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1.
Sci Rep ; 11(1): 17463, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465810

RESUMO

Spermine oxidase (SMOX) catalyzes the oxidation of spermine to spermidine. Observational studies have reported SMOX as a source of reactive oxygen species associated with cancer, implying that inhibition of SMOX could be a target for chemoprevention. Here we test causality of SMOX levels with cancer risk using a Mendelian randomization analysis. We performed a GWAS of spermidine/spermine ratio to identify genetic variants associated with regulation of SMOX activity. Replication analysis was performed in two datasets of SMOX gene expression. We then did a Mendelian randomization analysis by testing the association between the SMOX genetic instrument and neuroblastoma, gastric, lung, breast, prostate, and colorectal cancers using GWAS summary statistics. GWAS of spermidine/spermine ratio identified SMOX locus (P = 1.34 × 10-49) explaining 32% of the variance. The lead SNP rs1741315 was also associated with SMOX gene expression in newborns (P = 8.48 × 10-28) and adults (P = 2.748 × 10-8) explaining 37% and 6% of the variance, respectively. Genetically determined SMOX activity was not associated with neuroblastoma, gastric, lung, breast, prostate nor colorectal cancer (P > 0.05). A PheWAS of rs1741315 did not reveal any relevant associations. Common genetic variation in the SMOX gene was strongly associated with SMOX activity in newborns, and less strongly in adults. Genetic down-regulation of SMOX was not significantly associated with lower odds of neuroblastoma, gastric, lung, breast, prostate and colorectal cancer. These results may inform studies of SMOX inhibition as a target for chemoprevention.


Assuntos
Regulação Enzimológica da Expressão Gênica , Predisposição Genética para Doença , Análise da Randomização Mendeliana/métodos , Neoplasias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Adulto , Regulação Neoplásica da Expressão Gênica , Humanos , Recém-Nascido , Neoplasias/etiologia , Neoplasias/metabolismo , Fenótipo
2.
Cells ; 10(7)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34359834

RESUMO

Astrocytes act as neural stem cells (NSCs) that have the potential to self-renew and differentiate into other neuronal cells. The protein expression of these astrocytes depends on the stage of differentiation, showing sequential expression of multiple proteins such as octamer-binding transcription factor 4 (Oct4), nestin, glial fibrillary acidic protein (GFAP), and aldehyde dehydrogenase 1 family member L1 (aldh1L1). Photobiomodulation (PBM) affects cell apoptosis, proliferation, migration, and adhesion. We hypothesized that astrocyte proliferation and differentiation would be modulated by PBM. We used an optimized astrocyte culture method and a 660-nanometer light-emitting diode (LED) to enhance the biological actions of many kinds of cells. We determined that the 660-nanometer LED promoted the biological actions of cultured astrocytes by increasing the reactive oxygen species levels. The overall viability of the cultured cells, which included various cells other than astrocytes, did not change after LED exposure; however, astrocyte-specific proliferation was observed by the increased co-expression of GFAP and bromodeoxyuridine (BrdU)/Ki67. Furthermore, the 660-nanometer LED provides evidence of differentiation, as shown by the decreased Oct4 and GFAP co-expression and increased nestin and aldh1L1 expression. These results demonstrate that a 660-nanometer LED can modify astrocyte proliferation, which suggests the efficacy of the therapeutic application of LED in various pathological states of the central nervous system.


Assuntos
Astrócitos/efeitos da radiação , Proliferação de Células/efeitos da radiação , Expressão Gênica/efeitos da radiação , Neurônios/efeitos da radiação , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Adesão Celular/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Movimento Celular/efeitos da radiação , Técnicas de Cocultura , Embrião de Mamíferos , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Lasers Semicondutores , Luz , Nestina/genética , Nestina/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo
3.
Plant Mol Biol ; 106(6): 555-567, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34275101

