RESUMEN
Methionine is one of the main targets for biological oxidants. Its reaction with the majority of oxidants generates only methionine sulfoxide. However, when N-terminal methionine reacts with hypohalous acids (HOCl and HOBr) or singlet molecular oxygen (1O2), it can also generate a cyclic product called dehydromethionine (DHM). Previously, DHM was suggested as a biomarker of oxidative stress induced by hypohalous acids. However, DHM can also be generated by 1O2 -oxidation of methionine, and the contribution of this pathway of DHM formation in a context of a site-specific redox imbalance in an organism is unknown. In this work, a through comparison of the reactions of hypohalous acids and 1O2 with methionine, either free or inserted in peptides and proteins was undertaken. In addition, we performed methionine photooxidation in heavy water (H218O) to determine the influence of the pH in the mechanism of DHM formation. We showed that for free methionine, or methionine-containing peptides, the yields of DHM formation in the reactions with 1O2 were close to those achieved by HOBr oxidation, but much higher than the yields obtained with HOCl as the oxidant. This was true for all pH tested (5, 7.4, and 9). Interestingly, for the protein ubiquitin, DHM yields after reaction with 1O2 were higher than those obtained with both hypohalous acids. Our results indicate that 1O2 may also be an important source of DHM in biological systems.
Asunto(s)
Metionina , Oxígeno Singlete , Metionina/química , Oxidantes/química , Oxidación-Reducción , Oxígeno , Péptidos/química , Proteínas , Oxígeno Singlete/química , TiazolesRESUMEN
Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease (PD). Copper ions specifically bind at the N-terminus of AS, accelerating protein aggregation. Its protein homolog ß-synuclein (BS) is also a copper binding protein, but it inhibits AS aggregation. Here, a comparative spectroscopic study of the Cu2+ binding properties of AS and BS has been performed, using electronic absorption, circular dichroism (CD) and electronic paramagnetic resonance (EPR). Our comparative spectroscopic study reveals striking similarities between the Cu2+ binding features of the two proteins. The Cu2+ binding site at the N-terminal group of BS protein, modeled by the BS (1-15) fragment is identical to that of AS; however, its rate of reduction is three times faster as compared to the AS site, consistent with BS having an additional Met residue in its Met1-Xn-Met5-Xn-Met10 motif. The latter is also evident in the cyclic voltammetry studies of the Cu-BS complex. On the other hand, the Cu2+ binding features of the His site in both proteins, as modeled by AS(45-55) and BS(60-70), are identical, indicating that the shift in the His position does not affect its coordination features. Finally, replacement of Glu46 by Ala does not alter Cu2+ binding to the His site, suggesting that the familial PD E46K mutation would not impact copper-induced aggregation. While further studies of the redox activity of copper bound to His50 in AS are required to understand the role of this site in metal-mediated aggregation, our study contributes to a better understanding of the bioinorganic chemistry of PD.
Asunto(s)
Cobre/metabolismo , alfa-Sinucleína/metabolismo , Sinucleína beta/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Histidina/química , Histidina/metabolismo , Metionina/química , Metionina/metabolismo , Unión Proteica , alfa-Sinucleína/química , Sinucleína beta/químicaRESUMEN
Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and ß-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Maytenus/enzimología , Proteínas de Plantas/metabolismo , Triterpenos/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Sustitución de Aminoácidos , Vías Biosintéticas , Ciclización , Genes de Plantas , Leucina/química , Maytenus/genética , Metionina/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/biosíntesis , Triterpenos Pentacíclicos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Chronic myeloid leukemia (CML) is a pathological condition associated with the uncontrolled proliferation of white blood cells and respective loss of function. Imatinib was the first drug that could effectively treat this condition, but its use is hindered by the development of mutations of the BCR-ABL protein, which are the cause of resistance. Therefore, dasatinib and afatinib present similarities that can be explored to discover new molecules capable of overcoming the effects of imatinib. Afatinib exhibited electronic and docking behavior, indicating that a replacement with some minor modifications could design a new potential inhibitor. The amide group in each candidate is clearly of pharmacophoric importance, and it needs to concentrate a negative region. Sulfur group presents a good pharmacophoric profile, which was shown by dasatinib results, adding to the influence of the Met318 residue in the target protein active site configuration. This behavior suggests that the sulfur atom and other fragments that have an affinity for the methionine sidechain may provide a significant positive effect when present in TKI molecules such as afatinib or dasatinib.
