RESUMEN
To develop functional, sustainable and eco-friendly active packaging materials as alternatives to plastic films, we successfully prepared Ginkgo biloba leaf polysaccharide-stabilized selenium nanomaterials (Se-GBLP). Se-GBLP with glutathione peroxidase-like activity could efficiently remove harmful reactive oxygen species. As a functional additive, Se-GBLP was incorporated into degradable chitosan (CS) to fabricate CS/Se-GBLP films. The addition of Se-GBLP improved the mechanical properties, UV-visible light barrier performance, water vapor permeability, and antioxidant activity of the films. Preservation experiments demonstrated CS/Se-GBLP film could maintain quality and prolong the storage time of bananas and cherry tomatoes. It was the first time to use selenium-based nanozyme for fruit preservation. This work offered a cost-effective solution to reduce post-harvest losses, increasing sustainability and profitability. Future research should focus on more factors affecting freshness such as variety, maturity, harvest and storage conditions to improve preservation, as well as on the material's safety concern and environmental impact.
Asunto(s)
Embalaje de Alimentos , Conservación de Alimentos , Ginkgo biloba , Glutatión Peroxidasa , Polisacáridos , Selenio , Antioxidantes/química , Antioxidantes/metabolismo , Embalaje de Alimentos/instrumentación , Conservación de Alimentos/instrumentación , Conservación de Alimentos/métodos , Frutas/química , Ginkgo biloba/química , Glutatión Peroxidasa/metabolismo , Glutatión Peroxidasa/química , Musa/química , Nanoestructuras/química , Extractos Vegetales/química , Hojas de la Planta/química , Polisacáridos/química , Polisacáridos/metabolismo , Selenio/química , Solanum lycopersicum/químicaRESUMEN
Euglena gracilis, a fascinating organism in the scientific realm, exhibits characteristics of both animals and plants. It maintains redox homeostasis through a variety of enzymatic and non-enzymatic antioxidant molecules. In contrast to mammals, Euglena possesses nonselenocysteine glutathione peroxidase homologues that regulate its intracellular pools of reactive oxygen species. In the present study, a full-length cDNA of chloroplastic EgGPXL-1 was isolated and subjected to biochemical and functional characterization. Recombinant EgGPXL-1 scavenged H2O2 and t-BOOH, utilizing thioredoxin as an electron donor rather than glutathione. Despite its monomeric nature, EgGPXL-1 exhibits allosteric behavior with H2O2 as the electron acceptor and follows typical Michaelis-Menten kinetics with t-BOOH. Suppression of EgGPXL-1 gene expression under normal and high-light conditions did not induce critical situations in E. gracilis, suggesting the involvement of compensatory mechanisms in restoring normal conditions.
Asunto(s)
Euglena gracilis , Glutatión Peroxidasa , Tiorredoxinas , Euglena gracilis/enzimología , Euglena gracilis/genética , Euglena gracilis/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/química , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/química , Peróxido de Hidrógeno/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Cloroplastos/metabolismo , Cloroplastos/enzimología , Cloroplastos/genética , Secuencia de Aminoácidos , Cinética , Clonación Molecular , ADN Complementario/genéticaRESUMEN
The study included the purification of glutathione peroxidase enzyme (GPX) in the serum of women with breast cancer, which involved 60 samples of serum from women with breast cancer, and 30 samples from healthy individuals. The results of the study showed a significant decrease at a probability level of p<0.0001 for the activity of the GPX enzyme in the serum of women with breast cancer. Additionally, the GPX enzyme was purified from the serum of women with breast cancer through precipitation with ammonium sulfate and dialysis, and the use of DEAE-Cellulose ion exchange chromatography and gel filtration chromatography using Sephadex G-100, where a main protein band was separated, which was relied upon in determining the optimal conditions for the partially purified enzyme. The optimal conditions for the partially purified enzyme from the serum of women with breast cancer were determined and the highest activity was for the substrate concentration of 0.1 mM H2O2. The maximum speed Vmax was 3.125IU/L and the Michaelis-Menten constant Km was 0.0179 M using Lineweaver-Burk plot, the optimal pH was at 8.5, temperature at 37°C, and the highest activity time was at 5 minutes.