RESUMO

KEY MESSAGE: Root-specific expression of a cytokinin-degrading CKX gene in maize roots causes formation of a larger root system leading to higher element content in shoot organs. The size and architecture of the root system is functionally relevant for the access to water and soil nutrients. A great number of mostly unknown genes are involved in regulating root architecture complicating targeted breeding of plants with a larger root system. Here, we have explored whether root-specific degradation of the hormone cytokinin, which is a negative regulator of root growth, can be used to genetically engineer maize (Zea mays L.) plants with a larger root system. Root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene of Arabidopsis caused the formation of up to 46% more root dry weight while shoot growth of these transgenic lines was similar as in non-transgenic control plants. The concentration of several elements, in particular of those with low soil mobility (K, P, Mo, Zn), was increased in leaves of transgenic lines. In kernels, the changes in concentration of most elements were less pronounced, but the concentrations of Cu, Mn and Zn were significantly increased in at least one of the three independent lines. Our data illustrate the potential of an increased root system as part of efforts towards achieving biofortification. Taken together, this work has shown that root-specific expression of a CKX gene can be used to engineer the root system of maize and alter shoot element composition.


Assuntos
Proteínas de Arabidopsis/genética , Citocininas/metabolismo , Proteínas de Membrana/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Folhas de Planta/genética , Raízes de Plantas/genética , Zea mays/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cobre/metabolismo , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Manganês/metabolismo , Proteínas de Membrana/metabolismo , Minerais/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes/genética , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Zinco/metabolismo
4.
Genes (Basel) ; 12(5)2021 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066864

RESUMO

Glutaric aciduria type II (GA-II) is a rare autosomal recessive disease caused by defects in electron transfer flavoprotein (ETF), ultimately causing insufficiencies in multiple acyl-CoA dehydrogenase (MAD). 3-phosphoglycerate dehydrogenase (3-PHGDH) deficiency, is another rare autosomal disorder that appears due to a defect in the synthesis of L-serine amino acid. Several mutations of ETFDH and PHGDH genes have been associated with different forms of GA-II and serine deficiency, respectively. In this study, we report a unique case of GA-II with serine deficiency using biochemical, genetic, and in silico approaches. The proband of Syrian descent had positive newborn screening (NBS) for GA-II. At two years of age, the patient presented with developmental regression, ataxia, and intractable seizures. Results of amino acid profiling demonstrated extremely low levels of serine. Confirmatory tests for GA-II and whole exome sequencing (WES) were performed to determine the etiology of intractable seizure. Sequencing results indicated a previously reported homozygous missense mutation, c.679 C>A (p.Pro227Thr) in the ETFDH gene and a novel missense homozygous mutation c.1219 T>C (p.Ser407Pro) in the PHGDH gene. In silico tools predicted these mutations as deleterious. Here, the clinical and biochemical investigations indicate that ETFDH:p.Pro227Thr and PHGDH:p.Ser407Pro variants likely underlie the pathogenesis of GA-II and serine deficiency, respectively. This study indicates that two rare autosomal recessive disorders should be considered in consanguineous families, more specifically in those with atypical presentation.


Assuntos
Erros Inatos do Metabolismo dos Carboidratos/genética , Flavoproteínas Transferidoras de Elétrons/genética , Proteínas Ferro-Enxofre/genética , Microcefalia/genética , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Fosfoglicerato Desidrogenase/deficiência , Fosfoglicerato Desidrogenase/genética , Transtornos Psicomotores/genética , Convulsões/genética , Serina/deficiência , Erros Inatos do Metabolismo dos Carboidratos/sangue , Erros Inatos do Metabolismo dos Carboidratos/patologia , Pré-Escolar , Feminino , Humanos , Microcefalia/sangue , Microcefalia/patologia , Deficiência Múltipla de Acil Coenzima A Desidrogenase/patologia , Mutação de Sentido Incorreto , Fosfoglicerato Desidrogenase/sangue , Transtornos Psicomotores/sangue , Transtornos Psicomotores/patologia , Convulsões/sangue , Convulsões/patologia , Serina/sangue
5.
Langmuir ; 37(24): 7536-7547, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34102059

