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1.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879484

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribução , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribução , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribução , Vacúolos/metabolismo
2.
Am J Hum Genet ; 107(3): 473-486, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32781046

RESUMO

Africa contains more human genetic variation than any other continent, but the majority of the population-scale analyses of the African peoples have focused on just two of the four major linguistic groups, the Niger-Congo and Afro-Asiatic, leaving the Nilo-Saharan and Khoisan populations under-represented. In order to assess genetic variation and signatures of selection within a Nilo-Saharan population and between the Nilo-Saharan and Niger-Congo and Afro-Asiatic, we sequenced 50 genomes from the Nilo-Saharan Lugbara population of North-West Uganda and 250 genomes from 6 previously unsequenced Niger-Congo populations. We compared these data to data from a further 16 Eurasian and African populations including the Gumuz, another putative Nilo-Saharan population from Ethiopia. Of the 21 million variants identified in the Nilo-Saharan population, 3.57 million (17%) were not represented in dbSNP and included predicted non-synonymous mutations with possible phenotypic effects. We found greater genetic differentiation between the Nilo-Saharan Lugbara and Gumuz populations than between any two Afro-Asiatic or Niger-Congo populations. F3 tests showed that Gumuz contributed a genetic component to most Niger-Congo B populations whereas Lugabara did not. We scanned the genomes of the Lugbara for evidence of selective sweeps. We found selective sweeps at four loci (SLC24A5, SNX13, TYRP1, and UVRAG) associated with skin pigmentation, three of which already have been reported to be under selection. These selective sweeps point toward adaptations to the intense UV radiation of the Sahel.


Assuntos
Adaptação Fisiológica/genética , Variação Genética/genética , Seleção Genética/genética , Pigmentação da Pele/genética , Grupo com Ancestrais do Continente Africano/genética , Antiporters/genética , Gerenciamento de Dados , Etiópia/epidemiologia , Feminino , Genética Populacional , Genoma Humano/genética , Haplótipos/genética , Humanos , Masculino , Glicoproteínas de Membrana/genética , Oxirredutases/genética , Polimorfismo de Nucleotídeo Único/genética , Nexinas de Classificação/genética , Proteínas Supressoras de Tumor/genética , Uganda/epidemiologia
3.
Science ; 369(6507): 1094-1098, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855335

RESUMO

Bacterial production of gaseous hydrocarbons such as ethylene and methane affects soil environments and atmospheric climate. We demonstrate that biogenic methane and ethylene from terrestrial and freshwater bacteria are directly produced by a previously unknown methionine biosynthesis pathway. This pathway, present in numerous species, uses a nitrogenase-like reductase that is distinct from known nitrogenases and nitrogenase-like reductases and specifically functions in C-S bond breakage to reduce ubiquitous and appreciable volatile organic sulfur compounds such as dimethyl sulfide and (2-methylthio)ethanol. Liberated methanethiol serves as the immediate precursor to methionine, while ethylene or methane is released into the environment. Anaerobic ethylene production by this pathway apparently explains the long-standing observation of ethylene accumulation in oxygen-depleted soils. Methane production reveals an additional bacterial pathway distinct from archaeal methanogenesis.


Assuntos
Proteínas de Bactérias/química , Etilenos/biossíntese , Metano/biossíntese , Metionina/biossíntese , Oxirredutases/química , Rhodospirillum rubrum/enzimologia , Anaerobiose , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biocatálise , Vias Biossintéticas , Oxirredutases/classificação , Oxirredutases/genética , Microbiologia do Solo
4.
Nature ; 585(7824): 288-292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32641834

RESUMO

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.


Assuntos
Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquinona/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ciona intestinalis/enzimologia , Ciclo do Ácido Cítrico , Citosol/metabolismo , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Lactobacillus brevis/enzimologia , Masculino , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Neoplasias/enzimologia , Fosforilação Oxidativa , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ubiquinona/metabolismo
5.
Science ; 369(6499): 59-64, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32631887

RESUMO

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.