Asunto(s)
Afatinib/química , Dasatinib/química , Proteínas de Fusión bcr-abl/química , Afatinib/metabolismo , Afatinib/farmacología , Antineoplásicos/química , Antineoplásicos/metabolismo , Dominio Catalítico , Dasatinib/metabolismo , Dasatinib/farmacología , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/química , Mesilato de Imatinib/metabolismo , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Metionina/química , Simulación del Acoplamiento Molecular , Mutación , Teoría Cuántica , Azufre/químicaRESUMEN
The unavoidable presence of acrylamide in foods has fuelled the search for a suitable food additive, one that can successfully mitigate dietary acrylamide levels without changing food quality or compromising the health of consumers. The purpose of this study was to investigate the effect of a sulphur-based additive and amino acid, methionine, on acrylamide reduction. Differential scanning calorimetry, supported by chromatographic measurements, has shown that methionine interacts with acrylamide at a possible optimum temperature of 160°C, thereby disfavouring acrylamide polymerisation. Analysis of the methionine-acrylamide interaction via density functional theoretical modelling (DFT/6-31 + G(d)/RCAM-B3LYP) revealed that methionine's reducing effect may be driven by a Michael-type conjugation of the vinyl group of acrylamide at both the sulphur atom (∆Gf = -53 kJ mol-1) and the amino group (∆Gf = -11.84 kJ mol-1) of methionine. The former conjugation pathway results in a product that is more thermodynamically feasible.
Asunto(s)
Acrilamida/química , Aditivos Alimentarios/química , Contaminación de Alimentos/análisis , Metionina/química , Cromatografía Líquida de Alta Presión , Teoría Funcional de la Densidad , Alimentos , Análisis de los Alimentos/métodos , Calor , Humanos , Reacción de Maillard , Modelos Químicos , Oxidación-Reducción , TermodinámicaRESUMEN
BACKGROUND: In the therapy of cancer, several treatments have been designed using nanomaterials, among which gold nanoparticles (AuNPs) have been featured as a promising antitumoral agent. Our research group has developed the synthesis of gold nanoparticles L-AuNPs and D-AuNPs stabilized with zwitterions of imidazolium (L-1 and D-1) derived from L-methionine and D-methionine. Because the stabilizer agent is chiral, we observed through circular dichroism that AuNPs also present chirality; such chirality as well as the fact that the stabilizing agent contains fragments of methionine and imidazolium that are commonly involved in biological processes, opens up the possibility that this system may have biological compatibility. Additionally, the presence of methionine in the stabilizing agent opens the application of this system as a possible antitumor agent because methionine is involved in methylation processes of molecules such as DNA. OBJECTIVE: The aim of this research is the evaluation of the antitumor activity of gold nanoparticles stabilized with zwitterions of imidazolium (L-AuNPs) derived from L-methionine in the model of BALB/c mice with lymphoma L5178Y. METHODS: Taking as a parameter cell density, the evaluation of the inhibitory effect of L-AuNPs was carried out with a series of in vivo tests in BALB/c type mice; three groups of five mice each were formed (Groups 1, 2 and 3); all mice were i.p. inoculated with the lymphoblast murine L5178Y. Group 1 consisted of mice without treatment. In the Groups 2 and 3 the mice were treated with L-AuNPs at 0.3 mg/Kg on days 1, 7 and 14 by orally and intraperitonally respectively. RESULTS: These results show low antitumor activity of these gold nanoparticles (L-NPsAu) but interestingly, the imidazolium stabilizing agent of gold nanoparticle (L-1) displayed promising antitumor activity. On the other hand, the enantiomer of L-1, (D-1) as well as asymmetric imidazole derivate from L-methionine (L-2), do not exhibit the same activity as L-1. CONCLUSION: The imidazolium stabilizing agent (L-1) displayed promising antitumor activity. Modifications in the structure of L-1 showed that, the stereochemistry (like D-1) and the presence of methionine fragments (like L-2) are determinants in the antitumor activity of this compound.
