguladoras de Genes/efeitos dos fármacos , Leucemia/metabolismo , Proteínas Inativadoras de Ribossomos/farmacologia , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Lectinas/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , N-Glicosil Hidrolases/farmacologia , Toxina Shiga I/farmacologia , Fatores de Transcrição/metabolismo
3.
Tumour Biol ; 42(5): 1010428320918404, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32364878

RESUMO

Base excision repair, which is initiated by the DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. Several base excision repair genes are deregulated in cancer and affect cellular outcomes to chemotherapy and carcinogenesis. Endonuclease VIII-like 3 (NEIL3) is a DNA glycosylase protein that is involved in oxidative and interstrand crosslink DNA damage repair. Our previous work has showed that NEIL3 is required to maintain replication fork integrity. It is unknown whether NEIL3 overexpression could contribute to cancer phenotypes, and its prognostic value and use as potential drug target remain unexplored. Our analysis of cancer genomics data sets reveals that NEIL3 frequently undergoes overexpression in several cancers. Furthermore, patients who exhibited NEIL3 overexpression with pancreatic adenocarcinoma, lung adenocarcinoma, lower grade glioma, kidney renal clear cell carcinoma, and kidney papillary cell carcinoma had worse overall survival. Importantly, NEIL3 overexpressed tumors accumulate mutation and chromosomal variations. Furthermore, NEIL3 overexpressed tumors exhibit simultaneous overexpression of homologous recombination genes (BRCA1/2) and mismatch repair genes (MSH2/MSH6). However, NEIL3 overexpression is negatively correlated with tumor overexpressing nucleotide excision repair genes (XPA, XPC, ERCC1/2). Our results suggest that NEIL3 might be a potential prognosis marker for high-risk patients, and/or an attractive therapeutic target for selected cancers.


Assuntos
Biomarcadores Tumorais , Expressão Gênica , Variação Genética , N-Glicosil Hidrolases/genética , Neoplasias/genética , Neoplasias/mortalidade , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Dano ao DNA , Reparo do DNA , Humanos , Estimativa de Kaplan-Meier , Mutação , Neoplasias/diagnóstico , Prognóstico
4.
Cell Host Microbe ; 27(3): 345-357.e6, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32078802

RESUMO

Although the gut microbiome is generally symbiotic or commensal, some microbiome members become pathogenic under certain circumstances. However, the factors driving this pathogenic switch are largely unknown. Pathogenic bacteria can generate uracil that triggers host dual oxidase (DUOX) to produce antimicrobial reactive oxygen species (ROS). We show that pathogens generate uracil and ribose upon nucleoside catabolism of gut luminal uridine, which triggers not only host defenses but also inter-bacterial communication and pathogenesis in Drosophila. Uridine-derived uracil triggers DUOX-dependent ROS generation, whereas ribose induces bacterial quorum sensing (QS) and virulence gene expression. Genes implicated in nucleotide metabolism are found in pathogens but not commensal bacteria, and their genetic ablation blocks QS and the commensal-to-pathogen transition in vivo. Furthermore, commensal bacteria lack functional nucleoside catabolism, which is required to achieve gut-microbe symbiosis, but can become pathogenic by enabling nucleotide catabolism. These findings reveal molecular mechanisms governing the commensal-to-pathogen transition in different contexts of host-microbe interactions.


Assuntos
Bactérias/metabolismo , Bactérias/patogenicidade , Drosophila/microbiologia , Percepção de Quorum , Uracila/metabolismo , Virulência , Animais , Proteínas de Bactérias/metabolismo , Oxidases Duais/metabolismo , N-Glicosil Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribose/metabolismo , Simbiose , Uridina/metabolismo
5.
Nucleic Acids Res ; 48(6): 3014-3028, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-31980815

