RESUMEN

Objective: Obesity poses an increased risk for the onset of Nonalcoholic fatty liver disease (NAFLD). The influence of other factors, such as sex in the incidence and severity of this liver disease has not yet been fully elucidated. Thus, we aimed to identify the NAFLD serum metabolic signatures associated with sex in normal, overweight and obese patients and to associate the metabolite fluctuations across the increasing liver steatosis stages. Methods and results: Using nuclear magnetic resonance (NMR) serum samples of 210 NAFLD cases and control individuals diagnosed with liver U/S, our untargeted metabolomics enquiry provided a sex distinct metabolic bouquet. Increased levels of alanine, histidine and tyrosine are associated with severity of NAFLD in both men and women. Moreover, higher serum concentrations of valine, aspartic acid and mannose were positively associated with the progression of NAFLD among the male subjects, while a negative association was observed with the levels of creatine, phosphorylcholine and acetic acid. On the other hand, glucose was positively associated with the progression of NAFLD among the female subjects, while levels of threonine were negatively related. Fluctuations in ketone bodies acetoacetate and acetone were also observed among the female subjects probing a significant reduction in the circulatory levels of the former in NAFLD cases. A complex glycine response to hepatic steatosis of the female subjects deserves further investigation. Conclusion: Results of this study aspire to address the paucity of data on sex differences regarding NAFLD pathogenesis. Targeted circulatory metabolome measurements could be used as diagnostic markers for the distinct stages of NAFLD in each sex and eventually aid in the development of novel sex-related therapeutic options.

Asunto(s)

Enfermedad del Hígado Graso no Alcohólico , Humanos , Femenino , Masculino , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Metabolómica/métodos , Obesidad/metabolismo , Metaboloma

RESUMEN

Seasonal weight loss (SWL), is a major limitation to animal production. In the Canary Islands, there are two dairy goat breeds with different levels of tolerance to SWL: Majorera (tolerant) and Palmera (susceptible). Our team has studied the response of these breeds to SWL using different Omics tools. The objective of this study was to integrate such results in a data driven approach and using dedicated tools, namely the DIABLO method. The outputs of our analysis mainly separate unrestricted from restricted goats. Metabolites behave as "hub" molecules, grouping interactions with several genes and proteins. Unrestricted goats upregulated protein synthesis, along with arginine catabolism and adipogenesis pathways, which are related with higher anabolic rates and a larger proportion of secretory tissue, in agreement with their higher milk production. Contrarily, restricted goats seemingly increased the synthesis of acetyl-CoA through serine and acetate conversion into pyruvate. This may have occurred to increase fatty acid synthesis and/or to use them as an energy source in detriment to glucose, which was more available in the diet of unrestricted goats. Lastly, restricted Palmera upregulated the expression of PEBP4 and GPD1 genes compared to all other groups, which might support their use as putative biomarkers for SWL susceptibility. SIGNIFICANCE: Seasonal weight loss (SWL) is a major issue influencing animal production in the tropics and Mediterranean. By studying its impact on the mammary gland of tolerant and susceptible dairy goat breeds, using Omics, we aim at surveying the tissue for possible biomarkers that reflect these traits. In this study, data integration of three Omics (transcriptomics, proteomics and metabolomics) was performed using bioinformatic tools, to relate putative biomarkers and evaluate all three levels of information; in a novel approach. This information can enhance selection programs, lowering the impact of SWL on food production systems.

Asunto(s)

Cabras , Metabolómica , Animales , Femenino , Cabras/genética , Estaciones del Año , Biomarcadores/análisis , Pérdida de Peso , Glándulas Mamarias Animales/metabolismo , Leche/metabolismo , Lactancia