Assuntos
Cobre/metabolismo , Histonas/química , Oxirredutases/química , Multimerização Proteica , Animais , Biocatálise , Domínio Catalítico/genética , Mutação com Ganho de Função , Histonas/genética , Histonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/química , Fatores de Transcrição/metabolismo , Xenopus laevis
6.
J Am Mosq Control Assoc ; 36(1): 22-32, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32497474

RESUMO

In several insect species, resistance to pyrethroids and DDT (dichlorodiphenyltrichloroethane) is linked to point mutations in the voltage-gated sodium channel (VGSC) gene. Pyrethroid-based insecticides prolong the opening of sodium channels, causing paralysis known as a "knockdown" effect before mortality occurs. Point mutations in the VGSC gene result in decreased pyrethroid binding and reduced sensitivity to the insecticide-this resistance mechanism is known as knockdown resistance (kdr) as insects do not die but recover from paralysis with time. In Culex mosquito species loss of target site sensitivity to pyrethroids is linked to a number of substitutions, one of which is leucine (L) to phenylalanine (F) at residue 1014 (L1014F) in the VGSC gene. Here we report the identification of kdr-associated pyrethroid resistance and developing resistance in Cx. quinquefasciatus field collections from Collier County, FL. Evaluation of position 1014 of the VGSC in Cx. quinquefasciatus collections from 7 locations in Collier County, FL, revealed a wide range of genotypes from one part of the district to the other. Centers for Disease Control and Prevention bottle bioassay, linear regression analysis, and cage trial evaluations suggest that the L1014F mutation plays a role, at least in part, to the pyrethroid resistance status of Cx. quinquefasciatus collected in Collier County, FL. Furthermore, we identified resistance attributed to both oxidase and esterase activity, indicating that multiple mechanisms are responsible for pyrethroid resistance in Collier County Cx. quinquefasciatus.


Assuntos
Culex/genética , Esterases/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Oxirredutases/genética , Piretrinas/farmacologia , Animais , Culex/efeitos dos fármacos , Culex/enzimologia , Esterases/metabolismo , Feminino , Florida , Oxirredutases/metabolismo
7.
PLoS Genet ; 16(4): e1008629, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282858

RESUMO

Analyzing 12,361 all-cause cirrhosis cases and 790,095 controls from eight cohorts, we identify a common missense variant in the Mitochondrial Amidoxime Reducing Component 1 gene (MARC1 p.A165T) that associates with protection from all-cause cirrhosis (OR 0.91, p = 2.3*10-11). This same variant also associates with lower levels of hepatic fat on computed tomographic imaging and lower odds of physician-diagnosed fatty liver as well as lower blood levels of alanine transaminase (-0.025 SD, 3.7*10-43), alkaline phosphatase (-0.025 SD, 1.2*10-37), total cholesterol (-0.030 SD, p = 1.9*10-36) and LDL cholesterol (-0.027 SD, p = 5.1*10-30) levels. We identified a series of additional MARC1 alleles (low-frequency missense p.M187K and rare protein-truncating p.R200Ter) that also associated with lower cholesterol levels, liver enzyme levels and reduced risk of cirrhosis (0 cirrhosis cases for 238 R200Ter carriers versus 17,046 cases of cirrhosis among 759,027 non-carriers, p = 0.04) suggesting that deficiency of the MARC1 enzyme may lower blood cholesterol levels and protect against cirrhosis.