RESUMO

The NEIL3 DNA glycosylase is a base excision repair enzyme that excises bulky base lesions from DNA. Although NEIL3 has been shown to unhook interstrand crosslinks (ICL) in Xenopus extracts, how NEIL3 participants in ICL repair in human cells and its corporation with the canonical Fanconi anemia (FA)/BRCA pathway remain unclear. Here we show that the NEIL3 and the FA/BRCA pathways are non-epistatic in psoralen-ICL repair. The NEIL3 pathway is the major pathway for repairing psoralen-ICL, and the FA/BRCA pathway is only activated when NEIL3 is not present. Mechanistically, NEIL3 is recruited to psoralen-ICL in a rapid, PARP-dependent manner. Importantly, the NEIL3 pathway repairs psoralen-ICLs without generating double-strand breaks (DSBs), unlike the FA/BRCA pathway. In addition, we found that the RUVBL1/2 complex physically interact with NEIL3 and function within the NEIL3 pathway in psoralen-ICL repair. Moreover, TRAIP is important for the recruitment of NEIL3 but not FANCD2, and knockdown of TRAIP promotes FA/BRCA pathway activation. Interestingly, TRAIP is non-epistatic with both NEIL3 and FA pathways in psoralen-ICL repair, suggesting that TRAIP may function upstream of the two pathways. Taken together, the NEIL3 pathway is the major pathway to repair psoralen-ICL through a unique DSB-free mechanism in human cells.


Assuntos
Replicação do DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , N-Glicosil Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Proteínas de Transporte/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Fibroblastos/metabolismo , Ficusina/farmacologia , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Xenopus/genética
6.
Biochim Biophys Acta Gen Subj ; 1864(1): 129455, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669585

RESUMO

BACKGROUND: Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome. METHODS: Here we examine three unique 5'-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay. RESULTS: The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5'-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5'-alkylthio substitution increased. Non-hydrolysable nucleoside transition state analogs demonstrated sub-nanomolar enzyme inhibition constants. Lastly, two late stage transition state analogs exerted in vitro IC50 values of 0.3-0.4 µg/mL against cultured B. burgdorferi cells. CONCLUSION: B. burgdorferi is unusual in that it expresses three distinct MTNs (cytoplasmic, membrane bound, and secreted) that are effectively inactivated by nucleoside analogs. GENERAL SIGNIFICANCE: The Borrelia MTNs appear to be promising targets for developing new antibiotics to treat Lyme disease.


Assuntos
Antibacterianos/uso terapêutico , Borrelia burgdorferi/enzimologia , Doença de Lyme/tratamento farmacológico , N-Glicosil Hidrolases/genética , Borrelia burgdorferi/efeitos dos fármacos , Borrelia burgdorferi/patogenicidade , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Doença de Lyme/enzimologia , Doença de Lyme/microbiologia , N-Glicosil Hidrolases/antagonistas & inibidores , S-Adenosil-Homocisteína/metabolismo , Tionucleosídeos/metabolismo
7.
Biosci Biotechnol Biochem ; 84(1): 76-84, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31478783

RESUMO

The circadian clock enables plants to adapt to their environment and control numerous physiological processes, including plant-pathogen interactions. However, it is unknown if the circadian clock controls nonhost resistance (NHR) in plants. To find out, we analyzed microarray data with the web-based tool DIURNAL to reveal that NHR-related genes show rhythmic expression patterns in the absence of a pathogen challenge. Our clock mutant analyses found that cca1-1 lhy-11 double mutant showed compromised NHR to Pyricularia oryzae, suggesting that two components of the circadian clock, CCA1 and LHY, are involved in regulating penetration resistance in Arabidopsis thaliana. By analyzing pen2 double mutants, we revealed that CCA1 contributes to time-of-day-dependent penetration resistance as a positive regulator and that LHY regulates post-penetration resistance as a positive regulator. Taken together, our results suggest that the circadian clock regulates the time-of-day-dependent NHR to P. oryzae and thus enables A. thaliana to counteract pathogen attacks.Abbreviations: EE: evening element; ETI: effector-triggered immunity; NHR: nonhost resistance; PAMP: pathogen-associated molecular pattern; PTI: PAMP-triggered immunity; SAR: systemic acquired resistance.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/microbiologia , Relógios Circadianos/genética , Ritmo Circadiano/genética , Proteínas de Ligação a DNA/genética , Resistência à Doença/genética , Magnaporthe/fisiologia , Fatores de Transcrição/genética , Regulação da Expressão Gênica de Plantas , Genótipo , Interações Hospedeiro-Patógeno , Mutação , N-Glicosil Hidrolases/genética , Fotoperíodo , Folhas de Planta/microbiologia , Temperatura
8.
Food Chem ; 309: 125671, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31670129