RESUMO

Controlling enzyme orientation and location on surfaces is a critical step for their successful deployment in diverse applications from biosensors to lab-on-a-chip devices. Functional activity of the enzymes on the surface will largely depend on the spatial arrangement and orientation. Solid binding peptides have been proven to offer versatility for immobilization of biomolecules on inorganic materials including metals, oxides, and minerals. Previously, we demonstrated the utility of a gold binding peptide genetically incorporated into the enzyme putrescine oxidase (PutOx-AuBP), enabling self-enzyme assembly on gold substrates. PutOx is an attractive biocatalyst among flavin oxidases, using molecular oxygen as an electron acceptor without requiring a dissociable coenzyme. Here, we explore the selective self-assembly of this enzyme on a range of surfaces using atomic force microscopy (AFM) along with the assessment of functional activity. This work probes the differences in surface coverage, distribution, size, shape, and activity of PutOx-AuBP in comparison to those of native putrescine oxidase (PutOx) on multiple surfaces to provide insight for material-selective enzymatic assembly. Surfaces investigated include metal (templated-stripped gold (TSG)), oxide (native SiO2 on Si(111)), minerals (mica and graphite), and self-assembled monolayers (SAMs) with a range of hydrophobicity and charge. Supported by both the coverage and the dimensions of immobilized enzymes, our results indicate that of the surfaces investigated, material-selective binding takes place with orientation control only for PutOx-AuBP onto the TSG substrate. These differences are consistent with the measurements of surface-bound enzymatic activities. Substrate-dependent differences observed indicate significant variations in enzyme-surface interactions ranging from peptide-directed self-assembly to enzyme aggregation. The implications of this study provide insight for the fabrication of enzymatic patterns directed by self-assembling peptide tags onto localized surface regions. Enabling functional enzyme-based nanoscale materials offers a fascinating path for utilization of sustainable biocatalysts integrated into multiscale devices.


Assuntos
Ouro , Dióxido de Silício , Enzimas Imobilizadas , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Peptídeos , Propriedades de Superfície
6.
Int J Biol Macromol ; 182: 959-967, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33872614

RESUMO

Dihydromethanopterin reductase (DmrB), is a naturally occurring cage protein found in various archaeal and a few bacterial species. It exists as 24mer with cubic geometry where 8 trimeric subunits are present at the corners of each cube. Each trimer is made up of three monomeric units and six FMN, where two molecules of FMN are present at the interface of each monomer. DmrB is involved in the conversion of dihydromethanopterin to tetrahydromethanopterin using FMN as a redox equivalent. In the present study, we have used spectroscopic and biochemical techniques along with complementary bio-informatic work to understand the assembly principles of the DmrB. Our results show a concentration dependant self-assembly of DmrB which is mediated by ionic interactions. The co-factor FMN stabilizes and preserves the secondary and quaternary structure of DmrB against thermal insult, indicating that the higher order assembly of DmrB is very thermostable. Our work provides an interesting piece of information regarding the role of the co-factors in the thermostability of these classes of cage proteins. The understanding of the assembly and disassembly of this thermostable cage would enable the downstream usage of this system in various nano-biotechnological applications.


Assuntos
Proteínas de Bactérias/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Multimerização Proteica , Pterinas/química , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Concentração Osmolar
7.
Crit Rev Biochem Mol Biol ; 56(4): 360-372, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33823724

RESUMO

Electron transfer flavoprotein dehydrogenase, also called ETF-ubiquinone oxidoreductase (ETF-QO), is a protein localized in the inner membrane of mitochondria, playing a central role in the electron-transfer system. Indeed, ETF-QO mediates electron transport from flavoprotein dehydrogenases to the ubiquinone pool. ETF-QO mutations are often associated with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD, OMIM#231680), a multisystem genetic disease characterized by various clinical manifestations with different degrees of severity. In this review, we outline the clinical features correlated with ETF-QO deficiency and the benefits obtained from different treatments, such as riboflavin, L-carnitine and/or coenzyme Q10 supplementation, and a diet poor in fat and protein. Moreover, we provide a detailed summary of molecular and bioinformatic investigations, describing the mutations identified in ETFDH gene and highlighting their predicted impact on enzymatic structure and activity. In addition, we report biochemical and functional analysis, performed in HEK293 cells and patient fibroblasts and muscle cells, to show the relationship between the nature of ETFDH mutations, the variable impairment of enzyme function, and the different degrees of RR-MADD severity. Finally, we describe in detail 5 RR-MADD patients carrying different ETFDH mutations and presenting variable degrees of clinical symptom severity.