RESUMEN

The effect of dietary Spirulina (Arthrospira platensis) and CAZyme supplementation was assessed on the gut of weaned piglets, using an integrated NMR-metabolomics approach combined with Tandem Mass Tag labelled proteomics. Thirty weaned male piglets were assigned to one of the three following diets (n = 10): cereal and soybean meal basal diet (Control), basal diet with 10% Spirulina inclusion (SP) and SP diet supplemented with 0.01% lysozyme (SP + L). The experiment lasted 4 weeks and, upon slaughter, small intestine samples were collected for histological, metabolomic and proteomic analysis. No significant differences were found for the histology and metabolomics analysis between the three experimental groups. Lactate, glutamate, glycine and myo-inositol were the most abundant metabolites. Proteomics results showed 1502 proteins identified in the intestine tissue. A total of 23, 78, 27 differentially abundant proteins were detected respectively for the SP vs. Control, SP + L vs. Control and SP + L vs. SP comparisons. The incorporation of Spirulina and supplementation of lysozyme in the piglet's diets is associated to intestinal proteomic changes. These include increased protein synthesis and abundance of contractile apparatus proteins, related with increased nutrient availability, which has beneficial (increased glucose uptake) and detrimental (increased digesta viscosity) metabolic effects. SIGNIFICANCE: The use of conventional feedstuffs becomes increasingly prohibitive due to its environmental toll. To increase the sustainability of the livestock sector, novel feedstuffs such as microalgae need to be considered. However, its recalcitrant cell wall has antinutritional effects that can inhibit high dietary inclusion levels. The supplementation with CAZymes is a possible solution to this issue. The small intestine is a central piece in monogastric digestion and of particular importance for the weaned piglet. Studying the effect of dietary Spirulina and CAZyme supplementation on its histomorphology, metabolome and proteome allows studying relevant physiological adaptations to these diets.

Asunto(s)

Spirulina , Alimentación Animal/análisis , Animales , Dieta , Suplementos Dietéticos , Glucosa , Glutamatos , Glicina , Inositol , Lactatos , Masculino , Muramidasa , Proteoma , Proteómica , Porcinos

RESUMEN

Chronic kidney disease (CKD) is a progressive and irreversible disease. Although urine is an ideal biological sample for proteomics and metabolomics studies, sensitive and specific biomarkers are currently lacking in dogs. This study characterised dog urine proteome and metabolome aiming to identify and possibly quantify putative biomarkers of CKD in dogs. Twenty-two healthy dogs and 28 dogs with spontaneous CKD were selected and urine samples were collected. Urinary proteome was separated by SDS-PAGE and analysed by mass spectrometry, while urinary metabolome was analysed in protein-depleted samples by 1D 1H NMR spectra. The most abundant proteins in urine samples from healthy dogs were uromodulin, albumin and, in entire male dogs, arginine esterase. In urine samples from CKD dogs, the concentrations of uromodulin and albumin were significantly lower and higher, respectively, than in healthy dogs. In addition, these samples were characterised by a more complex protein pattern indicating mixed glomerular (protein bands ≥65 kDa) and tubular (protein bands <65 kDa) proteinuria. Urine spectra acquired by NMR allowed the identification of 86 metabolites in healthy dogs, belonging to 49 different pathways mainly involved in amino acid metabolism, purine and aminoacyl-tRNA biosynthesis or tricarboxylic acid cycle. Seventeen metabolites showed significantly different concentrations when comparing healthy and CKD dogs. In particular, carnosine, trigonelline, and cis-aconitate, might be suggested as putative biomarkers of CKD in dogs. SIGNIFICANCE: Urine is an ideal biological sample, however few proteomics and metabolomics studies investigated this fluid in dogs and in the context of CKD (chronic kidney disease). In this research, applying a multi-omics approach, new insights were gained regarding the molecular changes triggered by this disease in canine urinary proteome and metabolome. In particular, the involvement of the tubular component was highlighted, suggesting uromodulin, trigonelline and carnosine as possible biomarkers of CKD in dogs.

Asunto(s)

Insuficiencia Renal Crónica , Lobos , Animales , Biomarcadores/metabolismo , Perros , Masculino , Metaboloma , Metabolómica , Proteoma , Insuficiencia Renal Crónica/diagnóstico , Lobos/metabolismo