RESUMO

A pure glycoprotein (BGP4-I) was obtained from tartary buckwheat seeds by aqueous extraction followed by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The average molecular weight of BGP4-I, as determined by high performance gel permeation chromatography, was 123.43 kDa. The structure of BGP4-I was characterized based on Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy, etc. Based on the nano-liquid chromatography-coupled electrospray ionization mass spectrometry analysis of the amino acid sequence of BGP4-I, belongs unequivocally to the glycosyl hydrolase family 1 in the Carbohydrate Active Enzymes database by alignment studies. The specific activity of BGP4-I was 18.44 µmol/min/mg on the substrate p-nitrophenyl-ß-d-glucopyranoside. Furthermore, BGP4-I is unique in its specificity for some substrates. These results suggest that the BGP4-I from tartary buckwheat seeds is a novel specific ß-glucosidase setting the foundation for potential applications in the food industry.


Assuntos
Fagopyrum/metabolismo , Glicoproteínas/química , Proteínas de Plantas/química , Sementes/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Peso Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Espectrometria de Massas em Tandem
9.
Proc Natl Acad Sci U S A ; 116(35): 17563-17571, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31409710

RESUMO

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Domínio Catalítico , Desmetilação do DNA , Regulação da Expressão Gênica de Plantas , N-Glicosil Hidrolases/metabolismo , Transativadores/metabolismo , Arabidopsis/classificação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Epigênese Genética , Evolução Molecular , Heterocromatina/genética , Heterocromatina/metabolismo , Modelos Moleculares , N-Glicosil Hidrolases/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transativadores/química
10.
Molecules ; 24(13)2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31284667

RESUMO

Salinity stress limited the production in over 30% of irrigated crops and 7% of dryland agriculture worldwide. The objective was to evaluate the effects of NaCl-stress on the enzymatic activity in tomato. Two experiments were carried out in germination and early vegetative growth stages. The activity of proline and peroxidase of eight varieties (Missouri, Yaqui, Vita, Feroz, Rio Grande, Tropic, Ace, and Floradade) submitted to NaCl concentrations (0, 50, 100, 150 and 200 mM de NaCl) and the semi-quantitative activity of 19 enzymes APY ZYM® were measured under a completely randomized design with four replications. Data were analyzed using univariate-multivariate analysis of variance, Tukey's HSD (p = 0.05), canonical discriminant and cluster analysis. The results showed significant differences between varieties and NaCl in proline content. Proline increased as the NaCl concentration increased. Peroxidase did no show significant differences. Eight enzymes were included within the model to properly classify the varieties and NaCl. In shoots, varieties and NaCl showed that enzymatic activity was higher in the order of alkaline-phosphatase > leucine arylamidase > acid phosphatase > naphthol-AS-BI-phosphohydrolase > n-acetyl-ß-glucosaminidase > ß-galactosidase, while in roots was higher in the order of alkaline-phosphatase > naphthol-AS-BI-phosphohydrolase > acid phosphatase > n-acetyl-ß-glucosaminidase. Acid and alkali phosphatase, lipase, esterase, ß-galactosidase, and trypsin can be a potential biomarker for NaCl-stress tolerance in tomato.