Assuntos
Flavoproteínas Transferidoras de Elétrons , Proteínas Ferro-Enxofre , Mitocôndrias , Deficiência Múltipla de Acil Coenzima A Desidrogenase , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Animais , Carnitina/genética , Carnitina/metabolismo , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/genética , Deficiência Múltipla de Acil Coenzima A Desidrogenase/enzimologia , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/genética , Ubiquinona/metabolismo
8.
EMBO J ; 40(9): e106491, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33847380

RESUMO

Exercise can alter the skeletal muscle DNA methylome, yet little is known about the role of the DNA methylation machinery in exercise capacity. Here, we show that DNMT3A expression in oxidative red muscle increases greatly following a bout of endurance exercise. Muscle-specific Dnmt3a knockout mice have reduced tolerance to endurance exercise, accompanied by reduction in oxidative capacity and mitochondrial respiration. Moreover, Dnmt3a-deficient muscle overproduces reactive oxygen species (ROS), the major contributors to muscle dysfunction. Mechanistically, we show that DNMT3A suppresses the Aldh1l1 transcription by binding to its promoter region, altering its epigenetic profile. Forced expression of ALDH1L1 elevates NADPH levels, which results in overproduction of ROS by the action of NADPH oxidase complex, ultimately resulting in mitochondrial defects in myotubes. Thus, inhibition of ALDH1L1 pathway can rescue oxidative stress and mitochondrial dysfunction from Dnmt3a deficiency in myotubes. Finally, we show that in vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a-deficient mice. Together, we establish that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Músculo Esquelético/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Resistência Física/genética , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Camundongos , Mitocôndrias Musculares/metabolismo , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA
9.
FASEB J ; 35(5): e21473, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33811703

RESUMO

Pancreatic diseases including diabetes and exocrine insufficiency would benefit from therapies that reverse cellular loss and/or restore cellular mass. The identification of molecular pathways that influence cellular growth is therefore critical for future therapeutic generation. Deoxyhypusine synthase (DHPS) is an enzyme that post-translationally modifies and activates the mRNA translation factor eukaryotic initiation factor 5A (eIF5A). Previous work demonstrated that the inhibition of DHPS impairs zebrafish exocrine pancreas development; however, the link between DHPS, eIF5A, and regulation of pancreatic organogenesis remains unknown. Herein we identified that the conditional deletion of either Dhps or Eif5a in the murine pancreas results in the absence of acinar cells. Because DHPS catalyzes the activation of eIF5A, we evaluated and uncovered a defect in mRNA translation concomitant with defective production of proteins that influence cellular development. Our studies reveal a heretofore unappreciated role for DHPS and eIF5A in the synthesis of proteins required for cellular development and function.


Assuntos
Lisina/análogos & derivados , Organogênese , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/fisiologia , Pâncreas Exócrino/citologia , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Animais , Proliferação de Células , Feminino , Lisina/biossíntese , Masculino , Camundongos , Camundongos Knockout , Pâncreas Exócrino/metabolismo , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética
10.
Cell Death Dis ; 12(3): 225, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649354

RESUMO

Conversion of astrocytes into neurons in vivo offers an alternative therapeutic approach for neuronal loss after injury or disease. However, not only the efficiency of the conversion of astrocytes into functional neurons by single Neurog2, but also the conundrum that whether Neurog2-induced neuronal cells (Neurog2-iNs) are further functionally integrated into existing matured neural circuits remains unknown. Here, we adopted the AAV(2/8) delivery system to overexpress single factor Neurog2 into astrocytes and found that the majority of astrocytes were successfully converted into neuronal cells in multiple brain regions, including the midbrain and spinal cord. In the midbrain, Neurog2-induced neuronal cells (Neurog2-iNs) exhibit neuronal morphology, mature electrophysiological properties, glutamatergic identity (about 60%), and synapse-like configuration local circuits. In the spinal cord, astrocytes from both the intact and lesioned sources could be converted into functional neurons with ectopic expression of Neurog2 alone. Notably, further evidence from our study also proves that Neurog2-iNs in the intact spinal cord are capable of responding to diverse afferent inputs from dorsal root ganglion (DRG). Together, this study does not merely demonstrate the feasibility of Neurog2 for efficient in vivo reprogramming, it gives an indication for the Neurog2-iNs as a functional and potential factor in cell-replacement therapy.