RESUMEN

In pre-weaning calves, both leucine and threonine play important roles in growth and muscle metabolism. In this study, metabolomics, proteomics and clinical chemistry were used to assess the effects of leucine and threonine supplementation added to milk replacer on 14 newborn Holstein male calves: 7 were fed a control diet (Ctrl) and 7 were fed the Ctrl diet supplemented with 0.3% leucine and 0.3% threonine (LT) from 5.6 days of age to 53.6 days. At this time, blood and semitendinosus muscle biopsies were collected for analysis. Integrated metabolomics and proteomics showed that branched-chain amino acids (BCAA) degradation and mitochondrial oxidative metabolism (citrate cycle and respiratory chain) were the main activated pathways in muscle because of the supplementation. BCAA derivatives and metabolites related to lipid mobilization showed the major changes. The deleterious effects of activated oxidative phosphorylation were balanced by the upregulation of antioxidant proteins. An increase in protein synthesis was indicated by elevated aminoacyl-tRNA biosynthesis and increased S6 ribosomal protein phosphorylation in skeletal muscle. In conclusion, LT group showed greater BCAA availability and mitochondrial oxidative activity; as the muscle cells undergo greater aerobic metabolism, antioxidant defenses were activated to compensate for possible cell damage. Data are available via ProteomeXchange (PXD016098). SIGNIFICANCE: Leucine and threonine are essential amino acids for the pre-weaning calf, being of high importance for growth. In this study, we found that leucine and threonine supplementation of milk replacer to feed pre-weaning calves led to differences in the proteome, metabolome and clinical chemistry analytes in skeletal muscle and plasma, albeit no differences in productive performance were recorded. This study extends our understanding on the metabolism in dairy calves and helps optimizing their nutritional status.

Asunto(s)

Metaboloma , Proteoma , Alimentación Animal/análisis , Animales , Bovinos , Dieta , Suplementos Dietéticos , Leucina/metabolismo , Masculino , Leche , Músculo Esquelético/metabolismo , Proteoma/metabolismo , Treonina/metabolismo , Destete

RESUMEN

The effects of different amino acid (AA) supplementations of milk protein-based milk replacers in pre-ruminant calves from 3 days to 7 weeks of age were studied. Animals were divided into 4 groups: Ctrl) Control group fed with milk protein-based milk replacer without supplementation; GP) supplementation with 0.1% glycine and 0.3% proline; FY) supplementation with 0.2% phenylalanine and 0.2% tyrosine; MKT) supplementation with 0.62% lysine, 0.22% methionine and 0.61% threonine. For statistical analysis, t-test was used to compare AA-supplemented animals to the Ctrl group. At week 7, body weight and average daily gain (ADG) were measured and blood samples and skeletal muscle biopsies were taken. Blood biochemistry analytes related to energy metabolism were determined and it was shown that MKT group had higher serum creatinine and higher plasma concentration of three supplemented AAs as well as arginine compared with the Ctrl group. GP group had similar glycine/proline plasma concentration compared with the other groups while in FY group only plasma phenylalanine concentration was higher compared with Control. Although the AA supplementations in the GP and FY groups did not affect average daily gain and metabolic health profile from serum, the metabolome analysis from skeletal muscle biopsy revealed several differences between the GP-FY groups and the Ctrl-MKT groups, suggesting a metabolic adaptation especially in GP and FY groups.

Asunto(s)

Aminoácidos/farmacología , Análisis Químico de la Sangre , Industria Lechera , Suplementos Dietéticos/análisis , Metabolómica , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Alimentación Animal/análisis , Animales , Peso Corporal/efectos de los fármacos , Bovinos , Masculino

RESUMEN

Goat dairy products are an important source of animal protein in the tropics. During the dry season, pasture scarcity leads animals to lose up to 40% of their body weight, a condition known as Seasonal Weight Loss (SWL) that is one of the major constraints in ruminant production. Breeds with high tolerance to SWL are relevant to understand the physiological responses to pasture scarcity so they could be used in programs for animal breeding. In the Canary Islands there are two dairy goat breeds with different levels of tolerance to SWL: the Palmera, susceptible to SWL; and the Majorera, tolerant to SWL. Fat is one of the milk components most affected by environmental and physiological conditions. This study hypothesises that feed-restriction affects Majorera and Palmera breeds differently, leading to different fatty acid profiles in the mammary gland and milk. An interaction between breed and feed-restriction was observed in the mammary gland. Feed-restriction was associated with an increase in oleic acid and a decrease in palmitic acid percentage in the Palmera breed whereas no differences were observed in the Majorera breed. Palmitic and oleic acids together constituted around 60% of the total fatty acids identified, which suggests that Palmera breed is more susceptible to SWL. In milk, feed-restriction affected both breeds similarly. Regarding the interaction of the breed with the treatment, we also observed similar responses in both breeds, but this influence affects only around 2% of the total fatty acids. In general, Majorera breed is more tolerant to feed-restriction.