Asunto(s)
Neoplasias de la Mama , Glutatión Peroxidasa , Humanos , Femenino , Neoplasias de la Mama/enzimología , Glutatión Peroxidasa/sangre , Glutatión Peroxidasa/aislamiento & purificación , Glutatión Peroxidasa/química , Concentración de Iones de Hidrógeno , Peróxido de Hidrógeno/química , Persona de Mediana Edad , Cinética , Temperatura , Cromatografía por Intercambio Iónico , Cromatografía en Gel , AdultoRESUMEN
The synthesis of 1,3-benzoselenazoles was achieved by the reaction of corresponding bis[3-amino-N-(p-tolyl)benzamide-2-yl] diselenide, bis[3-amino-N-(4-methoxyphenyl)benzamide-2-yl] diselenide, and bis[3-amino-N-(4-(dimethylamino)phenyl) benzamide-2-yl] diselenide with aryl aldehydes. The 1,3-benzoselenazoles continued to exist as planar molecules due to the presence of secondary Se···O interactions as revealed by the single-crystal X-ray analysis. The presence of secondary Se···O interactions in 1,3-benzoselenazoles was confirmed using natural bond orbital (NBO) and atoms in molecules (AIM) calculations. Nucleus-independent chemical shift (NICS) values suggested the presence of aromatic character in a five-membered benzoselenazole heterocyclic ring. The glutathione peroxidase (GPx)-like antioxidant activity of all 1,3-benzoselenazoles was assessed using a thiophenol assay, exhibiting greater antioxidant activity than Ph2Se2 used as a reference. The most active catalyst carrying a strong electron-donating group (-NMe2) at the ortho-position to the benzoselenazole ring was further investigated at different concentrations of thiophenol, H2O2, and 1,3-benzoselenazoles as catalyst for determining their catalytic parameters. Moreover, the potential applications of all 1,3-benzoselenazoles against pancreatic lipase (PL) have been identified using in silico interactions between the active sites of the 1LPB protein as evaluated using a molecular docking study.
Asunto(s)
Antioxidantes , Peróxido de Hidrógeno , Antioxidantes/farmacología , Simulación del Acoplamiento Molecular , Glutatión Peroxidasa/química , Lipasa , BenzamidasRESUMEN
Selenocysteine (Sec)-derived cyclic selenenyl amides, formed by the intramolecular cyclization of Sec selenenic acids (Sec-SeOHs), have been postulated to function as protective forms in the bypass mechanism of glutathione peroxidase (GPx). However, their chemical properties have not been experimentally elucidated in proteins or small-molecule systems. Recently, we reported the first nuclear magnetic resonance observation of Sec-SeOHs and their cyclization to the corresponding cyclic selenenyl amides by using selenopeptide model systems incorporated in a molecular cradle. Herein, we elucidate the structures and reactivities of Sec-derived cyclic selenenyl amides. The crystal structures and reactions toward a cysteine thiol or a 1,3-diketone-type chemical probe indicated the highly electrophilic character of cyclic selenenyl amides. This suggests that they can serve not only as protective forms to suppress the inactivation of Sec-SeOHs in GPx but also as highly electrophilic intermediates in the reactions of selenoproteins.