Assuntos
Esterases/metabolismo , Lycopersicon esculentum/efeitos dos fármacos , Lycopersicon esculentum/fisiologia , N-Glicosil Hidrolases/metabolismo , Peptídeo Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Tolerância ao Sal , Cloreto de Sódio/farmacologia , Biomarcadores , Análise por Conglomerados , Ativação Enzimática , Brotos de Planta/fisiologia , Prolina/metabolismo , Proteoma , Proteômica , Plântula/fisiologia
11.
Elife ; 82019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31135340

RESUMO

Transposable elements (TEs), the movement of which can damage the genome, are epigenetically silenced in eukaryotes. Intriguingly, TEs are activated in the sperm companion cell - vegetative cell (VC) - of the flowering plant Arabidopsis thaliana. However, the extent and mechanism of this activation are unknown. Here we show that about 100 heterochromatic TEs are activated in VCs, mostly by DEMETER-catalyzed DNA demethylation. We further demonstrate that DEMETER access to some of these TEs is permitted by the natural depletion of linker histone H1 in VCs. Ectopically expressed H1 suppresses TEs in VCs by reducing DNA demethylation and via a methylation-independent mechanism. We demonstrate that H1 is required for heterochromatin condensation in plant cells and show that H1 overexpression creates heterochromatic foci in the VC progenitor cell. Taken together, our results demonstrate that the natural depletion of H1 during male gametogenesis facilitates DEMETER-directed DNA demethylation, heterochromatin relaxation, and TE activation.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Desmetilação do DNA , Elementos de DNA Transponíveis , Heterocromatina/metabolismo , Histonas/metabolismo , Células Vegetais/metabolismo , Proteínas de Arabidopsis/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , N-Glicosil Hidrolases/metabolismo , Transativadores/metabolismo
12.
Sci Transl Med ; 11(487)2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971455

RESUMO

Multidrug-resistant enterococcal strains emerged in the early 1980s and are now among the leading causes of drug-resistant bacterial infection worldwide. We used functional genomics to study an early bacterial outbreak in patients in a Wisconsin hospital between 1984 and 1988 that was caused by multidrug-resistant Enterococcus faecalis The goal was to determine how a clonal lineage of E. faecalis became adapted to growth and survival in the human bloodstream. Genome sequence analysis revealed a progression of increasingly fixed mutations and repeated independent occurrences of mutations in a relatively small set of genes. Repeated independent mutations suggested selection within the host during the course of infection in response to pressures such as host immunity and antibiotic treatment. We observed repeated independent mutations in a small number of loci, including a little studied polysaccharide utilization pathway and the cydABDC locus. Functional studies showed that mutating these loci rendered E. faecalis better able to withstand antibiotic pressure and innate immune defenses in the human bloodstream. We also observed a shift in mutation pattern that corresponded to the introduction of carbapenem antibiotics in 1987. This work identifies pathways that allow enterococci to survive the transition from the human gut into the bloodstream, enabling them to cause severe bacteremia associated with high mortality.


Assuntos
Adaptação Fisiológica , Antibacterianos/farmacologia , Enterococcus faecium/fisiologia , Infecções por Bactérias Gram-Positivas/sangue , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/efeitos dos fármacos , Adaptação Fisiológica/efeitos dos fármacos , Carbapenêmicos/farmacologia , Surtos de Doenças , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Enterococcus faecium/genética , Loci Gênicos , Variação Genética , Genoma Bacteriano , Infecções por Bactérias Gram-Positivas/microbiologia , Hospitais , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Lipopolissacarídeos/metabolismo , Mutação/genética , N-Glicosil Hidrolases/metabolismo , Óperon/genética , Estresse Fisiológico/efeitos dos fármacos , Ácidos Teicoicos/metabolismo
13.
J Microbiol Biotechnol ; 29(3): 401-409, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30939630

RESUMO

Heat-resistant microbial hosts are required for bioprocess development using high cell density cultivations at the industrial scale. We report that the thermotolerance of Escherichia coli can be enhanced by overexpressing ybeD, which was known to encode a hypothetical protein of unknown function. In the wild-type E. coli BL21(DE3), ybeD transcription level increased over five-fold when temperature was increased from 37°C to either 42°C or 46°C. To study the function of ybeD, a deletion strain and an overexpression strain were constructed. At 46°C, in comparison to the wild type, the ybeD-deletion reduced cell growth half-fold, and the ybeD-overexpression promoted cell growth over two-fold. The growth enhancement by ybeD-overexpression was much more pronounced at 46°C than 37°C. The ybeD-overexpression was also effective in other E. coli strains of MG1655, W3110, DH10B, and BW25113. These findings reveal that ybeD gene plays an important role in enduring high-temperature stress, and that ybeD-overexpression can be a prospective strategy to develop thermotolerant microbial hosts.