Assuntos
Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transdiferenciação Celular , Mesencéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Neurônios/metabolismo , Medula Espinal/metabolismo , Animais , Astrócitos/ultraestrutura , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Mesencéfalo/ultraestrutura , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/ultraestrutura , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fenótipo , Medula Espinal/ultraestrutura , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo
11.
PLoS Pathog ; 17(2): e1008909, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33592076

RESUMO

The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein and is essential in all eukaryotes. However, the specific roles of eIF5A in translation and in other biological processes remain elusive. In the present study, we described the role of eIF5A, its posttranslational modifications (PTM), and the biosynthetic pathway needed for the PTM in Entamoeba histolytica, the protozoan parasite responsible for amoebic dysentery and liver abscess in humans. E. histolytica encodes two isotypes of eIF5A and two isotypes of enzymes, deoxyhypusine synthase (DHS), responsible for their PTM. Both of the two eIF5A isotypes are functional, whereas only one DHS (EhDHS1, but not EhDHS2), is catalytically active. The DHS activity increased ~2000-fold when EhDHS1 was co-expressed with EhDHS2 in Escherichia coli, suggesting that the formation of a heteromeric complex is needed for full enzymatic activity. Both EhDHS1 and 2 genes were required for in vitro growth of E. histolytica trophozoites, indicated by small antisense RNA-mediated gene silencing. In trophozoites, only eIF5A2, but not eIF5A1, gene was actively transcribed. Gene silencing of eIF5A2 caused compensatory induction of expression of eIF5A1 gene, suggesting interchangeable role of the two eIF5A isotypes and also reinforcing the importance of eIF5As for parasite proliferation and survival. Furthermore, using a sibling species, Entamoeba invadens, we found that eIF5A1 gene was upregulated during excystation, while eIF5A2 was downregulated, suggesting that eIF5A1 gene plays an important role during differentiation. Taken together, these results have underscored the essentiality of eIF5A and DHS, for proliferation and potentially in the differentiation of this parasite, and suggest that the hypusination associated pathway represents a novel rational target for drug development against amebiasis.


Assuntos
Diferenciação Celular , Proliferação de Células , Entamoeba histolytica/crescimento & desenvolvimento , Entamebíase/parasitologia , Lisina/análogos & derivados , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Entamebíase/genética , Entamebíase/metabolismo , Humanos , Lisina/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética
12.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33397698

RESUMO

Nicotine and nicotinic acid (NA) are both considered to be representatives of N-heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the cooccurrence of these two pathways has only been observed in Pseudomonas sp. strain JY-Q. The nicotine pyrrolidine catabolism pathway of strain JY-Q consists of the functional modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenase (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm enzymes (namely, Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 were both in-frame deleted, the mutant still grew well in basic salt medium (BSM) supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains reorganized domains similar to those of Spm. When the JY-Q_nicAB gene (nicAB in strain JY-Q) was introduced into another Pseudomonas strain, one that is unable to degrade NA, the resultant recombinant strain exhibited the ability to transform SP to HSP, but without the ability to metabolize NA. Here, we conclude that NicAB in strain JY-Q exhibits an additional role in SP transformation. The other genes in the NA cluster, NicXDFE (Nic2 homolog), then also exhibit a role in subsequent HSP metabolism for energy yield. This finding also suggests that the cooccurrence of nicotine and NA degradation genes in strain JY-Q represents an advantage for JY-Q, making it more effective and flexible for the degradation of nicotine.IMPORTANCE 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) are both valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway, in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. strain JY-Q. However, when two homologous Spm genes (spm1 and spm2) were knocked out, the mutant retained the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have an alternative pathway for SP conversion. In this research, we showed that JY-Q_NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q_NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates.


