Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22.922
Filtrar
1.
Enzyme Microb Technol ; 150: 109880, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489033

RESUMO

The ene reductases (ERs) from the old yellow enzymes (OYEs) family have the ability to reduce activated alkenes to generate up to two stereocenters, therefore they have been received extensive attention as powerful biocatalysts. In this study, through gene mining, four ERs were identified from the genomes of Ensifer adhaerens, Pseudomonas fluorescens, and Pseudomonas veronil. The biocatalytic properties of these four ERs were identified, and their applications in the synthesis process of dihydrocarvone and profen derivatives were further evaluated. Among them, three ERs (EaER2, PvER1, and PvER2) belonging to the classic OYEs showed the best catalytic activity at 30 °C and pH 7.0 (100 mM potassium phosphate buffer) and the PfER2, which belongs to the thermophilic-like OYEs exhibited the best catalytic at 40 °C and pH 7.0 (100 mM potassium phosphate buffer). When exploring the influence of organic solvents on the catalytic efficiency, it was found that the four ERs were more sensitive to toluene and had tolerance to several other selected organic solvents. In addition, EaER2, PfER2, PvER1 and PvER2 showed excellent catalytic activity toward carvone, and the stereoselectivity of PvER2 toward carvone could reach up to 88.7 % de. EaER2 and PfER2 can catalyze the synthesis of a variety of profen derivatives with a stereoselectivity over 99 % ee. Moreover, through homology modeling and molecular docking, we preliminarily explained the mechanism of catalytic activity and stereoselectivity of the four ERs, which provided a solid base on the rational design of their stereo-preference in the future. The discovery of EaER2, PfER2, PvER1, and PvER2 provides four new enzyme sources for the study of the OYEs family and enriches the biocatalytic toolbox of ERs. Our exploration of the enzymatic properties of these four ERs will provide the sufficient data basis for future research and industrialization progress.


Assuntos
Oxirredutases , Biocatálise , Monoterpenos Cicloexânicos , Simulação de Acoplamento Molecular , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Rhizobiaceae
2.
Enzyme Microb Technol ; 150: 109884, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489037

RESUMO

Tyrosinase plays an essential role in melanin biosynthesis and inherently exhibits both monophenolase and diphenolase activity. A first derivative synchronous fluorometric assay was established for directly monitoring monophenolase activity. The zero-crossing point at 322 nm for the first-derivative under synchronous fluorescence with Δλ = 67 nm was utilized to selectively quantify tyrosine in the presence of the reaction product dihydroxyphenylalanine (DOPA). The limit of detection (LOD) for tyrosine was 0.54 µM. The fluorescence intensity of tyrosine was monitored at intervals of 30 s to establish the time course of tyrosine consumption. The LOD for the monophenolase activity was 0.0706 U⋅ mL-1. The Michaelis-Menten e constant and maximum speed were 21.83 µM and 1.12 µM min-1, respectively. Zinc ions competitively inhibited the monophenolase activity, with an IC50 value of 14.36 µM. This assay is easily and rapidly executed and is of great significance for analyzing the kinetics of enzymatic reactions and in fundamental research on monophenolase. This approach has potential applications in the discovery of tyrosinase inhibitors for medicine and cosmetics, as well as in the industrial synthesis of substituted o-diphenol intermediates.


Assuntos
Monofenol Mono-Oxigenase , Oxirredutases , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Oxirredutases/metabolismo , Tirosina/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445601

RESUMO

Ferroptosis, an iron-dependent form of programmed cell death, has excellent potential as an anti-cancer therapeutic strategy in different types of tumors, especially in RAS-mutated ones. However, the function of ferroptosis for inhibiting neuroblastoma, a common child malignant tumor with minimal treatment, is unclear. This study investigated the anti-cancer function of ferroptosis inducer Erastin or RSL3 in neuroblastoma N2A cells. Our results show that Erastin or RSL3 induces ROS level and cell death and, therefore, reduces the viability of RAS-proficient N2A cells. Importantly, inhibitors to ferroptosis, but not apoptosis, ameliorate the high ROS level and viability defect in Erastin- or RSL3-treated cells. In addition, our data also show that N2A cells are much more sensitive to ferroptosis inducers than primary mouse cortical neural stem cells (NSCs) or neurons. Moreover, a higher level of ROS and PARylation is evidenced in N2A, but not NSCs. Mechanically, ferritin heavy chain 1 (Fth), the ferroxidase function to oxidate redox-active Fe2+ to redox-inactive Fe3+, is likely responsible for the hypersensitivity of N2A to ferroptosis induction since its expression is lower in N2A compared to NSCs; ectopic expression of Fth reduces ROS levels and cell death, and induces expression of GPX4 and cell viability in N2A cells. Most importantly, neuroblastoma cell lines express a significantly low level of Fth than almost all other types of cancer cell lines. All these data suggest that Erastin or RSL3 induce ferroptosis cell death in neuroblastoma N2A cells, but not normal neural cells, regardless of RAS mutations, due to inadequate FTH. This study, therefore, provides new evidence that ferroptosis could be a promising therapeutic target for neuroblastoma.