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Amidas , Selenocisteína , Glutatión Peroxidasa/química , Selenocisteína/química , Amidas/química , Antioxidantes/química , SelenoproteínasRESUMEN
BACKGROUND: Neutrophils have a critical role in the pathogenesis of rheumatoid arthritis (RA) with immune system dysfunction. However, the molecular mechanisms of this process mediated by neutrophils still remain elusive. The purpose of the present study is to identify hub genes in neutrophils for diagnosis and treatment of RA utilizing publicly available datasets. METHODS: Gene expression profiles were downloaded from the Gene Expression Omnibus, and batch-corrected and normalized expression data were obtained using the ComBat package. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were used to conduct significantly functional analysis and crucial pathways. The resulting co-expression genes modules and hub genes were generated based on the weighted gene co-expression network analysis and visualization by Cytoscape. Flow cytometry was conducted to detect reactive oxygen species (ROS) levels in neutrophils. RESULTS: Neutrophils underwent transcriptional changes in synovial fluid (SF) of RA patients, different from peripheral blood of healthy controls or patients with RA. Especially, glycolysis, HIF-1 signaling, NADH metabolism, and oxidative stress were affected. These hub genes were strongly linked with classical glycolysis-related genes (ENO1, GAPDH, and PKM) responsible for ROS production. The antioxidant enzyme glutathione peroxidase 3 (GPX3), a ROS scavenger, was first identified as a hub gene in RA neutrophils. Neutrophils from patients with autoinflammatory and autoimmune diseases had markedly enhanced ROS levels, most notably in RA SF. CONCLUSION: This research recognized hub genes and explored the characteristics of neutrophils in RA. Our findings suggest that the novel hub gene GPX3 is involved in the neutrophil-driven oxidative stress-mediated pathogenesis of RA. It has the potency to be a target for neutrophil-directed RA therapy.
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Artritis Reumatoide , Glutatión Peroxidasa , Neutrófilos , Humanos , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/genética , Artritis Reumatoide/tratamiento farmacológico , Biomarcadores/metabolismo , Perfilación de la Expresión Génica/métodos , Glutatión Peroxidasa/química , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Glutathione peroxidases (Gpx) are a group of antioxidant enzymes that protect cells or tissues against damage from reactive oxygen species (ROS). The Gpx proteins identified in mammals exhibit high catalytic activity toward glutathione (GSH). In contrast, a variety of non-mammalian Gpx proteins from diverse organisms, including fungi, plants, insects, and rodent parasites, show specificity for thioredoxin (TRX) rather than GSH and are designated as TRX-dependent peroxiredoxins. However, the study of the properties of Gpx in the environmental microbiome or isolated bacteria is limited. In this study, we analyzed the Gpx sequences, identified the characteristics of sequences and structures, and found that the environmental microbiome Gpx proteins should be classified as TRX-dependent, Gpx-like peroxiredoxins. This classification is based on the following three items of evidence: i) the conservation of the peroxidatic Cys residue; ii) the existence and conservation of the resolving Cys residue that forms the disulfide bond with the peroxidatic cysteine; and iii) the absence of dimeric and tetrameric interface domains. The conservation/divergence pattern of all known bacterial Gpx-like proteins in public databases shows that they share common characteristics with that from the environmental microbiome and are also TRX-dependent. Moreover, phylogenetic analysis shows that the bacterial Gpx-like proteins exhibit a star-like radiating phylogenetic structure forming a highly diverse genetic pool of TRX-dependent, Gpx-like peroxidases.
Asunto(s)
Peroxidasas , Peroxirredoxinas , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/química , Glutatión Peroxidasa/metabolismo , Peroxidasas/genética , Peroxidasas/metabolismo , Filogenia , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Glutatión/metabolismo , Bacterias/genética , Bacterias/metabolismo , Oxidación-ReducciónRESUMEN
Bis(3-amino-1-hydroxybenzyl)diselenide containing two ortho groups was synthesized from 7-nitro-3H-2,1-benzoxaselenole and in situ generated sodium benzene tellurolate (PhTeNa). One-pot synthesis of 1,3-benzoselenazoles was achieved from bis(3-amino-1-hydroxybenzyl)diselenide and aryl aldehydes using acetic acid as a catalyst. The X-ray crystal structure of chloro-substituted benzoselenazole revealed a planar structure with T-shaped geometry around the Se atom. Both natural bond orbital and atoms in molecules calculations confirmed the presence of secondary Se···H interactions in bis(3-amino-1-hydroxybenzyl)diselenide and Se···O interactions in benzoselenazoles, respectively. The glutathione peroxidase (GPx)-like antioxidant activities of all compounds were evaluated using a thiophenol assay. Bis(3-amino-1-hydroxybenzyl)diselenide and benzoselenazoles showed better GPx-like activity compared to that of the diphenyl diselenide and ebselen, used as references, respectively. Based on 77Se{1H} NMR spectroscopy, a catalytic cycle for bis(3-amino-1-hydroxybenzyl)diselenide using thiophenol and hydrogen peroxide was proposed involving selenol, selenosulfide, and selenenic acid as intermediates. The potency of all GPx mimics was confirmed by their in vitro antibacterial properties against the biofilm formation of Bacillus subtilis and Pseudomonas aeruginosa. Additionally, molecular docking studies were used to evaluate the in silico interactions between the active sites of the TsaA and LasR-based proteins found in Bacillus subtilis and Pseudomonas aeruginosa.