Assuntos
Fígado Gorduroso/genética , Fígado Gorduroso/prevenção & controle , Predisposição Genética para Doença , Cirrose Hepática/genética , Cirrose Hepática/prevenção & controle , Proteínas Mitocondriais/genética , Mutação de Sentido Incorreto/genética , Oxirredutases/genética , Alelos , LDL-Colesterol/sangue , Doença da Artéria Coronariana/genética , Conjuntos de Dados como Assunto , Fígado Gorduroso/sangue , Fígado Gorduroso/enzimologia , Feminino , Homozigoto , Humanos , Fígado/enzimologia , Cirrose Hepática/sangue , Cirrose Hepática/enzimologia , Cirrose Hepática Alcoólica/sangue , Cirrose Hepática Alcoólica/enzimologia , Cirrose Hepática Alcoólica/genética , Cirrose Hepática Alcoólica/prevenção & controle , Mutação com Perda de Função/genética , Masculino , Pessoa de Meia-Idade
8.
Arch Microbiol ; 202(7): 1701-1708, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32296869

RESUMO

Responses to sunlight exposure of the oil-degrading Dietzia cinnamea P4 strain were evaluated by transcriptional levels of SOS genes, photoreactivation and genes involved in tolerance to high levels of reactive oxygen species. The P4 strain was exposed for 1 and 2 h and the magnitude of level changes in the mRNA was evaluated by qPCR. The results described the activation of the SOS system, with the decline of the repressor lexA gene levels and the concomitant increase of recA and uvrAD genes levels. The genes that participate in the photoreactivation process were also responsive to sunlight. The phrB gene encoding deoxyribodipyrimidine photo-lyase had its expression increased after 1-h exposure, while the phytAB genes showed a progressive increase over the studied period. The protective genes against reactive oxygen species, catalases, superoxides, peroxidases, and thioredoxins, had their expression rates detected under the conditions validated in this study. These results show a fast and coordinated response of genes from different DNA repair and tolerance mechanisms employed by strain P4, suggesting a complex concerted protective action against environmental stressors.


Assuntos
Actinobacteria/genética , Actinobacteria/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Luz Solar , Adaptação Fisiológica , Proteínas de Bactérias/genética , Reparo do DNA/genética , Hidrolases/genética , Oxirredutases/genética , Reação em Cadeia da Polimerase em Tempo Real
9.
Insect Biochem Mol Biol ; 120: 103347, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32114158

RESUMO

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.


Assuntos
Proteínas de Artrópodes/genética , Sistemas CRISPR-Cas , Edição de Genes , Mutagênese , Oxirredutases/genética , Tetranychidae/genética , Animais , Proteínas de Artrópodes/metabolismo , Oxirredutases/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-32155447

RESUMO

Plants can accumulate a large amount of reactive oxygen species under adverse conditions such as drought and high temperature, which seriously affect the normal growth and development of plants. The antioxidant system can scavenge the reactive oxygen species produced under drought conditions and so mitigate oxidative damage. However, the regulation patterns of many miRNAs under drought stress are still unclear. The content of antioxidant enzymes and the expression patterns of miRNAs and their target genes related to antioxidant systems were studied under drought stress in Zanthoxylum bungeanum. The results indicate that under drought stress, POD, CAT, APX, proline, MDA and related genes all show positive responses to drought, while SOD and its genes showed a negative response. It is indicated that in the antioxidant process of Z. bungeanum, POD, CAT, and APX play a major role, and SOD plays a supporting role. In addition, GUS histochemical and RT-qPCR experimental results show that the expression levels of miRNAs and their target genes are basically negatively correlated, indicating that miRNAs can inhibit the expression of related genes and are also important regulators in the antioxidant system of Z. bungeanum. According to the expression patterns of antioxidant enzymes, miRNA and its target genes under drought stress, combined with previous research results, a model of plant antioxidant mechanism was constructed to provide a reference for further understanding of plant antioxidant mechanism.


Assuntos
Secas , MicroRNAs , Oxirredutases , Estresse Fisiológico , Zanthoxylum , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , MicroRNAs/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Estresse Fisiológico/genética , Zanthoxylum/genética , Zanthoxylum/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(14): 7792-7798, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32209662

RESUMO

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer's and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1'-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1'-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of the TMEM189 gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivated Tmem189 gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the TMEM189 gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of Tmem189-deficient mice may be caused by a lack of plasmalogens.