Asunto(s)

Ácidos Grasos/análisis , Cabras/fisiología , Glándulas Mamarias Animales/química , Leche/química , Estaciones del Año , Pérdida de Peso , Alimentación Animal/provisión & distribución , Animales , Cruzamiento , Femenino , Glándulas Mamarias Animales/fisiología , Ácido Oléico/análisis , Ácido Palmítico/análisis , España , Especificidad de la Especie

RESUMEN

Sheep are a valuable resource for meat and wool production. During the dry summer, pastures are scarce and animals face Seasonal Weight Loss (SWL), which decreases production yields. The study of breeds tolerant to SWL is important to understand the physiological mechanisms of tolerance to nutritional scarcity, and define breeding strategies. Merino, Damara and Dorper sheep breeds have been described as having different levels of tolerance to SWL. In this work, we assess their liver and muscle metabolomes, and compare the responses to feed restriction. Ram lambs from each breed were divided into growth and feed restricted groups, over 42 days. Tissue metabolomes were assessed by 1H-NMR. The Dorper restricted group showed few changes in both tissues, suggesting higher tolerance to nutritional scarcity. The Merinos exhibited more differences between treatment groups. Major differences were related to fat and protein mobilization, and antioxidant activity. Between the Damara groups, the main differences were observed in amino acid composition in muscle and in energy-related pathways in the liver. Integration of present results and previous data on the same animals support the hypothesis that, Dorper and Damara breeds are more tolerant to SWL conditions and thus, more suitable breeds for harsh environmental conditions.

Asunto(s)

Hígado/química , Metabolómica/métodos , Músculo Esquelético/química , Pérdida de Peso , Adaptación Fisiológica , Animales , Cruzamiento , Espectroscopía de Protones por Resonancia Magnética/métodos , Estaciones del Año , Ovinos , Oveja Doméstica

RESUMEN

Goats are of special importance in the Mediterranean and tropical regions for producing a variety of dairy products. The scarcity of pastures during the dry season leads to seasonal weight loss (SWL), which affects milk production. In this work, we studied the effect of feed-restriction on two dairy goat breeds, with different tolerance levels to SWL: the Majorera breed (tolerant) and the Palmera breed (susceptible). Nuclear magnetic resonance (NMR) was used to compare the metabolome of an aqueous fraction of the mammary gland and milk serum from both breeds. Goats in mid-lactation were divided by breed, and each in two feed-regime groups: the control group and the restricted-fed group (to achieve 15-20% reduction of body weight at the end of the experiment). Milk and mammary gland samples were collected at the end of the experimental period (23rd day). (1)H NMR spectra were collected from the aqueous extract of the mammary gland biopsies and the milk serum. Profiling analysis has led to the identification of 46 metabolites in the aqueous extract of the mammary gland. Lactose, glutamate, glycine and lactate were found to be the most abundant. Analysis of milk serum allowed the identification of 50 metabolites, the most abundant being lactose, citrate and creatine. Significant differences were observed, in mammary gland biopsies and milk serum, between control and restricted-fed groups in both breeds, albeit with no differences between the breeds. Variations seem to be related to metabolism adaptation to the low-energy diet and are indicative of breed-specific microflora. Milk serum showed more metabolites varying between control and restricted groups, than the mammary gland. The Majorera breed also showed more variations than the Palmera breed in milk samples, which could be an indication of a prompt adaptation to SWL by the Majorera breed.

Asunto(s)

Glándulas Mamarias Animales/metabolismo , Metaboloma , Metabolómica , Leche/metabolismo , Resonancia Magnética Nuclear Biomolecular , Estaciones del Año , Pérdida de Peso , Adaptación Biológica , Animales , Cruzamiento , Femenino , Cabras , Metabolómica/métodos

RESUMEN

Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR. N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277±5 mV, at pH7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the ß-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment.