Asunto(s)
Imidazoles/química , Imidazoles/farmacología , Linfoma/patología , Metionina/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Oro/química , Imidazoles/uso terapéutico , Linfoma/tratamiento farmacológico , Nanopartículas del Metal/química , Metilación , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.
Asunto(s)
Citoplasma/química , Leptospira interrogans/genética , Metionina Sulfóxido Reductasas/genética , Metionina/metabolismo , Secuencia de Aminoácidos/genética , Catálisis , Citoplasma/enzimología , Genoma Bacteriano/genética , Humanos , Leptospira interrogans/enzimología , Metionina/química , Metionina/genética , Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/ultraestructura , Oxidación-Reducción , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por SustratoRESUMEN
The cis-[Ru(bpy)2(Met)](PF6)2 complex, where Met = L-methionine and bpy = 2,2'-bipyridine, was prepared and fully characterized. This complex was subjected to blue and green light photolysis (453 and 505 nm, respectively) in aqueous solution, leading to the release of methionine and formation of the cis-[Ru(bpy)2(H2O)2]2+ ion. This latter photoproduct was shown to subsequently interact with DNA, while DNA photocleavage was noticed. In agreement with these reactivities, this compound exhibited an exciting antibacterial action, particularly against Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis, which was enhanced upon blue light irradiation. Altogether, these results showed that our strategy was successful in producing light-triggered DNA-binding agents with pharmacological potential and a likely blocking reagent for efficient peptide chemistry formation.
Asunto(s)
Antibacterianos/farmacología , Complejos de Coordinación/farmacología , Metionina/farmacología , Rutenio/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus epidermidis/efectos de los fármacos , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , ADN/efectos de los fármacos , División del ADN , Luz , Masculino , Metionina/química , Pruebas de Sensibilidad Microbiana , Procesos Fotoquímicos , Rutenio/química , Salmón , Espermatozoides/químicaRESUMEN
An experiment was carried out to evaluate the requirement of digestible methionine for growing pullets at growth phase (7 to 12 weeks of age). A completely randomized design was distributed in five treatments, six replicates, and 15 pullets per experimental unit. 450 Dekalb White pullets from the 7th weeks of age, with an average initial weight of 313.14 ± 12.49 g were used. Dietary treatments consisted in five diets supplemented with DL-Methionine which resulted in five levels of digestible methionine (0.266, 0.294, 0.322, 0.350, and 0.378 %). Performance, serological blood, histopathology and histomorphometry data were evaluated. Quadratic responses were observed for final live weight (p < 0.0143), weight gain (p < 0.0073), feed conversion ratio (p < 0.0058), glycogen deposition in the liver (p < 0.0001), gamma-glutamyl transferase enzyme activity (p < 0.0008), and villus height (p < 0.0024) with digestible dMet levels. In conclusion, the use of 0.343 % dMet, corresponding to a dMet:dLys ratio 55, is recommended for white-egg pullets from 7 to 12 weeks of age.
