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10 p.- 5 fig. ; Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis ; This work was supported by European Union grant FP7-2008-FOOD-211441 BIAMFOOD and by grant AGL2012-40084-C03-01 from the Spanish Ministry of Economics and Competitiveness ; Peer reviewed

The analysis of Tumor Interstitial Fluid (TIF) composition is a valuable procedure to identify anti-metastatic targets, and different laboratories have set up techniques for TIF isolation and proteomic analyses. However, those methods had never been compared in samples from the same tumor and patient. In this work, we compared the two most used methods, elution and centrifugation, in pieces of the same biopsy samples of cutaneous squamous cell carcinoma (cSCC). First, we established that high G-force(10000g) was required to obtain TIF from cSCC by centrifugation. Secondly, we compared the centrifugation method with the elution method in pieces of three different cSCC tumors. We found that the mean protein intensities based in the number of peptide spectrum matches was significantly higher in the centrifuged samples than in the eluted samples. Regarding the robustness of the methods, we observed higher overlapping between both methods (77-80%) than among samples (50%). These results suggest that exists an elevated consistence of TIF composition independently of the method used. However, we observed a three-fold increase of extracellular proteins in non-overlapped proteome obtained by centrifugation. We therefore conclude that centrifugation is the method of choice to study the proteome of TIF from cutaneous biopsies. ; We thank Rosa Ayala for analyzing already published TIF dataset and the members of the dermatology department of HUAV to collaborate for the sample collection. This work was funded by Catalan Government (2017-SGR-569). C Matas is recipient of an intramural grant from Lleida Institute for Biomedical Research-Dr. Pifarré Foundation supported by Diputació de Lleida. M. Guasch was supported by a predoctoral fellowship from UdL. The proteomics analyses were performed in the IDIBELL Clinical Proteomic unit that is part of the Proteored, PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF. This article is dedicated to Glòria Nadal Xifra and Narcís Matas Ros

Glioblastoma multiforme (GBM) is the most common and aggressive type of malignant glioma. Oncolytic adenoviruses are being modified to exploit the aberrant expression of proteins in tumor cells to increase the antiglioma efficacy. E1A mutant adenovirus Delta-24-RGD (DNX-2401) has shown a favorable toxicity profile and remarkable efficacy in a first-in-human phase I clinical trial. However, the comprehensive modulation of glioma metabolism in response to Delta-24-RGD infection is poorly understood. Integrating mass spectrometry based-quantitative proteomics, physical and functional interaction data, and biochemical approaches, we conducted a cell-wide study of cytosolic, nuclear, and secreted glioma proteomes throughout the early time course of Delta-24-RGD infection. In addition to the severe proteostasis impairment detected during the first hours post-infection (hpi), Delta-24-RGD induces a transient inhibition of signal transducer and activator of transcription 3 (STAT3), and transcription factor AP-1 (c-JUN) between 3 and 10hpi, increasing the nuclear factor kappa B (NF-κB) activity at 6hpi. Furthermore, Delta-24-RGD specifically modulates the activation dynamics of protein kinase C (PKC), extracellular signal–regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) pathways early in infection. At extracellular level, Delta-24-RGD triggers a time –dependent dynamic production of multitasking cytokines, and chemotactic factors, suggesting potential pleiotropic effects on the immune system reactivation. Taken together, these data help us to understand the mechanisms used by Delta-24-RGD to exploit glioma proteome organization. Further mining of this proteomic resource may enable design and engineering complementary adenoviral based-vectors to increase the specificity and potency against glioma. ; This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R To ES), the Department of Economic Development from Government of Navarra (ref. PC025, PC023-24, PC81-82, PI059 to ES and PI031 to JFI), the Instituto de Salud Carlos III and Fondos Feder Europeos (PI16/00066 to MMA), the Spanish Ministry of Science and Innovation (IEDI2015-00638 to MMA), the Department of Health of the Government of Navarra (to MMA), the Basque Foundation for Health Research (BIOEF, BIO13/CI/005 to MMA), Asociación Pablo Ugarte-Fuerza, Julen (to MMA). AGM was supported by PEJ-2014-A-61949 (MINECO).

APID (Agile Protein Interactomes DataServer) is an interactive web server that provides unified generation and delivery of protein interactomes mapped to their respective proteomes. This resource is a new, fully redesigned server that includes a comprehensive collection of protein interactomes for more than 400 organisms (25 of which include more than 500 interactions) produced by the integration of only experimentally validated protein-protein physical interactions. For each protein-protein interaction (PPI) the server includes currently reported information about its experimental validation to allow selection and filtering at different quality levels. As a whole, it provides easy access to the interactomes from specific species and includes a global uniform compendium of 90,379 distinct proteins and 678,441 singular interactions. APID integrates and unifies PPIs from major primary databases of molecular interactions, from other specific repositories and also from experimentally resolved 3D structures of protein complexes where more than two proteins were identified. For this purpose, a collection of 8,388 structures were analyzed to identify specific PPIs. APID also includes a new graph tool (based on Cytoscape.js) for visualization and interactive analyses of PPI networks. The server does not require registration and it is freely available for use at http://apid.dep.usal.es. ; Spanish Government, Ministerio de Economía y Competitividad, Instituto de Salud Carlos III [PI12/00624 and PI15/00328 to Dr J. De Las Rivas group]; and EU Joint Programme, JPND [AC14/00024 to Dr J. De Las Rivas group]. Regional Government, Junta de Castilla y León [BIO/SA68/13 to Dr J. De Las Rivas group]. ; Peer Reviewed

Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research. ; We thank Alexander Johnson for providing the C. albicans strains and plasmids and Judith Berman, Csaba Pál, and Dieter Söll for their useful comments and suggestions on the manuscript. The study was funded by the European Union Framework Program 7 (EUFP7) Sybaris Consortium Project 242220 and the Portuguese Science Foundation through Fundo Europeu de Desenvolvimento Regional (FEDER/FCT) Project PTDC/BIA-MIC/099826/2008. ; published

Accumulation of chemicals in aquatic ecosystems remains a concern, despite extensive legislation to control discharges. In this study, the impacts of in vivo exposures of juvenile sea bass (Dicentrarchus labrax) to the antidepressant fluoxetine (FLX), and to the natural estrogen 17βestradiol (E2) were investigated. A significant increase in E2 and FLX plasma levels confirmed the effectiveness of exposure by injection. Plasma estrogen-responsive parameters including calcium, phosphorus and vitellogenin were significantly altered in response to E2 but not FLX, while enzyme activities related to mineral turnover in scales were not affected. The scale proteome of fish exposed to E2 and FLX for 5 days revealed that 213 proteins had significantly altered levels compared to the control group, 31 of which were altered by both E2 and FLX. In vitro transactivation assays revealed antiestrogenic activities of FLX via nuclear estrogen receptors when combined with E2 and indicated one of the potential mechanisms through which FLX may interfere with scales. ; Supported by FCT through projects PTDC/AAGGLO/4003/2012, UID/Multi/04326/2019 and UID/NEU/04539/2019, researcher contracts under "Norma Transitória"-DL57/2016/CP1361/CT0015 to PP and DL57/2016/CP1361/CT0011 to LA and grant SFRH/BD/88419/2012 to CS, and by the Spanish Ministry of Science through grant AGL2015-67477-C2-1-R to AG.

Background : Microglial cells are resident macrophages of the central nervous system and important cellular mediators of the immune response and neuroinflammatory processes. In particular, microglial activation and communication between microglia, astrocytes, and neurons are hallmarks of the pathogenesis of several neurodegenerative diseases. Membrane proteins and their N-linked glycosylation mediate this microglial activation and regulate many biological process including signal transduction, cell-cell communication, and the immune response. Although membrane proteins and N-glycosylation represent a valuable source of drug target and biomarker discovery, the knowledge of their expressed proteome in microglia is very limited. Results : To generate a large-scale repository, we constructed a membrane proteome and N-glycoproteome from BV-2 mouse microglia using a novel integrated approach, comprising of crude membrane fractionation, multienzyme-digestion FASP, N-glyco-FASP, and various mass spectrometry. We identified 6928 proteins including 2850 membrane proteins and 1450 distinct N-glycosylation sites on 760 N-glycoproteins, of which 556 were considered novel N-glycosylation sites. Especially, a total of 114 CD antigens are identified via MS-based analysis in normal conditions of microglia for the first time. Our bioinformatics analysis provides a rich proteomic resource for examining microglial function in, for example, cell-to-cell communication and immune responses. Conclusions : Herein, we introduce a novel integrated proteomic approach for improved identification of membrane protein and N-glycosylation sites. To our knowledge, this workflow helped us to obtain the first and the largest membrane proteomic and N-glycoproteomic datesets for mouse microglia. Collectively, our proteomics and bioinformatics analysis significantly expands the knowledge of the membrane proteome and N-glycoproteome expressed in microglia within the brain and constitutes a foundation for ongoing proteomic studies and drug development for various neurological diseases. ; This work was supported by the Proteogenomic Research Program through the National Research Foundation of Korea and a National Research Foundation of Korea [NRF] grant (No. 2011–0030740), funded by the Korea government [MSIP]. This work was also supported by the Industrial Strategic Technology Development Program (#10045352), funded by the Ministry of Knowledge Economy (MKE, Korea). ; Peer Reviewed

The differential production of transcript isoforms from gene loci is a key cellular mechanism. Yet, its impact in protein production remains an open question. Here, we describe ORQAS (ORF quantification pipeline for alternative splicing), a pipeline for the translation quantification of individual transcript isoforms using ribosome-protected mRNA fragments (ribosome profiling). We find evidence of translation for 40-50% of the expressed isoforms in human and mouse, with 53% of the expressed genes having more than one translated isoform in human, and 33% in mouse. Differential splicing analysis revealed that about 40% of the splicing changes at RNA level are concordant with changes in translation. Furthermore, orthologous cassette exons between human and mouse preserve the directionality of the change, and are enriched in microexons in a comparison between glia and glioma. ORQAS leverages ribosome profiling to uncover a widespread and evolutionarily conserved impact of differential splicing on translation, particularly of microexon-containing isoforms. ; We acknowledge funding from the Spanish Government and FEDER with grants BFU2015-65235-P, BIO2017-85364-R, and MDM-2014-0370, and by Catalan Government (AGAUR) with grant SGR2017-1020. MR-S had funding from an FI grant from the Catalan Government with reference 2018FI_B1_00126 for part of this work.

This publication is an output of the Projects financed by the Spanish Ministry of Economy and Competitiveness (AGL2014-54995-P) and by the Government of the Principality of Asturias (FC-15-GRUPIN14-055). J.P., M.E, and L.V. were respectively supported by FPU (AP2010-5857, Ministry of Education, Spain), Severo Ochoa (BP11117, Government of the Principality of Asturias), and Juan de la Cierva (JCI-2012-12444; Spanish Ministry of Economy and Competitiveness) fellows. M.N. was financed by FWF Grant P 26342.