Asunto(s)

Azurina/química , Cobre/química , Citocromo-c Peroxidasa/química , Electrones , Peróxido de Hidrógeno/química , Neisseria gonorrhoeae/química , Secuencia de Aminoácidos , Azurina/genética , Azurina/metabolismo , Clonación Molecular , Cobre/metabolismo , Citocromo-c Peroxidasa/genética , Citocromo-c Peroxidasa/metabolismo , Transporte de Electrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Neisseria gonorrhoeae/enzimología , Oxidación-Reducción , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato

RESUMEN

RodZ (also known as YfgA) is a component of the core bacterial morphogenic apparatus. RodZ is a key cell shape determinant in rod-shaped bacteria and it interacts with the actin-like cytoskeletal protein MreB. In Bacillus subtilis, this 304-residue transmembrane protein is composed of three distinct domains: a cytoplasmic domain (RodZn), a transmembrane domain, and an extra-cytoplasmic domain (RodZc). Here we report the (1)H, (13)C and (15)N backbone and side chain resonance assignments of the RodZc domain from B. subtilis by NMR spectroscopy, and the resulting secondary structure prediction.

Asunto(s)

Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectroscopía de Protones por Resonancia Magnética

RESUMEN

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.

Asunto(s)

AMP Cíclico/química , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Activación del Canal Iónico , Receptores Citoplasmáticos y Nucleares/química , Sitios de Unión , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Canales de Potasio/química , Canales de Potasio/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo

RESUMEN

In Saccharomyces cerevisiae, the transcription factor Yap8 is a key determinant in arsenic stress response. Contrary to Yap1, another basic region-leucine zipper (bZIP) yeast regulator, Yap8 has a very restricted DNA-binding specificity and only orchestrates the expression of ACR2 and ACR3 genes. In the DNA-binding basic region, Yap8 has three distinct amino acids residues, Leu26, Ser29 and Asn31, at sites of highly conserved positions in the other Yap family of transcriptional regulators and Pap1 of Schizosaccharomyces pombe. To evaluate whether these residues are relevant to Yap8 specificity, we first built a homology model of the complex Yap8bZIP-DNA based on Pap1-DNA crystal structure. Several Yap8 mutants were then generated in order to confirm the contribution of the residues predicted to interact with DNA. Using bioinformatics analysis together with in vivo and in vitro approaches, we have identified several conserved residues critical for Yap8-DNA binding. Moreover, our data suggest that Leu26 is required for Yap8 binding to DNA and that this residue together with Asn31, hinder Yap1 response element recognition by Yap8, thus narrowing its DNA-binding specificity. Furthermore our results point to a role of these two amino acids in the stability of the Yap8-DNA complex.

Asunto(s)

Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Secuencia Conservada , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Asociadas a Pancreatitis , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Elementos de Respuesta , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Homología de Secuencia , Factores de Transcripción/genética

RESUMEN

Automated methodologies to design synthetic proteins from first principles use energy computations to estimate the ability of the sequences to adopt a targeted structure. This approach is still far from systematically producing native-like sequences, due, most likely, to inaccuracies when modeling the interactions between the protein and its aqueous environment. This is particularly challenging when engineering small protein domains (with less polar pair interactions than with the solvent). We have re-designed a three-helix bundle, domain B, using a fixed backbone and a four amino acid alphabet. We have enlarged the rotamer library with conformers that increase the weight of electrostatic interactions within the design process without altering the energy function used to compute the folding free energy. Our synthetic sequences show less than 15% similarity to any Swissprot sequence. We have characterized our sequences in different solvents using circular dichroism and nuclear magnetic resonance. The targeted structure achieved is dependent on the solvent used. This method can be readily extended to larger domains. Our method will be useful for the engineering of proteins that become active only in a given solvent and for designing proteins in the context of hydrophobic solvents, an important fraction of the situations in the cell.

Asunto(s)

Aminoácidos/química , Biología Computacional/métodos , Ingeniería de Proteínas/métodos , Proteínas/química , Algoritmos , Secuencia de Aminoácidos , Dicroismo Circular , Simulación por Computador , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Electricidad Estática , Termodinámica

RESUMEN

Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer's disease (AD) and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau229₋237[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B241₋255 originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a ß-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

Asunto(s)

Aciltransferasas/química , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Péptidos/química , Vacunas/química , Proteínas tau/química , Aciltransferasas/síntesis química , Aciltransferasas/inmunología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/terapia , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/síntesis química , Proteínas Bacterianas/inmunología , Humanos , Mycobacterium tuberculosis/química , Resonancia Magnética Nuclear Biomolecular , Péptidos/síntesis química , Péptidos/inmunología , Fosforilación , Estructura Secundaria de Proteína , Vacunas/síntesis química , Vacunas/inmunología , Proteínas tau/síntesis química , Proteínas tau/inmunología

RESUMEN

Lipid-modified azurin (Laz) from Neisseria gonorrhoeae is a type 1 copper protein proposed to be the electron donor to several enzymes involved in the resistance mechanism to reactive oxygen and nitrogen species. Here we report the backbone and side-chain resonance assignment of Laz in the reduced form, which has been complete at 97%. The predicted secondary structure indicates that this protein belongs to the azurin subfamily of type 1 copper proteins.