Assuntos
Lipídeos/genética , Oxirredutases/genética , Plasmalogênios/genética , Enzimas de Conjugação de Ubiquitina/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Linhagem Celular , Humanos , Camundongos , Oxirredução , Oxirredutases/metabolismo , Fenótipo , Plasmalogênios/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Compostos de Vinila/metabolismo
12.
Aquat Toxicol ; 222: 105472, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32203794

RESUMO

ß-carotene is an efficient antioxidant and its accumulation is an oxidative response to stressors. Dunaliella salina strain GY-H13 is rich in ß-carotene under environmental stresses, which was selected as material to understand the molecular mechanism underlying ß-carotene biosynthesis. Seven full length cDNA sequences in ß-carotene biosynthesis pathway were cloned, including geranylgeranyl pyrophosphate synthase (GGPS), phytoene synthase (PSY), phytoene desaturase (PDS), 15-cis-zeta-carotene isomerase (ZISO), zeta-carotene desaturase (ZDS), prolycopene isomerase (CRTISO), lycopene beta-cyclase (LCYb). The seven protein sequences from the strain GY-H13 showed the highest similarity with other D. salina strains. Especially, PSY, PDS and LCYb protein sequences shared 100 % identity. Phylogenetic analysis indicated all proteins from GY-H13 firstly clustered with those from other D. salina strains with a bootstrap of 100 %. Multiple alignment indicated several distinct conserved motifs such as aspartate-rich domain (ARD), dinucleotide binding domain (DBD), and carotene binding domain (CBD). These motifs are located near ligand-binding pocket, which may be required for the activity of enzyme. Expression levels of these genes and ß-carotene content were measured over 24-h cycle, showing clear daily dynamics. All genes were dramatically up-regulated in the morning but the highest accumulation of ß-carotene was observed at noon, suggesting a lag-effect between gene transcription and biological response. Furthermore, the accumulation of ß-carotene increased under nitrogen deficiency, Cd exposure and high light and decreased under high salinity in a time-dependent manner. No gene of ß-carotene biosynthesis was up-regulated by high salinity while most genes were activated by the other stresses at the beginning stage of exposure. Growth inhibition and oxidative damage were also observed under high salinity. Overall, transcription activation of ß-carotene biosynthetic genes at the initial stage of stress exposure is a determinant of the increased accumulation of ß-carotene in microalgae, which help their survive under harsh environments. The newly isolated D. salina strain GY-H13 would be a promising microalgae model for investigating the molecular mechanism of stress-induced ß-carotene biosynthesis.


Assuntos
Cádmio/toxicidade , Microalgas/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , beta Caroteno/biossíntese , Sequência de Aminoácidos , Antioxidantes/metabolismo , Liases Intramoleculares/genética , Microalgas/genética , Microalgas/metabolismo , Oxirredutases/genética , Filogenia , Salinidade , beta Caroteno/genética
13.
Food Chem ; 318: 126483, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126468

RESUMO

In this study, the antioxidant activity of germinating Chinese wild rice was found to decline initially, after which it increased. The largest difference in antioxidant activity was observed between the 36-h (G36) and the 120-h germination (G120) stage. We further assessed the dynamic changes in metabolites, phenolic acids, flavonoids, and phenolic biosynthetic genes in germinating Chinese wild rice. Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry revealed that 315 metabolites were up-regulated and 28 were down-regulated between G36 and G120. Levels of p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and epigallocatechin increased significantly during germination. Gene expression of four phenylalanine ammonia-lyases, one 4-coumarate-CoA ligase, one cinnamoyl-CoA reductase, two cinnamyl alcohol dehydrogenases, one chalcone synthase, and one chalcone isomerase was significantly higher at G120 than at G36 and promoted phenolics accumulation. This study elucidated the biochemical mechanisms involved in antioxidant activity and phenolic profile changes during Chinese wild rice germination.