Asunto(s)
Femenino , Animales , Pruebas de Enzimas/clasificación , Pollos/metabolismo , Metionina/análisis , Metionina/química , Huevos/análisisRESUMEN
An experiment was carried out to evaluate the requirement of digestible methionine for growing pullets at growth phase (7 to 12 weeks of age). A completely randomized design was distributed in five treatments, six replicates, and 15 pullets per experimental unit. 450 Dekalb White pullets from the 7th weeks of age, with an average initial weight of 313.14 ± 12.49 g were used. Dietary treatments consisted in five diets supplemented with DL-Methionine which resulted in five levels of digestible methionine (0.266, 0.294, 0.322, 0.350, and 0.378 %). Performance, serological blood, histopathology and histomorphometry data were evaluated. Quadratic responses were observed for final live weight (p < 0.0143), weight gain (p < 0.0073), feed conversion ratio (p < 0.0058), glycogen deposition in the liver (p < 0.0001), gamma-glutamyl transferase enzyme activity (p < 0.0008), and villus height (p < 0.0024) with digestible dMet levels. In conclusion, the use of 0.343 % dMet, corresponding to a dMet:dLys ratio 55, is recommended for white-egg pullets from 7 to 12 weeks of age.(AU)
Asunto(s)
Animales , Femenino , Pollos/metabolismo , Metionina/análisis , Metionina/química , Huevos/análisis , Pruebas de Enzimas/clasificaciónRESUMEN
This study examined the influence of a diet enriched with free methionine (dl-Met) or methionine dipeptide (dl-MMet) on the intestinal health of Eimeria-challenged (EC) and unchallenged (UC) broilers. A non-supplemented, methionine-deficient diet (NS) was used as control. Treatments were arranged in a 2 × 3 factorial completely randomized design with eight replications. Broilers in the EC group were infected with sporulated oocysts of Eimeria spp. (E. acervulina, E. maxima, E. praecox, and E. mitis) at 14 d of age. Performance analysis, light and electron microscopy of the jejunum, analysis of genes related to apoptosis and cell proliferation in the jejunum, and blood tests were performed at 6 days post-inoculation (dpi). EC broilers had poorer performance than UC broilers, regardless of diet (P < 0.001). Broilers fed the dl-Met diet had greater weight gain (P = 0.004) and lower feed conversion ratio (P = 0.019) than broilers fed other diets. Jejunal sections from EC broilers fed the NS diet showed short (P = 0.001) and wide villi (P < 0.001) with increased crypt depth (P < 0.001) and reduced villus / crypt ratio (P = 0.001), jejunal absorptive surface area (P < 0.001), number of neutral goblet cells (Eimeria challenge: P = 0.048; diet P = 0.016), and mucin 2 (MUC2) gene expression (P = 0.018). EC birds fed the dl-MMet diet had higher enterocyte height (P < 0.001). Birds fed the dl-MMet diet had low lamina propria width (P = 0.009). UC broilers fed the dl-Met diet had the highest number of acidic goblet cells (P = 0.005), whereas EC broilers assigned the dl-MMet diet showed the highest number of intraepithelial lymphocytes (P = 0.033). Reduced expression of caspase-3 (CASP3) (P = 0.005), B-cell lymphoma 2 (BCL2) (P < 0.001), mechanistic target of rapamycin (MTOR) (P < 0.001), and ribosomal protein S6 kinase B1 (RPS6KB1) (P < 0.001) genes was observed in EC animals. MTOR expression levels were highest in birds fed the dl-MMet diet (P = 0.004). Plasma activities of aspartate aminotransferase (AST) was influenced by both diet (P = 0.002) and Eimeria challenge (P = 0.005), with EC broilers assigned the NS diet showing the highest levels. EC broilers fed the NS diet had higher creatine kinase (CK) activity (P = 0.049). EC broilers had lower plasma uric acid (P = 0.004) and higher serum mucoproteins level (P < 0.001). These results indicate that methionine dipeptide supplementation is able to mitigate the harmful intestinal effects of Eimeria spp. in broilers.