Assuntos
Ferritinas/metabolismo , Ferroptose , Células-Tronco Neurais/patologia , Neuroblastoma/patologia , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose , Feminino , Ferritinas/genética , Ferro/metabolismo , Peroxidação de Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Oxirredução , Oxirredutases/genética , Piperazinas/metabolismo , Proteínas ras/genética
4.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361015

RESUMO

The sacred lotus (Nelumbo nucifera) can maintain a stable floral chamber temperature between 30 and 35 °C when blooming despite fluctuations in ambient temperatures between about 8 and 45 °C, but the regulatory mechanism of floral thermogenesis remains unclear. Here, we obtained comprehensive protein profiles from receptacle tissue at five developmental stages using data-independent acquisition (DIA)-based quantitative proteomics technology to reveal the molecular basis of floral thermogenesis of N. nucifera. A total of 6913 proteins were identified and quantified, of which 3513 differentially abundant proteins (DAPs) were screened. Among them, 640 highly abundant proteins during the thermogenic stages were mainly involved in carbon metabolism processes such as the tricarboxylic acid (TCA) cycle. Citrate synthase was identified as the most connected protein in the protein-protein interaction (PPI) network. Next, the content of alternative oxidase (AOX) and plant uncoupling protein (pUCP) in different tissues indicated that AOX was specifically abundant in the receptacles. Subsequently, a protein module highly related to the thermogenic phenotype was identified by the weighted gene co-expression network analysis (WGCNA). In summary, the regulation mechanism of floral thermogenesis in N. nucifera involves complex regulatory networks, including TCA cycle metabolism, starch and sucrose metabolism, fatty acid degradation, and ubiquinone synthesis, etc.


Assuntos
Adaptação Fisiológica , Flores/metabolismo , Redes Reguladoras de Genes , Nelumbo/genética , Mapas de Interação de Proteínas , Proteoma/metabolismo , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico , Flores/genética , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nelumbo/crescimento & desenvolvimento , Nelumbo/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma/genética , Temperatura
5.
Int J Mol Sci ; 22(16)2021 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-34445240

RESUMO

Nitroaromatic compounds (ArNO2) maintain their importance in relation to industrial processes, environmental pollution, and pharmaceutical application. The manifestation of toxicity/therapeutic action of nitroaromatics may involve their single- or two-electron reduction performed by various flavoenzymes and/or their physiological redox partners, metalloproteins. The pivotal and still incompletely resolved questions in this area are the identification and characterization of the specific enzymes that are involved in the bioreduction of ArNO2 and the establishment of their contribution to cytotoxic/therapeutic action of nitroaromatics. This review addresses the following topics: (i) the intrinsic redox properties of ArNO2, in particular, the energetics of their single- and two-electron reduction in aqueous medium; (ii) the mechanisms and structure-activity relationships of reduction in ArNO2 by flavoenzymes of different groups, dehydrogenases-electrontransferases (NADPH:cytochrome P-450 reductase, ferredoxin:NADP(H) oxidoreductase and their analogs), mammalian NAD(P)H:quinone oxidoreductase, bacterial nitroreductases, and disulfide reductases of different origin (glutathione, trypanothione, and thioredoxin reductases, lipoamide dehydrogenase), and (iii) the relationships between the enzymatic reactivity of compounds and their activity in mammalian cells, bacteria, and parasites.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias , Citotoxinas , Elétrons , Flavoproteínas , Nitrocompostos , Oxirredutases , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citotoxinas/química , Citotoxinas/farmacologia , Flavoproteínas/química , Flavoproteínas/metabolismo , Humanos , Nitrocompostos/química , Nitrocompostos/farmacologia , Oxirredução , Oxirredutases/química , Oxirredutases/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445672

RESUMO

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid ß-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal ß-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid ß-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid ß-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.