Asunto(s)
Antioxidantes , Compuestos de Organoselenio , Simulación del Acoplamiento Molecular , Fenoles , Compuestos de Sulfhidrilo , Compuestos de Organoselenio/química , Glutatión Peroxidasa/químicaRESUMEN
Glutathione peroxidase 1 (GPx1) is an important cellular antioxidant enzyme that is found in the cytoplasm and mitochondria of mammalian cells. Like most selenoenzymes, it has a single redox-sensitive selenocysteine amino acid that is important for the enzymatic reduction of hydrogen peroxide and soluble lipid hydroperoxides. Glutathione provides the source of reducing equivalents for its function. As an antioxidant enzyme, GPx1 modulates the balance between necessary and harmful levels of reactive oxygen species. In this review, we discuss how selenium availability and modifiers of selenocysteine incorporation alter GPx1 expression to promote disease states. We review the role of GPx1 in cardiovascular and metabolic health, provide examples of how GPx1 modulates stroke and provides neuroprotection, and consider how GPx1 may contribute to cancer risk. Overall, GPx1 is protective against the development and progression of many chronic diseases; however, there are some situations in which increased expression of GPx1 may promote cellular dysfunction and disease owing to its removal of essential reactive oxygen species.
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Selenio , Selenocisteína , Animales , Antioxidantes/metabolismo , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Mamíferos/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Selenio/metabolismo , Selenocisteína/química , Glutatión Peroxidasa GPX1RESUMEN
Highly efficient stereoselective syntheses of novel bis(E-2-chlorovinyl) selenides and bis(E-2-bromovinyl) selenides in quantitative yields by reactions of selenium dichloride and dibromide with alkynes were developed. The reactions proceeded at room temperature as anti-addition giving products exclusively with (E)-stereochemistry. The glutathione peroxidase-like activity of the obtained products was estimated and compounds with high activity were found. The influence of substituents in the products on their glutathione peroxidase-like activity was discussed.
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Alquinos/química , Química Clic , Compuestos de Organoselenio/química , Alquinos/síntesis química , Catálisis , Glutatión Peroxidasa/química , Compuestos de Organoselenio/síntesis química , EstereoisomerismoRESUMEN
In the biological functions of selenoproteins, various highly reactive species formed by oxidative modification of selenocysteine residues have been postulated to play crucial roles. Representative examples of such species are selenocysteine selenenic acids (Sec-SeOHs) and selenocysteine selenenyl iodides (Sec-SeIs), which have been widely recognized as important intermediates in the catalytic cycle of glutathione peroxidase (GPx) and iodothyronine deiodinase, respectively. However, examples of even spectroscopic observation of Sec-SeOHs and Sec-SeIs in either protein or small-molecule model systems remain elusive so far, most likely due to their notorious instability. For the synthesis of small-molecule model compounds of these reactive species, it is essential to suppress their very facile bimolecular decomposition such as self-condensation and disproportionation. Here we outline a novel method for the synthesis of stable small-molecule model compounds of the selenocysteine-derived reactive species, in which a nano-sized molecular cavity is used as a protective cradle to accommodate the reactive selenocysteine unit. Stabilization by the molecular cradle led to the successful synthesis of Sec-SeOHs, which are stable in solution at low temperatures, and a Sec-SeI, which can be isolated as crystals. The catalytic cycle of GPx was investigated using the NMR-observable Sec-SeOH models, and all the chemical processes proposed for the catalytic cycle of GPx, including the bypass process from Sec-SeOH to the corresponding cyclic selenenyl amide, were experimentally confirmed. Detailed protocols for the syntheses of selenopeptide derivatives bearing the molecular cradle and for the spectroscopic monitoring of their reactions are provided.