Assuntos
Proteínas de Bactérias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Pseudomonas/enzimologia , Piridinas/metabolismo , Succinatos/metabolismo , Nicotina/metabolismo , Pseudomonas/genética
13.
Acta Biochim Pol ; 68(1): 29-31, 2021 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-33485289

RESUMO

Protein crystallographers are well aware of the trap of crystallizing E. coli proteins instead of the macromolecule of interest if heterologous recombinant protein expression in E. coli was part of the experimental pipeline. Among the well-known culprits are YodA metal-binding lipocalin (25 kDa) and YadF carbonic anhydrase (a tetramer of 25 kDa subunits). We report a novel crystal form of another such culprit, E. coli HPII catalase, which is a tetrameric protein of ~340 kDa molecular weight. HPII is likely to contaminate recombinant protein samples, co-purify, and then co-crystallize with the target proteins, especially if their masses in size exclusion chromatography are ~300-400 kDa. What makes this case more interesting but also parlous, is the fact that HPII can crystallize from very low concentrations, even well below 1 mg/mL.


Assuntos
Catalase/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Arabidopsis/enzimologia , Proteínas de Arabidopsis/química , Cromatografia em Gel/métodos , Cristalização , Glutamato Desidrogenase/química , Peso Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Difração de Raios X
14.
J Cereb Blood Flow Metab ; 41(5): 1080-1090, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32615885

RESUMO

In eukaryotes, the polyamine pathway generates spermidine that activates the hypusination of the translation factor eukaryotic initiation factor 5A (eIF5A). Hypusinated-eIF5A modulates translation, elongation, termination and mitochondrial function. Evidence in model organisms like drosophila suggests that targeting polyamines synthesis might be of interest against ischemia. However, the potential of targeting eIF5A hypusination in stroke, the major therapeutic challenge specific to ischemia, is currently unknown. Using in vitro models of ischemic-related stress, we documented that GC7, a specific inhibitor of a key enzyme in the eIF5A activation pathway, affords neuronal protection. We identified the preservation of mitochondrial function and thereby the prevention of toxic ROS generation as major processes of GC7 protection. To represent a thoughtful opportunity of clinical translation, we explored whether GC7 administration reduces the infarct volume and functional deficits in an in vivo transient focal cerebral ischemia (tFCI) model in mice. A single GC7 pre- or post-treatment significantly reduces the infarct volume post-stroke. Moreover, GC7-post-treatment significantly improves mouse performance in the rotarod and Morris water-maze, highlighting beneficial effects on motor and cognitive post-stroke deficits. Our results identify the targeting of the polyamine-eIF5A-hypusine axis as a new therapeutic opportunity and new paradigm of research in stroke and ischemic diseases.


Assuntos
Guanina/análogos & derivados , Lisina/análogos & derivados , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Acidente Vascular Cerebral/terapia , Animais , Comportamento Animal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Guanina/administração & dosagem , Guanina/farmacologia , Guanina/uso terapêutico , Injeções Intraperitoneais , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/prevenção & controle , Lisina/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/ultraestrutura , Modelos Animais , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/efeitos dos fármacos , Poliaminas/metabolismo , Proteínas de Ligação a RNA/efeitos dos fármacos , Espécies Reativas de Oxigênio/toxicidade , Acidente Vascular Cerebral/metabolismo
15.
Chembiochem ; 22(1): 124-128, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-32789939

RESUMO

Optically active ß-amino alcohols are very useful chiral intermediates frequently used in the preparation of pharmaceutically active substances. Here, a novel cyclohexylamine oxidase (ArCHAO) was identified from the genome sequence of Arthrobacter sp. TYUT010-15 with the R-stereoselective deamination activity of ß-amino alcohol. ArCHAO was cloned and successfully expressed in E. coli BL21, purified and characterized. Substrate-specific analysis revealed that ArCHAO has high activity (4.15 to 6.34 U mg-1 protein) and excellent enantioselectivity toward the tested ß-amino alcohols. By using purified ArCHAO, a wide range of racemic ß-amino alcohols were resolved, (S)-ß-amino alcohols were obtained in >99 % ee. Deracemization of racemic ß-amino alcohols was conducted by ArCHAO-catalyzed enantioselective deamination and transaminase-catalyzed enantioselective amination to afford (S)-ß-amino alcohols in excellent conversion (78-94 %) and enantiomeric excess (>99 %). Preparative-scale deracemization was carried out with 50 mM (6.859 g L-1 ) racemic 2-amino-2-phenylethanol, (S)-2-amino-2-phenylethanol was obtained in 75 % isolated yield and >99 % ee.


