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Milano, Italy

Lopez-Pedrouso M.,University of Santiago de Compostela | Alonso J.,Proteomics Laboratory | Zapata C.,University of Santiago de Compostela
Plant Molecular Biology

Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50 % of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride-pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation. © 2013 Springer Science+Business Media Dordrecht. Source

Perotti D.,Molecular Bases of Genetic Risk and Genetic Testing Unit | Hohenstein P.,Roslin Institute | Bongarzone I.,Proteomics Laboratory | Maschietto M.,University College London | And 3 more authors.
Molecular Cancer Therapeutics

The European Network for Cancer Research in Children and Adolescents consortium organized a workshop in Rome, in June 2012, on "Biology-Driven Drug Development Renal Tumors Workshop" to discuss the current knowledge in pediatric renal cancers and to recommend directions for further research. Wilms tumor is the most common renal tumor of childhood and represents a success of pediatric oncology, with cure rates of more than 85% of cases. However, a substantial minority (~25%) responds poorly to current therapies and requires "high-risk" treatment or relapse. Moreover, the successfully treated majority are vulnerable to the late effects of treatment, with nearly one quarter reporting severe chronic health conditions by 25 years of follow-up. Main purposes of this meeting were to advance our understanding on the molecular drivers in Wilms tumor, their heterogeneity and interdependencies; to provide updates on the clinical-pathologic associations with biomarkers; to identify eligible populations for targeted drugs; and to model opportunities to use preclinical model systems and prioritize targeted agents for early phase clinical trials. At least three different pathways are involved in Wilms tumor; this review represents the outcome of the workshop discussion on the WNT/b-catenin pathway in Wilms tumorigenesis. © 2013 AACR. Source

De La Fuente M.,Mision Biologica de Galicia CSIC | Borrajo A.,University of Santiago de Compostela | Bermudez J.,University of Santiago de Compostela | Lores M.,University of Santiago de Compostela | And 6 more authors.
Journal of Proteomics

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption. Proteomic studies in legumes have increased significantly in the last years but few studies have been performed to date in P. vulgaris. We report here a proteomic analysis of bean seeds by two-dimensional electrophoresis (2-DE). Three different protein extraction methods (TCA-acetone, phenol and the commercial clean-up kit) were used taking into account that the extractome can have a determinant impact on the level of quality of downstream protein separation and identification. To demonstrate the quality of the 2-DE analysis, a selection of 50 gel spots was used in protein identification by mass spectrometry (MALDI-TOF MS and MALDI-TOF/TOF). The results showed that a considerable proportion of spots (70%) were identified in spite of incomplete genome/protein databases for bean and other legume species. Most identified proteins corresponded to storage protein, carbohydrate metabolism, defense and stress response, including proteins highly abundant in the seed of P. vulgaris such as the phaseolin, the phytohemagglutinin and the lectin-related α-amylase inhibitor. © 2010 Elsevier B.V. Source

Bongarzone I.,Proteomics Laboratory
Seminars in Cancer Biology

Sequencing data show that both specific genes and a number of signaling pathways are recurrently mutated in various types of lymphoma. DNA sequencing analyses of lymphoma have identified several aberrations that might affect the interaction between malignant cells and the tumor microenvironment. Microenvironmental functions are essential to lymphoma; they provide survival and proliferation signals and license immune evasion. It is plausible that interventions that aim to destroy tumor-microenvironment interactions may improve responses to therapeutics. Accordingly, the identification of extrinsic factors and their downstream intracellular signaling targets has led to much progress in understanding tumor-microenvironment interactions. Lymphoma cells are differently influenced by cells' interactions with components of their microenvironment; these cell extrinsic factors include soluble and immobilized factors, the extracellular matrix, and signals presented by neighboring cells. Soluble factors, which are often cell-secreted autocrine and paracrine factors, comprise a significant fraction of targetable molecules. To begin to understand how intercellular communication is conducted in lymphoma, a first order of study is deciphering the soluble factors secreted by malignant cells and microenvironmental cells. These soluble factors are shed into the interstitial fluid in lymphoma and can be conveniently explored using mass spectrometry. Protein components can be detected and quantified, thus enabling the routine navigation of the soluble part of the microenvironment. Elucidating functional and signaling states affords a new paradigm for understanding cancer biology and devising new therapies. This review summarizes knowledge in this field and discusses the utility of studying tumor-secreted factors. © 2015 Elsevier Ltd. Source

Mato A.,University of Santiago de Compostela | Salgado F.J.,University of Santiago de Compostela | Lopez-Pedrouso M.,University of Santiago de Compostela | Carrera M.,CSIC - Institute of Marine Research | And 4 more authors.
Journal of Proteomics

Pre-slaughter stress has adverse effects on meat quality that can lead to the occurrence of Dark Firm Dry (DFD) meat in cattle. This study explores the previously uncharacterized proteome changes linked to pre-slaughter stress in the longissimus thoracis (LT) bovine muscle. Differential proteome profiles of DFD and normal (non-DFD) LT meat samples from male calves of the Rubia Gallega breed were assessed by 2-DE coupled to MS analysis (LC-MS/MS and MALDI TOF/TOF MS). A total of seven structural-contractile proteins (three different myosin light chain isoforms, two fast skeletal myosin light chain 2 isoforms, troponin C type 2 and cofilin-2) and three metabolism enzymes (triosephosphate isomerase, ATP synthase and beta-galactoside alpha-2,6-sialyltransferase) were found to have statistically significant differential abundance in sample groups. In addition, 2-DE in combination with the phosphoprotein-specific fluorescent dye Pro-Q DPS revealed that highly phosphorylated fast skeletal myosin regulatory light chain 2 isoforms underwent the most intense relative change in muscle conversion to DFD meat. Therefore, they appear to be the most sensitive biomarkers of stress just prior to slaughter in Rubia Gallega. Overall, these findings will facilitate a more integrative understanding of the biochemical processes associated with stress in cattle muscle and their effects in meat quality. Biological significance: Pre-slaughter stress is a crucial factor in meat production. Animals destined for slaughter are stressed by a variety of endogenous and exogenous factors that negatively affect the complex post-mortem biochemical events underlying the conversion of muscle into meat. The study of the muscle proteome has a great relevance for understanding the molecular mechanisms associated with stress. However, there is no information available on the molecular changes linked to pre-slaughter stress in cattle on the proteome scale. Our study led to the identification of a number of candidate proteins associated with the response to pre-slaughter stress in the LT bovine muscle of the Rubia Gallega breed. The functions of those significantly changed proteins have a clear biological relationship with stress response. These findings contribute to a deeper insight into the molecular pathways that respond to stress in cattle. © 2015 Elsevier B.V. Source

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