Producción de precursores esteroideos por mutantes en la cepa mycolicibacterium neoarum B-3805 alterados en el operón IGR

Mycolicibacterium neoaurum NRRL B-3805 es una bacteria saprófita presente en el suelo, capaz de convertir los fitoesteroles en androsta-4-ene-3,17-diona (AD) y otros productos esteroideos como androsta-1,4-dien-3,17-diona (ADD). Por este motivo, es considerada una cepa de referencia en la biotransformación de fitoesteroles, con el consiguiente interés para la industria farmacéutica. El principal objetivo de este trabajo fue la deleción de forma conjunta de los genes chsE1-chsE2 y chsH1-chsH2 presentes en el operón igr, el cual está involucrado en la degradación de la cadena lateral de los fitoesteroles por parte de la cepa M. neoaurum NRRL B-3805. Aunque se intentó la deleción de los genes mediante recombinación homóloga empleando diferentes plásmidos, no fue posible la obtención de mutantes. Como segundo objetivo, se puso a punto el método de la purificación de un compuesto con posible interés farmacológico producido por el mutante M. neoaurum ΔchsH1-H2. La purificación se llevó a cabo a partir del sobrenadante del medio de cultivo mediante extracción con diclorometano y evaporación del mismo

Estudio comparativo del secretoma de condrocitos articulares humanos analizados mediante las técnicas iTRAQ e SILAC

Comunicaciones a congreso

Sequential depletion of human serum for the search of osteoarthritis biomarkers

Background: The field of biomarker discovery, development and application has been the subject of intense interest and activity, especially with the recent emergence of new technologies, such as proteomics-based approaches. In proteomics, search for biomarkers in biological fluids such as human serum is a challenging issue, mainly due to the high dynamic range of proteins present in these types of samples. Methods for reducing the content of most highly abundant proteins have been developed, including immunodepletion or protein equalization. In this work, we report for the first time the combination of a chemical sequential depletion method based in two protein precipitations with acetonitrile and DTT, with a subsequent two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis for the search of osteoarthritis (OA) biomarkers in human serum. The depletion method proposed is non-expensive, of easy implementation and allows fast sample throughput. Results: Following this workflow, we have compared sample pools of human serum obtained from 20 OA patients and 20 healthy controls. The DIGE study led to the identification of 16 protein forms (corresponding to 14 different proteins) that were significantly (p < 0.05) altered in OA when compared to controls (8 increased and 7 decreased). Immunoblot analyses confirmed for the first time the increase of an isoform of Haptoglobin beta chain (HPT) in sera from OA patients. Conclusions: Altogether, these data demonstrate the utility of the proposed chemical sequential depletion for the analysis of sera in protein biomarker discovery approaches, exhibit the usefulness of quantitative 2D gel-based strategies for the characterization of disease-specific patterns of protein modifications, and also provide a list of OA biomarker candidates for validation

Cuantificación por SILAC del efecto de la nicotina sobre condrocitos articulares

Comunicaciones a congreso

Determinación del perfil proteico diferencial de líquido sinovial en pacientes con artrosis y artritis reumatoide mediante nanoLC acoplada a MALDI-TOF7TOF

Comunicaciones a congreso

Quantitative proteomics analysis of chondrogenic differentiation of mesenchymal stem cells by SILAC

Comunicaciones a congreso

An atlas of the knee joint proteins and their role in osteoarthritis defined by literature mining

[Abstract] Osteoarthritis (OA) is the most prevalent rheumatic pathology. However, OA is not simply a process of wear and tear affecting articular cartilage but rather a disease of the entire joint. One of the most common locations of OA is the knee. Knee tissues have been studied using molecular strategies, generating a large amount of complex data. As one of the goals of the Rheumatic and Autoimmune Diseases initiative of the Human Proteome Project, we applied a text-mining strategy to publicly available literature to collect relevant information and generate a systematically organized overview of the proteins most closely related to the different knee components. To this end, the PubPular literature-mining software was employed to identify protein-topic relationships and extract the most frequently cited proteins associated with the different knee joint components and OA. The text-mining approach searched over eight million articles in PubMed up to November 2022. Proteins associated with the six most representative knee components (articular cartilage, subchondral bone, synovial membrane, synovial fluid, meniscus, and cruciate ligament) were retrieved and ranked by their relevance to the tissue and OA. Gene ontology analyses showed the biological functions of these proteins. This study provided a systematic and prioritized description of knee-component proteins most frequently cited as associated with OA. The study also explored the relationship of these proteins to OA and identified the processes most relevant to proper knee function and OA pathophysiology.PI19/01206; Instituto de Salud Carlos IIIPI20/00793; Instituto de Salud Carlos IIIPI20/01409; Instituto de Salud Carlos IIIPI22/01155; Instituto de Salud Carlos IIIRICORS-REIRD21/0002/0009; Instituto de Salud Carlos IIIED431E2018/03; Xunta de GaliciaIN607A2021/07; Xunta de GaliciaIN607D2020/10; Xunta de Galici

Variable effect of 3 different chondroitin sulfate compounds on chondrocytes secreted proteins profile revealed by silac techinique

Comunicaciones a congreso

Identification of an acetyl esterase in the supernatant of the environmental strain Bacillus sp. HR21-6

Bacillus sp. HR21-6 is capable of the chemo- and regioselective synthesis of lipophilic partially acetylated phenolic compounds derived from olive polyphenols, which are powerful antioxidants important in the formulation of functional foods. In this work, an acetyl esterase was identified in the secretome of this strain by non-targeted proteomics, and classified in the GDSL family (superfamily SGNH). The recombinant protein was expressed and purified from Escherichia coli in the soluble form, and biochemically characterized. Site-directed mutagenesis was performed to understand the role of different amino acids that are conserved among GDSL superfamily of esterases. Mutation of Ser-10, Gly-45 or His-185 abolished the enzyme activity, while mutation of Asn-77 or Thr-184 altered the substrate specificity of the enzyme. This new enzyme is able to perform chemoselective conversions of olive phenolic compounds with great interest in the food industry, such as hydroxytyrosol, 3,4-dihydroxyphenylglycol, and oleuropein.Junta de Andalucía P11-CVI-7427 M