Asunto(s)
Coccidiosis/veterinaria , Metionina/farmacología , Enfermedades de las Aves de Corral/prevención & control , Alimentación Animal/análisis , Animales , Pollos , Coccidiosis/prevención & control , Dieta/veterinaria , Suplementos Dietéticos/análisis , Eimeria , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Masculino , Metionina/administración & dosificación , Metionina/química , Enfermedades de las Aves de Corral/metabolismo , Enfermedades de las Aves de Corral/parasitologíaRESUMEN
Two performance studies were conducted to investigate the effects of 3 different sources of Cu on production parameters of piglets. A total of 256 piglets weaned at 24 ± 2 d were randomly allocated into 4 treatments with 10 or 8 replicates per treatment of 4 or 3 piglets per pen in Exp. 1 and 2, respectively. The experimental period was divided into 3 feeding phases: Phase 1 (24 to 35 d), Phase 2 (36 to 49 d), and Phase 3 (50 to 70 d). Treatments included a Control group (fed 10 mg/kg of Cu from CuSO4), a group fed 160 mg/kg of either CuSO4 (CuSO4-160) or tri-basic copper chloride (TBCC), and a group fed Cu methionine hydroxy analogue chelated (Cu-MHAC) at 150, 80, and 50 mg/kg in Phases 1, 2, and 3, respectively. The methionine value of Cu-MHAC was accounted during diet formulation to achieve the same levels of methionine across treatments. Phases 1 and 2 diets contained 2,200 and 1,500 ppm of ZnO, respectively; and antibiotics were used as growth promoters. Performance parameters were analyzed as completely randomized block design, in which each experiment was considered as a block. In trial 2, blood serum and mucosal samples, from the fundic region of the stomach, were collected from 1 piglet per replicate at day 70 and tested for serum growth hormone levels (GH) and ghrelin mRNA expression, respectively. The contrast between Cu-MHAC vs. CuSO4-160 + TBCC showed that piglets fed Cu-MHAC exhibited better feed conversion ratio (FCR) in all feeding phases compared with feeding inorganic Cu (P < 0.05). Overall, feeding Cu-MHAC improved body weight (BW), BW gain, feed intake (FI), and FCR vs. Control diet fed piglets; yet, it improved BW and FCR vs. TBCC fed piglets, and improved BW, BW gain, and FI vs. CuSO4-160 fed piglets (P < 0.05). Feeding TBCC promoted similar performance than feeding CuSO4-160, regardless of age (P > 0.05). Both ghrelin expression and growth hormone serum levels were significantly increased by feeding Cu-MHAC vs. Control diet fed animals (P < 0.01). Feeding CuSO4-160 upregulated ghrelin expression vs. Control (P < 0.01) while GH serum levels and ghrelin expression did no change by feeding TBCC compared with Control diet fed animals (P > 0.05). It was concluded that feeding Cu-MHAC at the levels tested herein can improve growth performance of piglets beyond feeding 160 ppm of either CuSO4 or TBCC, which may be partially explained by the increased expression of ghrelin and GH serum levels.
Asunto(s)
Alimentación Animal/análisis , Cobre/administración & dosificación , Suplementos Dietéticos/análisis , Ghrelina/genética , Hormona del Crecimiento/sangre , Porcinos/fisiología , Animales , Peso Corporal/efectos de los fármacos , Dieta/veterinaria , Femenino , Masculino , Metionina/análogos & derivados , Metionina/química , ARN Mensajero/genética , Distribución Aleatoria , Estómago/fisiología , Porcinos/genética , Porcinos/crecimiento & desarrollo , Destete , Aumento de Peso/efectos de los fármacosRESUMEN
Misfolded prion protein (PrPSc) is known for its role in fatal neurodegenerative conditions, such as Creutzfeldt-Jakob disease. PrP fragments and their mutants represent important tools in the investigation of the neurotoxic mechanisms and in the evaluation of new compounds that can interfere with the processes involved in neuronal death. Metal-catalyzed oxidation of PrP has been implicated as a trigger for the conformational changes in protein structure, which, in turn, lead to misfolding. Targeting redox-active biometals copper and iron is relevant in the context of protection against the oxidation of biomolecules and the generation of oxidative stress, observed in several conditions and considered an event that might promote sporadic prion diseases as well as other neurodegenerative disorders. In this context, ortho-pyridine aroylhydrazones are of interest, as they can act as moderate tridentate ligands towards divalent metal ions such as copper(II). In the present work, we explore the potentiality of this chemical class as peptide protecting agents against the deleterious metal-catalyzed oxidation in the M112A mutant fragment of human PrP, which mimics relevant structural features that may play an important role in the neurotoxicity observed in prion pathologies. The compounds inhere studied, especially HPCFur, showed an improved stability in aqueous solution compared to our patented lead hydrazone INHHQ, displaying a very interesting protective effect toward the oxidation of methionine and histidine, processes that are related to both physiological and pathological aging.