Assuntos
Amino Álcoois/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Transaminases/metabolismo , Amino Álcoois/química , Arthrobacter/enzimologia , Biocatálise , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Estereoisomerismo , Transaminases/genética
16.
Mol Plant ; 14(2): 344-351, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33220510

RESUMO

Under conditions of labor or resource scarcity, direct seeding, rather than transplantation, is the preferred mode of rice (Oryza sativa) cultivation. This approach requires varieties that exhibit uniform seedling emergence. Mesocotyl elongation (ME), the main driver of rapid emergence of rice seedlings from soil, is enhanced by darkness and inhibited by light. Plant polyamine oxidases (PAOs) oxidize polyamines (PAs) and release H2O2. Here, we established that OsPAO5 expression in rice seedlings is increased in the presence of light and inhibited by darkness. To determine its role in ME, we created OsPAO5 mutants using CRISPR/Cas9. Compared with the wild type, pao5 mutants had longer mesocotyls, released less H2O2, and synthesized more ethylene. The mutant seedlings emerged at a higher and more uniform rate, indicating their potential for use in direct seeding. Nucleotide polymorphism analysis revealed that an SNP (PAO5-578G/A) located 578 bp upstream of the OsPAO5 start codon alters its expression, and was selected during rice mesocotyl domestication. The PAO5-578G genotype conferring a long mesocotyl mainly exists in wild rice, most Aus accessions, and some Geng (Japonica) accessions. Intriguingly, knocking out OsPAO5 can remarkably increase the grain weight, grain number, and yield potential. In summary, we developed a novel strategy to obtain elite rice with higher emergence vigor and yield potential, which can be conveniently and widely used to breed varieties of direct-seeding rice.


Assuntos
Cotilédone/crescimento & desenvolvimento , Mutagênese/genética , Oryza/crescimento & desenvolvimento , Oryza/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Sementes/crescimento & desenvolvimento , Biomassa , Etilenos/biossíntese , Mutação/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/metabolismo , Plântula , Solo
17.
J AOAC Int ; 104(3): 693-711, 2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-33367653

RESUMO

BACKGROUND: BioSystems has developed a histamine kit for automated procedure, through the use of a Y15 analyzer, for quantification of histamine in fish and fishery products. OBJECTIVE: Validate the method under the specific guidelines of the AOAC Research Institute Performance Tested MethodSM (PTM) program. METHOD: Samples are extracted with boiling water. The enzymatic method is based on histamine dehydrogenase that catalyzes the oxidation of histamine in the presence of an electron mediator that reduces a dye that is measured at 420 nm. The increase of absorbance is proportional to the histamine concentration. Dispensing of reagents and sample, absorbance readings, calibration, and calculation of results are performed automatically in the analyzer BioSystems Y15. RESULTS: The linearity ranges from 0 to 200 mg/kg (r2 > 0.99). The LOQ is 10 mg/kg in all the matrixes. Recoveries range from 75 to 107% at concentrations from 5 to 200 mg/kg, with repeatability precision values between 0.8 and 5.5%. Comparisons with the HPLC reference method shows a good correlation. Cross-reactivity of the assay is negligible for all biogenic amines tested except for agmatine (6.3%). Product consistency was verified by validating lot-to-lot variations and variations within the same lot. Shelf life was verified by real-time stability testing during 40 months at 2-8°C. No differences in histamine detection were observed in robustness testing, in which minor changes are introduced to the assay protocol. CONCLUSIONS: The automated, simple, and rapid BioSystems Y15 Histamine Dehydrogenase Kit has been successfully validated. HIGHLIGHTS: The method is qualified for PTM certification No. 072001.