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Selenocisteína , Catálisis , Glutatión Peroxidasa/química , Glutatión Peroxidasa/metabolismo , Modelos Moleculares , Oxidación-Reducción , Selenocisteína/química , Selenocisteína/metabolismoRESUMEN
Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, Mw = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo, and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo. Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.
Asunto(s)
Antioxidantes/farmacología , Glutatión Peroxidasa/metabolismo , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Línea Celular , Diseño de Fármacos , Escherichia coli , Glutatión Peroxidasa/química , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutación , Miocitos Cardíacos , Polietilenglicoles/química , Conformación Proteica , Estabilidad Proteica , Ratas , Ratas Sprague-Dawley , Succinimidas/química , Temperatura , Glutatión Peroxidasa GPX1RESUMEN
The review presents current concepts of the molecular mechanisms of oxidative stress development and describes main stages of the free-radical reactions in oxidative stress. Endogenous and exogenous factors of the oxidative stress development, including dysfunction of cell oxidoreductase systems, as well as the effects of various external physicochemical factors, are discussed. The review also describes the main components of the antioxidant defense system and stages of its evolution, with a special focus on peroxiredoxins, glutathione peroxidases, and glutathione S-transferases, which share some phylogenetic, structural, and catalytic properties. The substrate specificity, as well as the similarities and differences in the catalytic mechanisms of these enzymes, are discussed in detail. The role of peroxiredoxins, glutathione peroxidases, and glutathione S-transferases in the regulation of hydroperoxide-mediated intracellular and intercellular signaling and interactions of these enzymes with receptors and non-receptor proteins are described. An important contribution of hydroperoxide-reducing enzymes to the antioxidant protection and regulation of such cell processes as growth, differentiation, and apoptosis is demonstrated.
Asunto(s)
Antioxidantes/metabolismo , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo/fisiología , Animales , Antioxidantes/química , Radicales Libres/metabolismo , Glutatión/metabolismo , Glutatión Peroxidasa/química , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Humanos , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , FilogeniaRESUMEN
Although selenocysteine selenenic acids (Sec-SeOHs) have been recognized as key intermediates in the catalytic cycle of glutathione peroxidase (GPx), examples of the direct observation of Sec-SeOH in either protein or small-molecule systems have remained elusive so far, mostly due to their instability. Here, we report the first direct spectroscopic (1H and 77Se NMR) evidence for the formation of Sec-SeOH in small-molecule selenocysteine and selenopeptide model systems with a cradle-type protective group. The catalytic cycle of GPx was investigated using NMR-observable Sec-SeOH models. All the hitherto proposed chemical processes, i.e., not only those of the canonical catalytic cycle but also those involved in the bypass mechanism, including the intramolecular cyclization of Sec-SeOH to the corresponding five-membered ring selenenyl amide, were examined in a stepwise manner.
Asunto(s)
Ácidos Carboxílicos/química , Glutatión Peroxidasa/química , Compuestos de Organoselenio/química , Selenocisteína/química , Ácidos Carboxílicos/metabolismo , Catálisis , Cristalografía por Rayos X , Glutatión Peroxidasa/metabolismo , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Compuestos de Organoselenio/metabolismo , Selenocisteína/metabolismoRESUMEN
Emerging artificial enzymes with reprogrammed and augmented catalytic activity and substrate selectivity have long been pursued with sustained efforts. The majority of current candidates have rather poor catalytic activity compared with natural molecules. To tackle this limitation, we design artificial enzymes based on a structurally well-defined Au25 cluster, namely clusterzymes, which are endowed with intrinsic high catalytic activity and selectivity driven by single-atom substitutions with modulated bond lengths. Au24Cu1 and Au24Cd1 clusterzymes exhibit 137 and 160 times higher antioxidant capacities than natural trolox, respectively. Meanwhile, the clusterzymes demonstrate preferential enzyme-mimicking catalytic activities, with Au25, Au24Cu1 and Au24Cd1 displaying compelling selectivity in glutathione peroxidase-like (GPx-like), catalase-like (CAT-like) and superoxide dismutase-like (SOD-like) activities, respectively. Au24Cu1 decreases peroxide in injured brain via catalytic reactions, while Au24Cd1 preferentially uses superoxide and nitrogenous signal molecules as substrates, and significantly decreases inflammation factors, indicative of an important role in mitigating neuroinflammation.
