Rosana Navajas

Spanish National Research Council, Hispalis, Andalusia, Spain

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Publications (10)52.84 Total impact

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    ABSTRACT: T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that β-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the β-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of β-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.
    The EMBO Journal 02/2014; · 9.82 Impact Factor
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    ABSTRACT: Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labelled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM). This article is part of a Special Issue entitled: Standardization and Quality Control. Significance The HUPO Proteomics Standards Initiative (HUPO-PSI) has invested considerable efforts to improve the standardization of proteomics data handling, representation and sharing through the development of data standards, reporting guidelines, controlled vocabularies and tooling. In this manuscript, we describe a key output from the HUPO-PSI - namely the MIAPE Quant guidelines, which have developed in parallel with the corresponding data exchange format mzQuantML [1]. The MIAPE Quant guidelines describe the HUPO-PSI proposal concerning the minimum information to be reported when a quantitative data set, derived from mass spectrometry (MS), is submitted to a database or as supplementary information to a journal. The guidelines have been developed with input from a broad spectrum of stakeholders in the proteomics field to represent a true consensus view of the most important data types and metadata, required for a quantitative experiment to be analysed critically or a data analysis pipeline to be reproduced. It is anticipated that they will influence or be directly adopted as part of journal guidelines for publication and by public proteomics databases and thus may have an impact on proteomics laboratories across the world.
    Journal of proteomics 03/2013; · 5.07 Impact Factor
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    ABSTRACT: Deficiency in the translocase complex (SecG mutant strain) or in the major type I signal peptidase (SipY mutant strain) function in Streptomyces lividans resulted, as expected, in a drastic reduction of secretory protein production and in a bald phenotype. The transcriptional profiling of both strains showed that the expression of a set of genes involved in the morphological differentiation process was down regulated in both mutant strains (bldG, bldN and bldM), whereas bldA and bldH were only down-regulated in the SipY mutant strain. Consistently, low temperature scanning electron microscopy revealed that the disruption of sipY had a more noticeable effect in the growth/morphological aspect of the mycelium than that of secG, suggesting that in the sipY mutant, the blockage of the export process might have more severe consequences than in the secG mutant. In both cases, the likely degradation of the proteins that cannot be secreted might provide nutrients that might be responsible for the lack of induction of the bald cascade, which is thought to be triggered under conditions of nutritional limitation.
    Journal of Biotechnology 04/2012; 160(3-4):112-22. · 3.18 Impact Factor
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    ABSTRACT: The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. First, it can verify that the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Second, the toolkit can convert several XML-based data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing, or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at http://www.proteored.org/MIAPE/.
    Molecular &amp Cellular Proteomics 10/2011; 10(10):M111.008334. · 7.25 Impact Factor
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    ABSTRACT: The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope stress. Signal transmission occurs from the sensor RcsC to the transcriptional regulator RcsB. Accessory proteins, such as IgaA, are known to adjust the response level. In a previous transcriptomic study, we uncovered 85 genes differentially expressed in Salmonella enterica serovar Typhimurium igaA mutants. Here, we extended these observations to proteomics by performing differential isotope-coded protein labeling (ICPL) followed by liquid chromatography-electrospray ionization tandem mass spectrometry. Five-hundred five proteins were identified and quantified, with 75 of them displaying significant changes in response to alterations in the RcsCDB system. Divergent expression at the RNA and protein level was observed for the metabolic genes pckA and metE, involved in gluconeogenesis and methionine synthesis, respectively. When analyzed in diverse environmental conditions, including the intracellular niche of eukaryotic cells, inverse regulation was more evident for metE and in bacteria growing in defined minimal medium or to stationary phase. The RcsCDB system was also shown to repress the synthesis of the small RNA FnrS, previously reported to modulate metE expression. Collectively, these findings provide new insights into post-transcriptional regulatory mechanisms involving the RcsCDB system and its control over metabolic functions.
    Journal of Proteome Research 06/2011; 10(8):3386-98. · 5.06 Impact Factor
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    ABSTRACT: The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. Firstly, it can verify the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Secondly, the toolkit can convert several XML-based data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at http://www.proteored.org/MIAPE/.
    Molecular &amp Cellular Proteomics 06/2011; · 7.25 Impact Factor
  • Haemophilia 02/2011; 17(4):709-10. · 3.17 Impact Factor
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    ABSTRACT: Major difficulties in phosphoprotein analysis relate to the presence of a huge number of nonphosphorylated proteins and to the wide concentration dynamic range among them. In order to overcome the analysis complexity, specific clean-up and highly efficient enrichment procedures are mandatory prior to the -chromatographic separation and identification by tandem mass spectrometry. In this chapter, a procedure based on immobilized metal affinity chromatography (IMAC)/reversed-phase phosphopeptide purification and analysis by nanoHPLC-ESI-MS/MS with ion trap is described in detail. CID (collision-induced -dissociation) and ETD (electron-transfer dissociation) fragmentation techniques are used in combination to specifically determine phosphorylation sites inside the peptide sequences, through the analysis of MS/MS spectra.
    Methods in molecular biology (Clifton, N.J.) 01/2011; 681:337-48. · 1.29 Impact Factor
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    ABSTRACT: An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE "classical" approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification.
    Talanta 02/2010; 80(4):1496-502. · 3.50 Impact Factor
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    ABSTRACT: Tandem mass spectrometry-based proteomics is currently in great demand of computational methods that facilitate the elimination of likely false positives in peptide and protein identification. In the last few years, a number of new peptide identification programs have been described, but scores or other significance measures reported by these programs cannot always be directly translated into an easy to interpret error rate measurement such as the false discovery rate. In this work we used generalized lambda distributions to model frequency distributions of database search scores computed by MASCOT, X!TANDEM with k-score plug-in, OMSSA, and InsPecT. From these distributions, we could successfully estimate p values and false discovery rates with high accuracy. From the set of peptide assignments reported by any of these engines, we also defined a generic protein scoring scheme that enabled accurate estimation of protein-level p values by simulation of random score distributions that was also found to yield good estimates of protein-level false discovery rate. The performance of these methods was evaluated by searching four freely available data sets ranging from 40,000 to 285,000 MS/MS spectra.
    Molecular &amp Cellular Proteomics 06/2008; 7(9):1748-54. · 7.25 Impact Factor