BlaSTorP : herramienta NCBI-BLAST a nivel local

Tesis dirigida por Antonio Jesús Pérez Pulido y Juan Falgueras Cano. Se ha desarrollado una nueva herramienta bioinformática con el fin de resolver los problemas que el investigador novel en proteómica y bioinformática tiene a la hora de llevar a cabo la identificación masiva de secuencias peptídicas provenientes de secuenciación “de novo” mediante espectrometría de masas en las bases de datos más comúnmente utilizadas por los investigadores en Internet. Estas bases de datos son muy complejas y contienen demasiada información que no siempre se usa en su totalidad. Por ejemplo, la página web de uniprot ofrece información muy extensa sobre la proteína que estemos buscando en varios subapartados: nombres y orígenes, atributos de la proteína, comentarios de la anotación, ontología, anotaciones sobre la secuencia, secuencia en formato fasta y propiedades como longitud y masa teóricas, referencias cruzadas, referencias bibliográficas, etc. La creación de este programa constituye una herramienta eficaz para la realización de este trabajo y ayuda a ahorrar tiempo en la identificación y búsqueda de las funciones más comunes de las proteínas que estemos tratando de identificar, además de resultar una herramienta sencilla y disponible por la web en el caso de montar un servidor público o bien de forma local. // A new bioinformatics tool has been developed to help the new researchers in proteomics and bioinformatics to carry out the identification of sequences obtained by "de novo" sequencing using the most common mass spectrometry databases. Databases are very complex and contain too much information that is not always enterely used. Thus, UniProt offers extensive information about proteins divided into several subsections: names and origins, attributes of the protein, commentaries, ontology, annotations on the sequence, sequence in fasta format and theoretical properties like length and mass, cross references, bibliographic references, etc. This new program is an effective tool for carrying out this work, saving time in protein identifications and recruiting the most common functions of the proteins we are trying to identify, as well as being a simple tool

The NtrYX Two-Component System of Paracoccus denitrificans Is Required for the Maintenance of Cellular Iron Homeostasis and for a Complete Denitrification under Iron-Limited Conditions

Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd1-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins

Proteómica para la identificación de biomarcadores en estudios medioambientales

Comunicaciones a congreso

Omic approaches in environmental risk assessment

Resumen de la comunicación presentada al XX Congreso de la Sociedad Española de Mutagénesis Ambiental – Córdoba 201

Hepatic proteome changes in solea senegalensis exposed to contaminated estuarine sediments: a laboratory and in situ survey

Assessing toxicity of contaminated estuarine sediments poses a challenge to ecotoxicologists due to the complex geochemical nature of sediments and to the combination of multiple classes of toxicants. Juvenile Senegalese soles were exposed for 14 days in the laboratory and in situ (field) to sediments from three sites (a reference plus two contaminated) of a Portuguese estuary. Sediment characterization confirmed the combination of metals, polycyclic aromatic hydrocarbons and organochlorines in the two contaminated sediments. Changes in liver cytosolic protein regulation patterns were determined by a combination of two-dimensional electrophoresis with de novo sequencing by tandem mass spectrometry. From the forty-one cytosolic proteins found to be deregulated, nineteen were able to be identified, taking part in multiple cellular processes such as anti-oxidative defence, energy production, proteolysis and contaminant catabolism (especially oxidoreductase enzymes). Besides a clear distinction between animals exposed to the reference and contaminated sediments, differences were also observed between laboratory- and in situ-tested fish. Soles exposed in the laboratory to the contaminated sediments failed to induce, or even markedly down-regulated, many proteins, with the exception of a peroxiredoxin (an anti-oxidant enzyme) and a few others, when compared to reference fish. In situ exposure to the contaminated sediments revealed significant up-regulation of basal metabolism-related enzymes, comparatively to the reference condition. Down-regulation of basal metabolism enzymes, related to energy production and gene transcription, in fish exposed in the laboratory to the contaminated sediments, may be linked to sedimentbound contaminants and likely compromised the organisms’ ability to deploy adequate responses against insult.info:eu-repo/semantics/publishedVersio

Sensitivity to anti-Fas is independent of increased cathepsin D activity and adrenodoxin reductase expression occurring in NOS-3 overexpressing HepG2 cells