Assuntos
Antioxidantes/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Fenóis/metabolismo , Proteínas de Plantas/genética , Poaceae/fisiologia , Aciltransferases/genética , Cromatografia Líquida de Alta Pressão , Coenzima A Ligases/genética , Germinação , Hidroxibenzoatos/metabolismo , Liases Intramoleculares/genética , Espectrometria de Massas , Oxirredutases/genética , Fenilalanina Amônia-Liase/genética , Poaceae/química , Poaceae/genética , Sementes/química , Sementes/genética , Sementes/fisiologia
14.
Plant Biol (Stuttg) ; 22(4): 679-690, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32003103

RESUMO

Melatonin has emerged as an essential molecule in plants, due to its role in defence against metal toxicity. Aluminium (Al) and cadmium (Cd) toxicity inhibit rapeseed seedling growth. In this study, we applied different doses of melatonin (50 and 100 µm) to alleviate Al (25 µm) and Cd (25 µm) stress in rapeseed seedlings. Results show that Al and Cd caused toxicity in rapeseed seedling, as evidenced by a decrease in height, biomass and antioxidant enzyme activity. Melatonin increased the expression of melatonin biosynthesis-related Brassica napus genes for caffeic acid O-methyl transferase (BnCOMT) under Al and Cd stress. The genes BnCOMT-1, BnCOMT-5 and BnCOMT-8 showed up-regulated expression, while BnCOMT-4 and BnCOMT-6 were down-regulated during incubation in water. Melatonin application increased the germination rate, shoot length, root length, fresh and dry weight of seedlings. Melatonin supplementation under Al and Cd stress increased superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, proline, chlorophyll and anthocyanin content, as well as photosynthesis rate. Both Cd and Al treatments significantly increased hydrogen peroxide and malondialdehyde levels in rapeseed seedlings, which were strictly counterbalanced by melatonin. Analysis of Cd and Al in different subcellular compartments showed that melatonin enhanced cell wall and soluble fractions, but reduced the vacuolar and organelle fractions in Al- and Cd-treated seedlings. These results suggest that melatonin-induced improvements in antioxidant potential, biomass, photosynthesis rate and successive Cd and Al sequestration play a pivotal role in plant tolerance to Al and Cd stress. This mechanism may have potential implications in safe food production.


Assuntos
Alumínio , Brassica napus , Cádmio , Regulação da Expressão Gênica de Plantas , Melatonina , Alumínio/toxicidade , Antioxidantes/farmacologia , Brassica napus/efeitos dos fármacos , Brassica napus/enzimologia , Cádmio/toxicidade , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Melatonina/farmacologia , Oxirredutases/genética , Oxirredutases/metabolismo , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/crescimento & desenvolvimento , Poluentes do Solo/toxicidade
15.
Nat Commun ; 11(1): 1098, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107375

RESUMO

The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.


Assuntos
Proteínas de Bactérias/genética , Reatores Biológicos , Caulobacter crescentus/genética , Engenharia Metabólica/métodos , Modelos Químicos , Proteínas de Bactérias/metabolismo , Biocombustíveis , Metabolismo dos Carboidratos/genética , Caulobacter crescentus/enzimologia , Simulação por Computador , Hidroliases/genética , Hidroliases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Redes e Vias Metabólicas/genética , NADP/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Xilose/metabolismo
16.
Nat Commun ; 11(1): 864, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054833

RESUMO

Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, siroheme is produced by a trifunctional enzyme, siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD+-dependent dehydrogenation and iron chelation. How this active site performs such different chemistries is unknown. Here, we report the structures of CysG bound to precorrin-2, the initial substrate; sirohydrochlorin, the dehydrogenation product/chelation substrate; and a cobalt-sirohydrochlorin product. We identified binding poses for all three tetrapyrroles and tested the roles of specific amino acids in both activities to give insights into how a bifunctional active site catalyzes two different chemistries and acts as an iron-specific chelatase in the final step of siroheme synthesis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Heme/análogos & derivados , Metiltransferases/química , Metiltransferases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Eletroquímica , Ferroquelatase/química , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/biossíntese , Heme/química , Metiltransferases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidade por Substrato , Tetrapirróis/química , Tetrapirróis/metabolismo , Uroporfirinas/química , Uroporfirinas/metabolismo
17.
Nat Commun ; 11(1): 867, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054832