Assuntos
Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , N-Glicosil Hidrolases/biossíntese , N-Glicosil Hidrolases/genética , Termotolerância/genética , Contagem de Células , DNA Bacteriano/análise , Escherichia coli/crescimento & desenvolvimento , Genes Bacterianos/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Deleção de Sequência , Termotolerância/fisiologia
14.
Genes (Basel) ; 10(4)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018584

RESUMO

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.


Assuntos
DNA Glicosilases/metabolismo , Replicação do DNA , N-Glicosil Hidrolases/genética , Ciclo Celular , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Modelos Genéticos , Timina/análogos & derivados , Timina/metabolismo , Uracila/análogos & derivados , Uracila/metabolismo
15.
Protein Expr Purif ; 161: 40-48, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31034876

RESUMO

Leishmaniasis represents an important public health problem in several countries. The main target in this study is the nucleoside hydrolase Leishmania chagasi (LcNH) that is responsible for causing visceral leishmaniasis, principally in Brazil. Nucleoside hydrolase enzymes are members of this pathway, hydrolyzing the N-glycosidic bond of ribonucleosides for the synthesis of nucleic acids. We present here for the first time, the expression and purification protocols to obtain the enzymes LcNH1 and LcNH2 that can be employed to explore novel strategies to produce nucleoside hydrolase inhibitors for use in chemotherapy. Protein integrity was also confirmed by SDS-PAGE gel, mass spectrometry and enzymatic activity.


Assuntos
Leishmania/enzimologia , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/isolamento & purificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Clonagem Molecular , Leishmania/genética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo
16.
Mol Cell ; 74(6): 1239-1249.e4, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31023582

RESUMO

The stringent response alarmones pppGpp and ppGpp are essential for rapid adaption of bacterial physiology to changes in the environment. In Escherichia coli, the nucleosidase PpnN (YgdH) regulates purine homeostasis by cleaving nucleoside monophosphates and specifically binds (p)ppGpp. Here, we show that (p)ppGpp stimulates the catalytic activity of PpnN both in vitro and in vivo causing accumulation of several types of nucleobases during stress. The structure of PpnN reveals a tetramer with allosteric (p)ppGpp binding sites located between subunits. pppGpp binding triggers a large conformational change that shifts the two terminal domains to expose the active site, providing a structural rationale for the stimulatory effect. We find that PpnN increases fitness and adjusts cellular tolerance to antibiotics and propose a model in which nucleotide levels can rapidly be adjusted during stress by simultaneous inhibition of biosynthesis and stimulation of degradation, thus achieving a balanced physiological response to constantly changing environments.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato/química , Guanosina Tetrafosfato/química , N-Glicosil Hidrolases/química , Regulação Alostérica , Sequência de Aminoácidos , Antibacterianos/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Cinética , Modelos Moleculares , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estresse Fisiológico , Especificidade por Substrato
17.
Nature ; 567(7747): 267-272, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842657

RESUMO

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , DNA/química , DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , DNA/biossíntese , Replicação do DNA , Feminino , Humanos , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , N-Glicosil Hidrolases/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitinação , Xenopus
18.
Plant Cell ; 31(3): 734-751, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30787180