Asunto(s)
Quelantes/química , Cobre/química , Hidrazonas/química , Proteínas Priónicas/efectos de los fármacos , Piridinas/química , Quelantes/síntesis química , Humanos , Hidrazonas/síntesis química , Ligandos , Metionina/química , Mutación , Oxidación-Reducción , Proteínas Priónicas/química , Proteínas Priónicas/genética , Piridinas/síntesis químicaRESUMEN
The formation of neurotoxic oligomers of the presynaptic protein α-Synuclein (aSyn) is suggested to be associated with Parkinson's disease neurodegeneration. In this respect, it was demonstrated that the aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), a product from the enzymatic oxidation of dopamine, is capable of stabilizing potentially toxic aSyn oligomers via formation of covalent adducts with Lys residues of the protein. In addition, DOPAL-induced production of reactive oxygen species (ROS) leads to the oxidation of aSyn's Met residues to Met-sulfoxide. Recently, our group pointed out that the pre-oxidation of all-four Met residues of aSyn, upon treatment with H2O2, decreases the formation of large aSyn-DOPAL oligomers, which are suggested to be more toxic to neurons than the corresponding small oligomers (Carmo-Gonçalves et al., Biochem. Biophys. Res. Comm. 505, 295-301. 2018). By using a series of Met to Val mutants of aSyn, we demonstrated that the ability of aSyn to scavenge ROS/H2O2 generated from DOPAL oxidation is primarily dependent on Met residues located at the C-terminal domain of the protein, which contrasts with the reactivity of aSyn against H2O2 itself in which N-terminal Met residues (notably Met5) were more readily oxidized. Interestingly, the substitution of C-terminal Met residues (particularly Met127) by Val increased the formation of DOPAL-induced large oligomers in comparison with the wild-type protein. In this context, we demonstrated that the hydrophobicity of aSyn monomer, which is affected distinctively by the oxidation of N- versus C-terminal methionines, is correlated with the formation of large (but not small) oligomers of aSyn mediated by DOPAL.
Asunto(s)
Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Peróxido de Hidrógeno/química , Metionina/química , Valina/química , alfa-Sinucleína/química , Ácido 3,4-Dihidroxifenilacético/química , Ácido 3,4-Dihidroxifenilacético/metabolismo , Sustitución de Aminoácidos , Naftalenosulfonatos de Anilina/química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Metionina/metabolismo , Mutación , Oxidación-Reducción , Dominios Proteicos , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Valina/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.
Asunto(s)
Ácido 3,4-Dihidroxifenilacético/farmacología , Metionina/química , Neuronas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/toxicidad , Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Peróxido de Hidrógeno/farmacología , Metionina/genética , Ratones , Mutación , Neuronas/citología , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
Since the 1863 discovery of a new green hemoglobin derivative called "sulfhemoglobin", the nature of the characteristic 618 nm absorption band has been the subject of several hypotheses. The experimental spectra are a function of the observation time and interplay between two major sulfheme isomer concentrations (a three- and five-membered ring adduct), with the latter being the dominant isomer at longer times. Thus, time-dependent density functional theory (TDDFT) was used to calculate the sulfheme excited states and visualize the highest occupied molecular orbitals (HOMOs) and lowest unoccupied MOs (LUMOs) of both isomers in order to interpret the transitions between them. These two isomers have distinguishable a1u and a2u HOMO energies. Formation of the three-membered ring SA isomeric structure decreases the energy of the HOMO a1u and a2u orbitals compared to the unmodified heme due to the electron-withdrawing, sulfur-containing, three-membered ring. Conversely, formation of the SC isomeric structure decreases the energy of the HOMO a1u and a2u orbitals due to the electron-withdrawing, sulfur-containing, five-membered ring. The calculations reveal that the absorption spectrum within the 700 nm region arises from a mixture of MOs but can be characterized as π to π* transitions, while the 600 nm region is characterized by π to dπ (d yz, d xz) transitions having components of a deoxy-like derivative.