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Enzimas/química , Inflamación , Neuronas/enzimología , Compuestos Organometálicos/química , Animales , Antioxidantes , Encéfalo/enzimología , Catalasa , Catálisis , Línea Celular , Glutatión Peroxidasa/química , Masculino , Metales/química , Ratones Endogámicos C57BL , Modelos Moleculares , Neuronas/inmunología , Superóxido Dismutasa/química , SuperóxidosRESUMEN
The so-called peroxidatic cysteines and selenocysteines in proteins reduce hydroperoxides through a dual attack to the peroxide bond in a two-step mechanism. First, a proton dislocation from the thiol/selenol to a close residue of the enzymatic pocket occurs. Then, a nucleophilic attack of the anionic cysteine/selenocysteine to one O atom takes place, while the proton is shuttled back to the second O atom, promoting the formation of a water molecule. In this computational study, we use a molecular model of GPx to demonstrate that the enzymatic environment significantly lowers the barrier of the latter SN 2 step. Particularly, in our Se-based model the energy barriers for the two steps are 29.82 and 2.83â kcal mol-1 , both higher than the corresponding barriers computed in the enzymatic cluster, i. e., 21.60 and null, respectively. Our results, obtained at SMD-B3LYP-D3(BJ)/6-311+G(d,p), cc-pVTZ//B3LYP-D3(BJ)/6-311G(d,p), cc-pVTZ level of theory, show that the mechanistic details can be well reproduced using an oversimplified model, but the energetics is definitively more favorable in the GPx active site. In addition, we pinpoint the role of the chalcogen in the peroxide reduction process, rooting the advantages of the presence of selenium in its acidic and nucleophilic properties.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Compuestos de Selenio/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Dominio Catalítico , Teoría Funcional de la Densidad , Glutatión Peroxidasa/química , Humanos , Simulación de Dinámica Molecular , Oxidación-Reducción , Protones , Compuestos de Selenio/química , Compuestos de Sulfhidrilo/química , TermodinámicaRESUMEN
Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H2O2 and t-BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H2O2 better than t-BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at Kmapp = 11.7 mM, Vmaxapp = 10.9 IU/µg TrGPx, kcat = 19 s-1 and a catalytic efficiency of 1.6 mM-1 s-1 to H2O2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0-12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications.
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Hypocreales/enzimología , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Secuencia de Aminoácidos , Antioxidantes/química , Antioxidantes/farmacología , Fraccionamiento Químico , Clonación Molecular , Activación Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Concentración de Iones de Hidrógeno , Hypocreales/genética , Modelos Moleculares , Peroxirredoxinas/genética , Peroxirredoxinas/aislamiento & purificación , Conformación Proteica , Relación Estructura-Actividad , TemperaturaRESUMEN
Catalpol has gained increasing attention for its potential contributions in controlling glycolipid metabolism and diabetic complications, which makes used as a very promising scaffold for seeking new anti-diabetic drug candidates. Acylation derivatives of catalpol crotonate (CCs) were designed as drug ligands of glutathione peroxidase (GSH-Px) based on molecular docking (MD) using Surfex-Docking method. Catalpol hexacrotonate (CC-6) was synthesized using microwave assisted method and characterized by FT-IR, NMR, HPLC and HRMS. The MD results indicate that with the increasing of esterification degree of hydroxyl, the C log P of CCs increased significantly, and the calculated total scores (Total_score) of CCs are all higher than that of catalpol. It shows that CCs maybe served as potential lead compounds for neuroprotective agents. It was found that the maximum Total_score of isomers in one group CCs is often not that the molecule with minimum energy. MD calculations show that there are five hydrogen bonds formed between CC-6 and the surrounding amino acid residues. Molecular dynamics simulation results show that the binding of CC-6 with GSH-Px is stable. CC-6 was screened for SH-SY5Y cells viability by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, the result indicates CC-6 can effectively reverse SZT induced cells apoptosis with dose-dependent manner, which can indirectly show that CC-6 is a potential neuroprotective agent.