Assuntos
Ácidos Graxos/metabolismo , PPAR alfa/metabolismo , Peroxissomos/metabolismo , Acil-CoA Oxidase/metabolismo , Animais , Humanos , Fígado/metabolismo , Oxirredução , Oxirredutases/metabolismo , PPAR alfa/fisiologia , Proliferadores de Peroxissomos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta/genética , Receptores X de Retinoides/metabolismo , Ativação Transcricional/genética
7.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445771

RESUMO

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.


Assuntos
Corynebacterium glutamicum/metabolismo , Ácido Diaminopimélico/metabolismo , Lisina/metabolismo , Transdução de Sinais/fisiologia , Aciltransferases/metabolismo , Compostos de Amônio/metabolismo , Oxirredutases/metabolismo
8.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360597

RESUMO

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC50 of 0.822 µM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites.


Assuntos
Cumarínicos/metabolismo , Malatos/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Ubiquinona/metabolismo , Animais , Humanos , Proteínas Mitocondriais/genética , Oxirredutases/genética , Proteínas de Protozoários/genética , Especificidade por Substrato
9.
Sci Rep ; 11(1): 14748, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285303

RESUMO

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose-response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.


Assuntos
Candida albicans/enzimologia , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Lipossomos/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Cinética , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Fish Shellfish Immunol ; 116: 52-60, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34216786

RESUMO

The aim of this study was to investigate the effects of dietary bile acids (BAs) on intestinal healthy status of tongue sole in terms of immunity, antioxidant status, digestive ability, mucosal barrier-related genes expression and microbiota. Three experimental diets were prepared with BA levels at 0 mg/kg (CT), 300 mg/kg (BA1) and 900 mg/kg (BA2) in a commercial basal diet. Each diet was fed to three replicates with 120 fish (10.87 ± 0.32 g) in each tank. After an 8-week feeding trial, growth parameters were significantly enhanced in both BAs supplementary groups (P < 0.05), and compared with CT group, survival rate in BA2 group was significantly improved (P < 0.05). Intestinal lysozyme activity and contents of immunoglobulin M and complement 3 were significantly increased in both BAs supplementary groups (P < 0.05), suggesting an enhancement effect on the non-specific immune response. BAs inclusion also significantly improved intestinal antioxidant capabilities by increasing antioxidase activities and decreasing malondialdehyde levels. In addition, compared with CT group, intestinal digestive ability was substantially enhanced as indicated by the significantly increased lipase activity in BA2 group (P < 0.05) and significantly increased amylase activity in BA1 and BA2 groups (P < 0.05). Coincidentally, BAs inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes (P < 0.05). Further, dietary BAs distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. In conclusion, dietary BAs supplementation is an effective way to improve the intestinal healthy status of tongue sole.


Assuntos
Ácidos e Sais Biliares/farmacologia , Suplementos Nutricionais , Linguados , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Fosfatase Alcalina/imunologia , Amilases/metabolismo , Animais , Complemento C3/imunologia , Dieta/veterinária , Proteínas de Peixes/metabolismo , Linguados/genética , Linguados/imunologia , Linguados/metabolismo , Linguados/microbiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina M/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Lipase/metabolismo , Muramidase/imunologia , Oxirredutases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Junções Íntimas/genética
11.
Nat Microbiol ; 6(9): 1129-1139, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34267357

RESUMO

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/genética , Domínio Catalítico , Microscopia Crioeletrônica , Cristalografia por Raios X , Cinética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Oxirredução , Oxirredutases/genética
12.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299202

RESUMO

Rice spotted leaf mutants are helpful to investigate programmed cell death (PCD) and defense response pathways in plants. Using a map-based cloning strategy, we characterized novel rice spotted leaf mutation splHM143 that encodes a 7-hydroxymethyl chlorophyll a reductase (OsHCAR). The wild-type (WT) allele could rescue the mutant phenotype, as evidenced by complementation analysis. OsHCAR was constitutively expressed at all rice tissues tested and its expression products localized to chloroplasts. The mutant exhibited PCD and leaf senescence with increased H2O2 (hydrogen peroxide) accumulation, increased of ROS (reactive oxygen species) scavenging enzymes activities and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling) -positive nuclei, upregulation of PCD related genes, decreased chlorophyll (Chl) contents, downregulation of photosynthesis-related genes, and upregulation of senescence-associated genes. Besides, the mutant exhibited enhanced bacterial blight resistance with significant upregulation of defense response genes. Knockout lines of OsHCAR exhibited spotted leaf phenotype, cell death, leaf senescence, and showed increased resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) coupled with upregulation of five pathogenesis-related marker genes. The overexpression of OsHCAR resulted in increased susceptibility to Xoo with decreased expression of pathogenesis-related marker genes. Altogether, our findings revealed that OsHCAR is involved in regulating cell death and defense response against bacterial blight pathogen in rice.