Stable overexpression of endothelial nitric oxide synthase (NOS-3) in HepG2 cells (4TO-NOS) leads to increased nitro-oxidative stress and upregulation of the cell death mediators p53 and Fas. Thus, NOS-3 overexpression has been suggested as a useful antiproliferative mechanism in hepatocarcinoma cells. We aimed to identify the underlying mechanism of cell death induced by NOS-3 overexpression at basal conditions and with anti-Fas treatment. The intracellular localization of NOS-3, the nitro-oxidative stress and the mitochondrial activity were analysed. In addition, the protein expression profile in 4TO-NOS was screened for differentially expressed proteins potentially involved in the induction of apoptosis. NOS-3 localization in the mitochondrial outer membrane was not associated with changes in the respiratory cellular capacity, but was related to the mitochondrial biogenesis increase and with a higher protein expression of mitochondrial complex IV. Nitro-oxidative stress and cell death in NOS-3 overexpressing cells occurred with the expression increase of pro-apoptotic genes and a higher expression/activity of the enzymes adrenodoxin reductase mitochondrial (AR) and cathepsin D (CatD). CatD overexpression in 4TO-NOS was related to the apoptosis induction independently of its catalytic activity. In addition, CatD activity inhibition by pepstatin A was not effective in blocking apoptosis induced by anti-Fas. In summary, NOS-3 overexpression resulted in an increased sensitivity to anti-Fas induced cell death, independently of AR expression and CatD activityThis study was supported by the Instituto de Salud Carlos III (FIS 09/00185). G. Ferrín was supported by the Networked Biomedical Research Center Hepatic and Digestive Diseases (CIBEREHD

Development of a prediction model for short-term remission of patients with Crohn’s disease treated with anti-TNF drugs

Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn’s disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins (p ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins (p < 0.001), whose differential expression was confirmed by ELISA (p = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR

Spatial and Temporal Protein Modules Signatures Associated with Alzheimer Disease in 3xTg-AD Mice Are Restored by Early Ubiquinol Supplementation

Despite its robust proteopathic nature, the spatiotemporal signature of disrupted protein modules in sporadic Alzheimer's disease (AD) brains remains poorly understood. This considered oxidative stress contributes to AD progression and early intervention with coenzyme Q10 or its reduced form, ubiquinol, delays the progression of the disease. Using MALDI-MSI and functional bioinformatic analysis, we have developed a protocol to express how deregulated protein modules arise from hippocampus and cortex in the AD mice model 3xTG-AD in an age-dependent manner. This strategy allowed us to identify which modules can be efficiently restored to a non-pathological condition by early intervention with ubiquinol. Indeed, an early deregulation of proteostasis-related protein modules, oxidative stress and metabolism has been observed in the hippocampus of 6-month mice (early AD) and the mirrored in cortical regions of 12-month mice (middle/late AD). This observation has been validated by IHC using mouse and human brain sections, suggesting that these protein modules are also affected in humans. The emergence of disrupted protein modules with AD signature can be prevented by early dietary intervention with ubiquinol in the 3xTG-AD mice model.A pesar de su robusta naturaleza proteopática, la firma espaciotemporal de los módulos de proteínas interrumpidos en los cerebros de la enfermedad de Alzheimer (EA) esporádica sigue siendo poco conocida. Este considerado estrés oxidativo contribuye a la progresión de la EA y la intervención precoz con coenzima Q10 o su forma reducida, el ubiquinol, retrasa la progresión de la enfermedad. Usando MALDI-MSI y análisis bioinformático funcional, hemos desarrollado un protocolo para expresar cómo surgen módulos de proteína desregulados del hipocampo y la corteza en el modelo de ratones AD 3xTG-AD de una manera dependiente de la edad. Esta estrategia nos permitió identificar qué módulos se pueden restaurar de manera eficiente a una condición no patológica mediante una intervención temprana con ubiquinol. De hecho, una desregulación temprana de los módulos proteicos relacionados con la proteostasis, Se ha observado estrés oxidativo y metabolismo en el hipocampo de ratones de 6 meses (EA temprana) y se refleja en regiones corticales de ratones de 12 meses (EA media/tardía). Esta observación ha sido validada por IHC utilizando secciones de cerebro humano y de ratón, lo que sugiere que estos módulos de proteína también se ven afectados en humanos. La aparición de módulos de proteínas interrumpidos con la firma AD puede prevenirse mediante una intervención dietética temprana con ubiquinol en el modelo de ratones 3xTG-AD