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Crotonatos/farmacología , Glutatión Peroxidasa/antagonistas & inhibidores , Hipoglucemiantes/farmacología , Glucósidos Iridoides/farmacología , Fármacos Neuroprotectores/farmacología , Sitios de Unión , Encefalopatías/tratamiento farmacológico , Encefalopatías/enzimología , Encefalopatías/etiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Crotonatos/síntesis química , Complicaciones de la Diabetes/tratamiento farmacológico , Complicaciones de la Diabetes/enzimología , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/enzimología , Glutatión Peroxidasa/química , Glutatión Peroxidasa/metabolismo , Humanos , Enlace de Hidrógeno , Hipoglucemiantes/síntesis química , Glucósidos Iridoides/síntesis química , Microondas , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/síntesis química , Unión ProteicaRESUMEN
In addition to their own antioxidants, human cells feed on external antioxidants, such as the phenolic compounds of fruits and vegetables, which work together to keep oxidative stress in check. Sechium edule, an edible species of chayote, has phenolic compounds with antioxidant activity and antineoplastic activity. A Sechium hybrid shows one thousand times greater antineoplastic activity than edible species, but its antioxidant and anti-inflammatory activities and the content of phenolic compounds are unknown. The aim of this study was to determine the antioxidant and anti-inflammatory capacity of the extract of fruits of the Sechium hybrid in vitro and in vivo. Phytochemical analysis using HPLC showed that the extract of the Sechium hybrid has at least 16 phenolic compounds; galangin, naringenin, phloretin and chlorogenic acid are the most abundant. In an in vitro assay, this extract inhibited 2,2-diphenyl-L-picrylhydrazyl (DPPH) activity and protected the dimyristoylphosphatidylethanolamine (DMPE) phospholipid model cell membrane from oxidation mediated by hypochlorous acid (HClO). In vivo, it was identified that the most abundant metabolites in the extract enter the bloodstream of the treated mice. On the other hand, the extract reduces the levels of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin-6 (IL-6) but increases interleukin-10 (IL-10) and glutathione peroxidase levels. Our findings indicate that intake of the fruits of the Sechium hybrid leads to antioxidant and anti-inflammatory effects in a mouse model. Therefore, these results support the possibility of exploring the clinical effect of this hybrid in humans.
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Antioxidantes/química , Frutas/química , Interleucina-10/química , Factor de Necrosis Tumoral alfa/química , Animales , Compuestos de Bifenilo/química , Glutatión Peroxidasa/química , Humanos , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Ratones , Fosfatidiletanolaminas/química , Picratos/química , Extractos Vegetales/químicaRESUMEN
This study was conducted to determine the serum and milk levels of thiobarbturic acid-reac- tive substances (TBARS), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxi- dase (GSH-Px), vitamin E and selenium, IL-4 and IL-6 in lactating dairy cows affected with bloody milk using commercially available ELISA kits. Milk and whole blood samples were collected from 60 cows affected with bloody milk and 20 apparently healthy cows for control. In the serum, levels of GSH-Px and SOD were significantly (pË0.05) higher in healthy cows compared to cows affected with bloody milk while the levels of TBARS and NO were significantly (pË0.05) higher in affected cows. In the milk, levels of SOD, TBARS and NO were significantly (pË0.05) higher in affected cows. In the serum, levels of vitamin E were significantly (pË0.05) lower in affected cows compared to healthy cows, while no significant changes were observed in the levels of this vitamin in the milk between healthy and affected cows. In the serum, levels of selenium were significantly (pË0.05) lower in affected cows while in milk, selenium levels were significantly (pË0.05) higher in affected cows compared to healthy ones. Levels of IL-4 were significantly (pË0.05) lower in the serum and milk of affected cows compared to healthy cows while levels of IL-6 were significantly (pË0.05) higher in both serum and milk of affected cows. Results of this study suggest a possible role of oxidative stress in the pathogenesis of bloody milk in dairy cows.