Asunto(s)
Hemo/análogos & derivados , Hemoglobinas/química , Metionina/química , Hemo/química , Hemoglobinas/genética , Hemoglobinas/metabolismo , Isomerismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Teoría Cuántica , EspectrofotometríaRESUMEN
Chlorinase SalL halogenate S-adenosyl-l-methionine (SAM) reacts with chloride to generate 5'-chloro-5'-deoxyadenosine and l-methionine through a nucleophilic substitution mechanism. Although it is known that chlorinase enhances the rate of reaction by a factor of 1.2 × 1017 fold, it is not entirely clear how this is accomplished. The search for the origin of the catalysis of chlorinase and other enzymes has led to a desolvation hypothesis. In the present work, we have used well defined computational simulations in order to evaluate the origin of the catalytic efficiency of chlorinase. The results demonstrate that the catalytic effect of chlorinase is associated with the fact that Cl- is "solvated" by the protein more than by the reference solution reaction, which is not in accordance with proposed catalysis by desolvation. It is found that chlorinase SalL active sites provide electrostatic stabilization of the transition state which is the origin of its catalytic effect.
Asunto(s)
Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Biocatálisis , Dominio Catalítico , Cladribina/química , Cladribina/metabolismo , Enlace de Hidrógeno , Metionina/química , Metionina/metabolismo , Metiltransferasas/química , Metiltransferasas/genética , Mutagénesis Sitio-Dirigida , S-Adenosilmetionina/química , Electricidad Estática , Termodinámica , Agua/químicaRESUMEN
Multiparous Holstein cows were assigned in a randomized complete block design into four treatments from 21 d before calving to 30 d in milk (DIM). Treatments were: MET [n = 19, fed the basal diet + rumen-protected methionine at a rate of 0.08% (w/w) of the dry matter, Smartamine® M], CHO (n = 17, fed the basal diet + choline 60 g/d, Reashure®), MIX (n = 21, fed the basal diet + Smartamine® M at a rate of 0.08% (w/w) of the dry matter and 60 g/d Reashure®), and CON (n = 20, no supplementation, fed the close-up and fresh cow diets). Follicular development was monitored via ultrasound every 2 d starting at 7 DIM until ovulation (n = 37) or aspiration (n = 40) of the first postpartum dominant follicle (DF). Follicular fluid from 40 cows was aspirated and cells were retrieved immediately by centrifugation. Gene expression of TLR4, TNF, IL1-ß, IL8, IL6, LHCGR, STAR, 3ß-HSD, P450scc, CYP19A1, IRS1, IGF, MAT1A, and SAHH, was measured in the follicular cells of the first DF. Cows in CON had higher TNF, TLR4, and IL1-ß mRNA expression (11.70 ± 4.6, 21.29 ± 10.4, 6.28 ± 1.4, respectively) than CHO (2.77 ± 0.9, 2.16 ± 0.9, 2.29 ± 0.7, respectively), and MIX (2.23 ± 0.7, 1.46 ± 0.6, 2.92 ± 0.8, respectively). Cows in CON had higher IL1-ß expression (6.27 ± 1.4) than cows in MET (3.28 ± 0.6). Expression of IL8 mRNA was lower for cows in CHO (0.98 ± 0.3) than cows in CON (4.90 ± 0.7), MET (6.10 ± 1.7), or MIX (5.05 ± 1.8). Treatments did not affect mRNA expression of LHCGR, STAR, P450scc, CYP19A, SAHH, MAT1A, or IL6 however, 3ß-HSD expression was higher for cows in MET (1.46 ± 0.3) and MIX (1.25 ± 0.3) than CON (0.17 ± 0.04) and CHO (0.26 ± 0.1). Supplementation of methionine, choline, and both methionine and choline during the transition period did not affect days to first ovulation or number of cows that ovulated the first follicular wave. Plasma and follicular fluid estradiol and progesterone concentrations were not different among treatments. Methionine concentrations in the follicular fluid of the first postpartum DF was higher for cows in MET (18.2 ± 0.1 µM) than cows in CON (11.1 ± 0.9 µM). In conclusion, supplementing choline and methionine during the transition period changed mRNA expression in follicular cells and dietary methionine supplementation increased plasma and follicular fluid concentrations of methionine of the first postpartum DF in Holstein cows.