Assuntos
Resistência à Doença/imunologia , Oryza/imunologia , Oxirredutases/metabolismo , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Proteínas de Plantas/metabolismo , Xanthomonas/fisiologia , Clorofila/análogos & derivados , Clorofila/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Oxirredutases/genética , Doenças das Plantas/microbiologia , Folhas de Planta/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética
13.
Front Immunol ; 12: 673692, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34305903

RESUMO

In a perspective entitled 'From plant survival under severe stress to anti-viral human defense' we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named 'ReprogVirus' was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the 'ReprogVirus platform' was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to 'RegroVirus' complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called 'CoV-MAC-TED'. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target 'CoV-MAC-TED' in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that 'de-stressing' disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.


Assuntos
Reprogramação Celular/genética , Herança Multifatorial/genética , SARS-CoV-2/patogenicidade , Acetilserotonina O-Metiltransferasa/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Ciclo Celular/genética , Bases de Dados Genéticas , Daucus carota/genética , Daucus carota/crescimento & desenvolvimento , Fermentação , Perfilação da Expressão Gênica , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tubulina (Proteína)/genética , Vírus/patogenicidade
14.
Front Immunol ; 12: 673723, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34211468

RESUMO

Reprogramming of primary virus-infected cells is the critical step that turns viral attacks harmful to humans by initiating super-spreading at cell, organism and population levels. To develop early anti-viral therapies and proactive administration, it is important to understand the very first steps of this process. Plant somatic embryogenesis (SE) is the earliest and most studied model for de novo programming upon severe stress that, in contrast to virus attacks, promotes individual cell and organism survival. We argued that transcript level profiles of target genes established from in vitro SE induction as reference compared to virus-induced profiles can identify differential virus traits that link to harmful reprogramming. To validate this hypothesis, we selected a standard set of genes named 'ReprogVirus'. This approach was recently applied and published. It resulted in identifying 'CoV-MAC-TED', a complex trait that is promising to support combating SARS-CoV-2-induced cell reprogramming in primary infected nose and mouth cells. In this perspective, we aim to explain the rationale of our scientific approach. We are highlighting relevant background knowledge on SE, emphasize the role of alternative oxidase in plant reprogramming and resilience as a learning tool for designing human virus-defense strategies and, present the list of selected genes. As an outlook, we announce wider data collection in a 'ReprogVirus Platform' to support anti-viral strategy design through common efforts.


Assuntos
COVID-19/prevenção & controle , Técnicas de Reprogramação Celular/métodos , Técnicas de Embriogênese Somática de Plantas/métodos , SARS-CoV-2/genética , COVID-19/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/genética , Humanos , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Desenvolvimento Vegetal/genética , Proteínas de Plantas/metabolismo , Plantas/embriologia , Plantas/genética , Espécies Reativas de Oxigênio/metabolismo
15.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 1827-1844, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227279

RESUMO

Vitamin C is an essential vitamin for human beings. It has a huge market in the fields of food and pharmaceuticals. 2-keto-L-gulonic acid is an important precursor to produce vitamin C by microbial fermentation in industrial. In microbial fermentations, the L-sorbose pathway and the D-gluconate pathway have been the focus of research because of high yield. This article aims at stating recent research progress in dehydrogenases related to biosynthesis of vitamin C in the L-sorbose pathway and the D-gluconate pathway. The properties of dehydrogenase in terms of localization, substrate specificity, cofactors, and electron transport carrier are elaborated. And then, the main problems and strategies are reviewed in the L-sorbose pathway and in the D-gluconate pathway. Finally, future research on the dehydrogenases in the biosynthesis of vitamin C through L-sorbose pathway and D-gluconate pathway is discussed.


Assuntos
Ácido Ascórbico/biossíntese , Oxirredutases , Fermentação , Gluconatos , Oxirredutases/metabolismo , Sorbose
16.
Nat Commun ; 12(1): 4621, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330928

RESUMO

Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.