Asunto(s)

Azurina/química , Lípidos/química , Neisseria gonorrhoeae/metabolismo , Resonancia Magnética Nuclear Biomolecular , Protones , Secuencia de Aminoácidos , Isótopos de Carbono , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Estructura Secundaria de Proteína , Solubilidad

RESUMEN

Mannosylglycerate is a compatible solute typical of thermophilic marine microorganisms that has a remarkable ability to protect proteins from thermal denaturation. This ionic solute appears to be a universal stabilizing agent, but the extent of protection depends on the specific protein examined. To understand how mannosylglycerate confers protection, we have been studying its influence on the internal motions of a hyperstable staphylococcal nuclease (SNase). Previously, we found a correlation between the magnitude of protein stabilization and the restriction of fast backbone motions. We now report the effect of mannosylglycerate on the fast motions of side-chains and on the slower unfolding motions of the protein. Side-chain motions were assessed by (13)CH(3) relaxation measurements and model-free analysis while slower unfolding motions were probed by H/D exchange measurements at increasing concentrations of urea. Side-chain motions were little affected by the presence of different concentrations of mannosylglycerate or even by the presence of urea (0.25M), and show no correlation with changes in the thermodynamic stability of SNase. Native hydrogen exchange experiments showed that, contrary to reports on other stabilizing solutes, mannosylglycerate restricts local motions in addition to the global motions of the protein. The protein unfolding/folding pathway remained undisturbed in the presence of mannosylglycerate but the solute showed a specific effect on the local motions of ß-sheet residues. This work reinforces the link between solute-induced stabilization and restriction of protein motions at different timescales, and shows that the solute preferentially affects specific structural elements of SNase.

Asunto(s)

Excipientes/metabolismo , Ácidos Glicéricos/metabolismo , Manosa/análogos & derivados , Nucleasa Microcócica/química , Nucleasa Microcócica/metabolismo , Staphylococcus aureus/enzimología , Manosa/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Desnaturalización Proteica , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Termodinámica

RESUMEN

Enzymes produced by psychrophilic organisms have successfully overcome the low temperature challenge and evolved to maintain high catalytic rates in their permanently cold environments. As an initial step in our attempt to elucidate the cold-adaptation strategies used by these enzymes we report here the (1)H, (15)N and (13)C assignments for the reduced form of a thiol-disulphide oxidoreductase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Asunto(s)

Resonancia Magnética Nuclear Biomolecular , Proteína Disulfuro Reductasa (Glutatión)/química , Pseudoalteromonas/enzimología , Regiones Antárticas , Isótopos de Carbono , Hidrógeno , Isótopos de Nitrógeno , Estructura Secundaria de Proteína , Temperatura

RESUMEN

Attempts are made to efficiently decouple (13)C nuclei without significant loss of coherence during the application of the decoupling package. Such attempts are based on the S(3)E spin-state selection method. A newly developed double S(3)E (DS(3)E) is particularly efficient for C(alpha) detection for proteins as large as 480 kDa.

Asunto(s)

Ferritinas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Péptidos Cíclicos/química , Superóxido Dismutasa/química , Animales , Isótopos de Carbono , Isótopos de Nitrógeno , Rana catesbeiana

RESUMEN

Molecular size has limited solution NMR analyses of proteins. We report (13)C-(13)C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting (13)C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C-C connectivities between nuclei separated by 1-4 bonds. These results show the potential of (13)C-(13)C NOESY for solution studies of molecular assemblies >100 kDa.

Asunto(s)

Carbono/análisis , Ferritinas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Aminoácidos/química , Animales , Carbono/química , Isótopos de Carbono , Peso Molecular , Estructura Secundaria de Proteína , Rana catesbeiana , Soluciones , Factores de Tiempo