RESUMO

Alzheimer's disease (AD) is defined by progressive neurodegeneration, with oligomerization and aggregation of amyloid-ß peptides (Aß) playing a pivotal role in its pathogenesis. In recent years, the yeast Saccharomyces cerevisiae has been successfully used to clarify the roles of different human proteins involved in neurodegeneration. Here, we report a genome-wide synthetic genetic interaction array to identify toxicity modifiers of Aß42, using yeast as the model organism. We find that FMN1, the gene encoding riboflavin kinase, and its metabolic product flavin mononucleotide (FMN) reduce Aß42 toxicity. Classic experimental analyses combined with RNAseq show the effects of FMN supplementation to include reducing misfolded protein load, altering cellular metabolism, increasing NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios and increasing resistance to oxidative stress. Additionally, FMN supplementation modifies Htt103QP toxicity and α-synuclein toxicity in the humanized yeast. Our findings offer insights for reducing cytotoxicity of Aß42, and potentially other misfolded proteins, via FMN-dependent cellular pathways.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Mononucleotídeo de Flavina/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Genes Sintéticos , Genoma Fúngico , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Modelos Genéticos , Mutação , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Dobramento de Proteína , Proteólise , RNA-Seq , Riboflavina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
18.
Environ Pollut ; 259: 113907, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32023790

RESUMO

B cells contribute to produce inflammatory cytokines and antibodies, to present autoantigens, and to interact with T cells, which lead to body defense and disease control. Nuclear factor (erythroid-derived 2)-like 2(Nrf2) is responsible for gene expression of antioxidant enzymes to protect cells from oxidative stress by reactive oxygen species(ROS) production. Bisphenol A(BPA) may not be safe due to the effect on body's physiological functions. The chemicals that substitute for BPA may still have similar effects in the body. Tritan™ copolyester is a novel plastic form using BPA substitutes, 1,4-cyclohexanedimethanol(CHDM), dimethyl terephthalate(DMT), and 2,2,4,4-tetramethyl-1,3-cyclobutanediol(TMCD). Isosorbide(ISO) was also used as a substitute for TMCD and DMT. Here, we investigated whether B cell viability is influenced by BPA and its substitutes via Nrf2 induction using WiL2-NS human B lymphoblast cells. When cytotoxicity was measured by using assays with MTT, CellTiter-Glo, trypan blue and propidium iodide, cytotoxicity by BPA was higher than that by substitutes. BPA and its substitutes showed significant cytotoxicity and ROS production, which were attenuated by the treatment with N-acetylcysteine(NAC), a ROS scavenger. In addition, BPA treatment enhanced gene expression of antioxidant enzymes, heme oxygenase(HO)-1, catalase, superoxide dismutase(SOD) 1 and 2. As H2O2 treatment induced cell death and Nrf2 amount in WiL2-NS cells, BPA treatment increased Nrf2. Cell death by H2O2 was increased in doxycycline-inducible Nrf2-knockdown(KD) cells. In Cytotoxicity by the treatment with BPA or its substitutes was also enhanced in Nrf2-KD cells but that was reduced by Nrf2 overexpression compared to control cells. Taken together, these results implicate that B cell cytotoxicity by substitutes should be lower than BPA and Nrf2 can prevent B cells from BPA- or BPA substitutes-induced cytotoxicity via ROS production. Data suggest that the comprehensive studies or evaluation could be necessary to replace BPA in manufacture by other substitutes.