Asunto(s)
Bovinos/fisiología , Colina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Metionina/farmacología , Folículo Ovárico/efectos de los fármacos , Animales , Bovinos/sangre , Bovinos/inmunología , Colina/administración & dosificación , Colina/química , Formas de Dosificación , Estradiol/sangre , Femenino , Regulación de la Expresión Génica/inmunología , Metionina/administración & dosificación , Metionina/química , Leche , Periodo Posparto , EmbarazoRESUMEN
Introducción: diversos estudios han demostrado la relación de varios factores de riesgo muy conocidos con la aparición de enfermedades cardiovasculares; sin embargo, en algunos pacientes con dolencias cardiovasculares incipientes no es posible encontrar tal relación, por lo que se requiere identificar nuevos factores predictivos, entre los que se encuentra el nivel plasmático elevado de homocisteína. Objetivo: describir la homocisteína como marcador de riesgo vascular. Método: se revisó la bibliografía nacional e internacional sobre hematología, correspondiente a los últimos cinco años, disponible en la Biblioteca Virtual de Salud de Cuba, en español e inglés; para la búsqueda se emplearon los términos homocisteína y aterosclerosis, homocisteína y uso diagnóstico, metionina y uso diagnóstico. A partir de los artículos seleccionados se elaboró una reseña estructurada sobre el tema. Desarrollo: se deben medir los niveles de homocisteína plasmática a todos los pacientes con historia de enfermedad arterial coronaria, tromboembolismo pulmonar, trombosis venosa, aterosclerosis inexplicable (sin factores de riesgo o causas identificables para el aumento de los niveles de homocisteína) y con insuficiencia renal crónica o trasplante renal. Conclusiones: el papel de la homocisteína como factor independiente de riesgo aterogénico ha sido confirmado por estudios y datos epidemiológicos que demuestran que la homocisteína total circulante elevada es un potente marcador pronóstico de enfermedad cardiovascular y mortalidad en pacientes con factores de riesgo preexistentes; no obstante, la homocisteína es un factor de riesgo modificable puesto que la administración de vitaminas a los pacientes, en niveles adecuados, disminuye sus niveles plasmáticos(AU)
Introduction: several studies have demonstrated the relationship of several well-known risk factors with the occurrence of cardiovascular diseases; however, in some patients with incipient cardiovascular diseases, it is not possible to find such a relationship, so it is necessary to identify new predictive factors, such as elevated plasma homocysteine level. Objective: to describe homocysteine as a marker of vascular risk. Method: the national and international bibliography on hematology, corresponding to the last five years, available in the Virtual Health Library of Cuba, in Spanish and English was revised; for the search were used terms such as homocysteine and atherosclerosis, homocysteine and diagnostic use, methionine and diagnostic use. From the selected articles a structured review on the subject was elaborated.Development: plasma homocysteine levels should be measured in all patients with a history of coronary artery disease, pulmonary thromboembolism, venous thrombosis, unexplained atherosclerosis (with no risk factors or identifiable causes for increased levels of homocysteine), and chronic renal insufficiency or renal transplantation.Conclusions: the role of homocysteine as an independent factor in atherogenic risk has been confirmed by studies and epidemiological data showing that elevated circulating total homocysteine is a potent prognostic marker of cardiovascular disease and mortality in patients with preexisting risk factors; however, homocysteine is a modifiable risk factor since the administration of vitamins to patients, at appropriate levels, decreases their plasma levels(AU)
Asunto(s)
Humanos , Masculino , Femenino , Aterosclerosis/sangre , Factores de Riesgo , Biomarcadores/sangre , Metionina/química , Metionina/metabolismo , Homocisteína/química , Homocisteína/metabolismo , Literatura de Revisión como AsuntoRESUMEN
Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1-6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1-6) fragment, and in the redox activity of the Cu-AS(1-6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)-AS(1-6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu-AS(1-6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu-AS complex.