Assuntos
Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Grupo dos Citocromos b/ultraestrutura , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Mycobacterium smegmatis/enzimologia , Oxirredutases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Heme/química , Heme/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/genética , Oxirredutases/química , Oxirredutases/metabolismo , Oxigênio/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato
17.
Nat Commun ; 12(1): 4402, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285231

RESUMO

Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/-) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis.


Assuntos
Injúria Renal Aguda/patologia , Ferroptose/fisiologia , Túbulos Renais/patologia , Traumatismo por Reperfusão/patologia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/etiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cisplatino/administração & dosagem , Cisplatino/toxicidade , Modelos Animais de Doenças , Células Epiteliais , Feminino , Ferroptose/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HT29 , Transplante de Coração/efeitos adversos , Humanos , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/química , Indóis/farmacologia , Indóis/uso terapêutico , Masculino , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos , Proteínas Mitocondriais/metabolismo , Células NIH 3T3 , Necrose/tratamento farmacológico , Necrose/etiologia , Necrose/patologia , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Cultura Primária de Células , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/etiologia
18.
Methods Mol Biol ; 2277: 371-389, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080163

RESUMO

In vitro experiments using permeabilized cells and/or isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across multiple scientific fields to shed valuable insight on mitochondrial-linked pathologies. The present chapter is intended to serve as a methodological blueprint for comprehensively phenotyping peripheral blood cell mitochondria. While primarily adapted for peripheral blood cells, the protocols outlined herein could easily be made amenable to most all cell types with minimal modifications.


Assuntos
Bioquímica/métodos , Leucócitos Mononucleares/citologia , Mitocôndrias/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/química , Citrato (si)-Sintase/análise , Citrato (si)-Sintase/metabolismo , Creatina Quinase/metabolismo , Humanos , Mitocôndrias/química , Oxirredutases/metabolismo , Fenótipo , Fluxo de Trabalho
19.
Plant Cell Rep ; 40(9): 1631-1646, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34146141

RESUMO

KEY MESSAGE: Studying RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes) in 5ß-cardenolide formation. Progesterone 5ß-reductases (P5ßR) are assumed to catalyze the reduction of progesterone to 5ß-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5ß-cardenolides. P5ßRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5ßRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5ßR1 (AY585867.1) and DlP5ßR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5ß-cardenolide formation, we here established several RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5ßR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5ßR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5ßR2-RNAi lines but not in Dl P5ßR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5ßR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).


Assuntos
Cardenolídeos/metabolismo , Digitalis/genética , Digitalis/metabolismo , Oxirredutases/genética , Proteínas de Plantas/genética , Butanonas/farmacologia , Butionina Sulfoximina/farmacologia , Digitalis/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Glutationa/farmacologia , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/genética , Plantas Geneticamente Modificadas , Progesterona/farmacologia , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
ChemSusChem ; 14(15): 3219-3225, 2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34138524

RESUMO

Cyanobacteria have the capacity to use photosynthesis to fuel their metabolism, which makes them highly promising production systems for the sustainable production of chemicals. Yet, their dependency on visible light limits the cell-density, which is a challenge for the scale-up. Here, it was shown with the example of a light-dependent biotransformation that internal illumination in a bubble column reactor equipped with wireless light emitters (WLEs) could overcome this limitation. Cells of the cyanobacterium Synechocystis sp. PCC 6803 expressing the gene of the ene-reductase YqjM were used for the reduction of 2-methylmaleimide to (R)-2-methylsuccinimide with high optical purity (>99 % ee). Compared to external source of light, illumination by floating wireless light emitters allowed a more than two-fold rate increase. Under optimized conditions, product formation rates up to 3.7 mm h-1 and specific activities of up to 65.5 U gDCW -1 were obtained, allowing the reduction of 40 mm 2-methylmaleimide with 650 mg isolated enantiopure product (73 % yield). The results demonstrate the principle of internal illumination as a means to overcome the intrinsic cell density limitation of cyanobacterial biotransformations, obtaining high reaction rates in a scalable photobioreactor.


Assuntos
Synechocystis/química , Synechocystis/metabolismo , Biocatálise , Biotransformação , Contagem de Células , Técnicas de Cultura de Células , Iluminação , Maleimidas/química , Oxirredução , Oxirredutases/metabolismo , Fotossíntese , Succinimidas/química , Synechocystis/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...