Assuntos
Linfócitos B , Compostos Benzidrílicos , Regulação da Expressão Gênica , Peróxido de Hidrogênio , Fator 2 Relacionado a NF-E2 , Fenóis , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Compostos Benzidrílicos/química , Compostos Benzidrílicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/genética , Fenóis/química , Fenóis/toxicidade , Espécies Reativas de Oxigênio
19.
Plant Biol (Stuttg) ; 22(3): 425-432, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32052535

RESUMO

Cyanide-resistant respiration in potato mitochondria is an important pathway for energy dissipation. It can be activated by high light; however, it is unclear what roles cyanide-resistant respiration plays in the response to high light stress in potato. We designed a CRISPR vector for the functional gene StAOX of the potato cyanide-resistant respiratory pathway. Agrobacterium tumefaciens GV3101 was transformed into potato. Hydrogen peroxide level, MDA content, antioxidant activity and cyanide-resistant respiratory capacity of potato leaves under high light stress were determined. Photosynthetic efficiency and chlorophyll content were determined. In addition, the operation of the malate-oxaloacetate shuttle route and transcription level of photorespiration-related enzymes were also examined. The results showed that two base substitutions occurred at the sequencing target site on leaves of the transformed potato. Accumulation of ROS and increased membrane lipid peroxidation were detected in the transformed potato leaves and lower photosynthetic efficiency was observed. The transcription level of the malate-oxaloacetate shuttle route and photorespiration-related enzymes also significantly increased. These results indicate that the cyanide-resistant respiration is an important physiological pathway in potato in response to high light stress. It also suggests that plant cyanide-resistant respiration is closely related to photosynthesis. This implies the unexplored importance of plant cyanide-resistant respiration in plant photosynthesis, energy conversion and carbon skeleton formation.


Assuntos
Respiração Celular , Cianetos , Resistência a Medicamentos , Luz , Folhas de Planta , Solanum tuberosum , Agrobacterium tumefaciens/genética , Respiração Celular/efeitos dos fármacos , Respiração Celular/efeitos da radiação , Clorofila , Cianetos/toxicidade , Oxirredutases/genética , Fotossíntese , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Solanum tuberosum/efeitos da radiação
20.
Photochem Photobiol Sci ; 19(2): 274-280, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32002529

RESUMO

In the present study, we aimed to purify and characterize LuxG obtained from Photobacterium leiognathi YL and examine its improvement for NADH detection. To this end, we cloned and expressed the putative luxG gene of P. leiognathi YL in the Escherichia coli BL21 strain. The product of luxG is a flavin reductase that consists of 206 amino acids, corresponding to a subunit molecular mass of ∼26 kDa. Phylogenetic analysis demonstrated that P. leiognathi YL LuxG has a rather distant evolutionary relationship with Frase I of Aliivibrio fischeri and Frp of Vibrio harveyi, but a close evolutionary relationship with Fre from Escherichia coli, which are all enzymes related to oxido-reductase. Further comparison shows that the changes in the functionally conserved sites may contribute to the functional divergence of LuxG and Fre. LuxG could supply reduced flavin mononucleotide (FMN) for bacterial luminescence by catalyzing the oxidation of nicotinamide adenine dinucleotide hydrogen (NADH). Based on this, a coupled pure enzyme bioluminescent system was established and used for NADH detection. The NADH samples with concentrations of 0.1-1 nM were used to validate the linear relationship, and it was found that the logarithmic deviations were less than 3%, which showed more sensitive and stable results than the NADH detection by recombinant E. coli including the exogenously expressed luciferase and intrinsic Fre. Investigation of P. leiognathi YL LuxG would provide a basic understanding of its evolution, and structural and functional properties, which might contribute to the development of a NADH detection kit in the future.


Assuntos
Proteínas de Bactérias/metabolismo , Medições Luminescentes , NAD/análise , Oxirredutases/metabolismo , Photobacterium/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/metabolismo , Evolução Molecular , Oxirredutases/classificação , Oxirredutases/genética , Filogenia , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência
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