Assuntos
Histamina , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Animais , Pesqueiros , Peixes
18.
Bioelectrochemistry ; 138: 107719, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33333456

RESUMO

Histamine released from mast cells plays an important role as not only a physiological active substance for the trigger of allergic reactions but also a neurotransmitter in a nervous system. To detect histamine directly, we immobilized oxygen-independent histamine dehydrogenase (HmDH) onto the surface of cup-stacked carbon nanofibers (CSCNFs), which work both as an electrical nanowire and an enzyme support, and investigated the direct electron transfer reaction from HmDH reduced by histamine to CSCNF. Current responses of histamine oxidation at the HmDH-modified CSCNF electrode showed a linear relationship between 0.3 µM and 300 µM with detection limit of 0.1 µM in a Briton Robinson buffer (pH 9.4) and was about 25 times larger than those at a flat glassy carbon electrode modified with HmDH because of its three-dimensional network. Using the HmDH-modified CSCNF electrode, we successfully observed histamine release from rat basophilic leukemia cell line RBL-2H3 after stimulation with degranulation agents, such as antigen 2,4-dinitrophenylated bovine serum albumin and calcium ionophore A23187. The present sensing system might be applied to at least advanced in vitro allergic diagnostic methods.


Assuntos
Basófilos/patologia , Técnicas Biossensoriais/métodos , Carbono/química , Liberação de Histamina , Leucemia/patologia , Nanofibras/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Linhagem Celular Tumoral , Eletroquímica , Humanos , Limite de Detecção
19.
Protein Expr Purif ; 178: 105767, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32987121

RESUMO

Nicotine contamination in tobacco waste effluent (TWE) from tobacco industry is a serious threat to public health and environment. Microbial degradation is an impending approach to remove nicotine and transform it into some other high value chemicals. Pseudomonas sp. JY-Q exhibits high efficiency of degradation, which can degrade 5 g/L of nicotine within 24 h. In strain JY-Q, we found the co-occurrence of two homologous key enzymes NicA2 and Nox, which catalyze nicotine to N-methylmyosmine, and then to pseudooxylnicotine via simultaneous hydrolysis. In this study, recombinant NicA2 and Nox were expressed in E. coli BL21(DE3) and purified. In vitro, the activity of recombinant NicA2 and Nox was accelerated by adding co-factor NAD+, suggesting that they worked as dehydrogenases. The optimal reaction conditions, substrate affinity, catabolism efficiency, pH-stability and thermal-stability were determined. Nox showed lower efficiency, but at a higher stability level than NicA2. Nox exhibited wider pH range and higher temperature as optimal conditions for the enzymatic reaction. In addition, The Nox showed higher thermo-stability and acid-stability than that of NicA2. The study on enzymatic reaction kinetics showed that Nox had a lower Km and higher substrate affinity than NicA2. These results suggest that Nox plays more significant role than NicA2 in nicotine degradation in TWE, which usually is processed at low pH (4-5) and high temperature (above 40 °C). Genetic engineering is required to enhance the affinity and suitability of NicA2 for an increased additive effect on homologous NicA2 and Nox in strain JY-Q.


Assuntos
Proteínas de Bactérias , Nicotina/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Pseudomonas/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Pseudomonas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
20.
Mult Scler Relat Disord ; 48: 102689, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33383363

RESUMO

We report a case of late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) with recurrent abdominal pain, vomiting, and impaired consciousness as the initial symptoms in Yemen; the case showed distinctive characteristics from those of Asian or Caucasian patients. Initially, he was misdiagnosed with pancreatitis, acute disseminated encephalomyelitis(ADEM), and fatty liver. Final diagnosis was further confirmed by electromyography, muscle biopsy, uric organic acid analysis, and a novel missense mutation in exon 7 (c.807A>C) of ETFDH was identified by next-generation sequencing. To our knowledge, we report this mutation in an adult MADD patient as well as late-onset MADD in a Middle East country for the first time. MADD is characterised by varied genotypes and broad spectrum of clinical manifestations among different populations and ages, which requires more attention and awareness in the clinic.


Assuntos
Encefalomielite Aguda Disseminada , Proteínas Ferro-Enxofre , Deficiência Múltipla de Acil Coenzima A Desidrogenase , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Adulto , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Erros de Diagnóstico , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Proteínas Ferro-Enxofre/genética , Masculino , Oriente Médio , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Iêmen
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