RESUMO

Plants can fully catabolize purine nucleotides. A firmly established central intermediate is the purine base xanthine. In the current widely accepted model of plant purine nucleotide catabolism, xanthine can be generated in various ways involving either inosine and hypoxanthine or guanosine and xanthosine as intermediates. In a comprehensive mutant analysis involving single and multiple mutants of urate oxidase, xanthine dehydrogenase, nucleoside hydrolases, guanosine deaminase, and hypoxanthine guanine phosphoribosyltransferase, we demonstrate that purine nucleotide catabolism in Arabidopsis (Arabidopsis thaliana) mainly generates xanthosine, but not inosine and hypoxanthine, and that xanthosine is derived from guanosine deamination and a second source, likely xanthosine monophosphate dephosphorylation. Nucleoside hydrolase 1 (NSH1) is known to be essential for xanthosine hydrolysis, but the in vivo function of a second cytosolic nucleoside hydrolase, NSH2, is unclear. We demonstrate that NSH1 activates NSH2 in vitro and in vivo, forming a complex with almost two orders of magnitude higher catalytic efficiency for xanthosine hydrolysis than observed for NSH1 alone. Remarkably, an inactive NSH1 point mutant can activate NSH2 in vivo, fully preventing purine nucleoside accumulation in nsh1 background. Our data lead to an altered model of purine nucleotide catabolism that includes an NSH heterocomplex as a central component.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Guanosina Monofosfato/metabolismo , Ribonucleosídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Mutação , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Nucleosídeo Desaminases/genética , Nucleosídeo Desaminases/metabolismo , Plantas Geneticamente Modificadas
19.
ACS Infect Dis ; 5(3): 345-352, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30701958

RESUMO

Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis and is the most prevalent, nonviral sexually transmitted disease. The parasite has shown increasing resistance to the current 5-nitroimidazole therapies indicating the need for new therapies with different mechanisms. T. vaginalis is an obligate parasite that scavenges nucleosides from host cells and then uses salvage pathway enzymes to obtain the nucleobases. The adenosine/guanosine preferring nucleoside ribohydrolase was screened against a 2000-compound diversity fragment library using a 1H NMR-based activity assay. Three classes of inhibitors with more than five representatives were identified: bis-aryl phenols, amino bicyclic pyrimidines, and aryl acetamides. Among the active fragments were 10 compounds with ligand efficiency values greater than 0.5, including five with IC50 values <10 µM. Jump-dilution and detergent counter screens validated reversible, target-specific activity. The data reveals an emerging SAR that is guiding our medicinal chemistry efforts aimed at discovering compounds with nanomolar potency.


Assuntos
Antiprotozoários/química , Inibidores Enzimáticos/química , N-Glicosil Hidrolases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Trichomonas vaginalis/enzimologia , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Ligantes , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/química , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/genética
20.
FEBS J ; 286(6): 1214-1229, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633437

RESUMO

The ammonium-dependent posttranslational regulation of nitrogenase activity in Azospirillum brasilense requires dinitrogenase reductase ADP-ribosyl transferase (DraT) and dinitrogenase reductase ADP-glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP-ribosylated Fe-protein was used as substrate to study the mechanism involved in the regulation of A. brasilense DraG in vitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG-GlnZ-AmtB complex in vitro. Previous structural data have revealed that when the DraG-GlnZ complex associates with AmtB, the flexible T-loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG-GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T-loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T-loop (GlnZΔ42-54). However, although the GlnZΔ42-54 variant was more effective in inhibiting DraG in vitro, it bound to DraG with a slightly lower affinity than does wild-type GlnZ and was not competent to completely inhibit DraG activity either in vitro or in vivo. We, therefore, conclude that the formation of a ternary complex between DraG-GlnZ-AmtB is necessary for the inactivation of DraG.


Assuntos
ADP Ribose Transferases/metabolismo , Compostos de Amônio/metabolismo , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , N-Glicosil Hidrolases/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , ADP Ribose Transferases/genética , Azospirillum brasilense/genética , Azospirillum brasilense/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Regulação Bacteriana da Expressão Gênica , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , Proteínas PII Reguladoras de Nitrogênio/genética , Ligação Proteica , Conformação Proteica , Transdução de Sinais
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