Proteomics Unit

Barcelona, Spain

Proteomics Unit

Barcelona, Spain
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Pellinen T.,University of Helsinki | Sanchez S.,Microscopy and Dynamic Imaging Unit | Sanchez S.,University of Concepción | Wang Y.,University of Helsinki | And 2 more authors.
Developmental Cell | Year: 2015

Nuclear membrane microdomains are proposed to act as platforms for regulation of nuclear function, but little is known about the mechanisms controlling their formation. Organization of the plasma membrane is regulated by actin polymerization, and the existence of an actin pool in the nucleus suggests that a similar mechanism might operate here. Weshow that nuclear membrane organization and morphology are regulated by the nuclear level of active Rac1 through actin polymerization-dependent mechanisms. Rac1 nuclear export is mediated by two internal nuclear export signals and through its interaction with nucleophosmin-1 (B23), which acts as a Rac1 chaperone inside the nucleus. Rac1 nuclear accumulation alters the balance between cytosolic Rac1 and Rho, increasing RhoA signaling in the cytoplasm and promoting a highly invasive phenotype. Nuclear Rac1 shuttling is a finely tuned mechanism for controlling nuclear shape and organization and cell invasiveness. © 2015 Elsevier Inc.

Goetz J.G.,CNRS Institute of Genetics and of Molecular and Cellular Biology | Samaniego R.,Hospital General Universitario Gregorio Maranon | Calvo E.,Proteomics Unit | Tello M.,CSIC - National Center of Microelectronics | And 5 more authors.
Cell | Year: 2011

Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment. PaperFlick: © 2011 Elsevier Inc.

Lopez-Contreras A.J.,Genomic Instability Group | Ruppen I.,Proteomics Unit | Nieto-Soler M.,Genomic Instability Group | Murga M.,Genomic Instability Group | And 8 more authors.
Cell Reports | Year: 2013

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance. © 2013 The Authors.

Lomsadze K.,Tbilisi State University | Salgado A.,CSIC - National Center for Metallurgical Research | Calvo E.,Proteomics Unit | Antonio Lopez J.,Proteomics Unit | Chankvetadze B.,Tbilisi State University
Electrophoresis | Year: 2011

In our recent studies, the reversal of the enantiomer migration order (EMO) was observed with heptakis (2,3-dimethyl-6-sulfo)-β-CD (HDMS-β-CD) when aqueous electrolyte was changed with nonaqueous electrolyte in CE. One-dimensional rotating frame nuclear Overhauser effect spectroscopy experiments prevailed that an inclusion complex was formed between the analyte and the chiral selector in the aqueous buffer, whereas an external complex resulted when a methanolic electrolyte was employed. In the case of the similarly substituted heptakis (2,3-diacetyl-6-sulfo)-β-CD (HDAS-β-CD), the external complex was observed in the aqueous buffer but an inclusion complex was formed in methanolic electrolyte. In contrast to heptakis (2,3-dimethyl-6-sulfo)-β-CD, no reversal of the enantiomer migration order was observed with HDAS-β-CD. In the present study, further mechanisms of enantioselective recognition and separation of propranolol enantiomers with HDAS-β-CD were investigated by using different techniques of nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. To the best of our knowledge, enantioselective nuclear Overhauser effect was observed for the first time in this study. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Altelaar A.F.M.,University Utrecht | Altelaar A.F.M.,Netherlands Proteomics Center | Munoz J.,University Utrecht | Munoz J.,Netherlands Proteomics Center | And 3 more authors.
Nature Reviews Genetics | Year: 2013

Next-generation sequencing allows the analysis of genomes, including those representing disease states. However, the causes of most disorders are multifactorial, and systems-level approaches, including the analysis of proteomes, are required for a more comprehensive understanding. The proteome is extremely multifaceted owing to splicing and protein modifications, and this is further amplified by the interconnectivity of proteins into complexes and signalling networks that are highly divergent in time and space. Proteome analysis heavily relies on mass spectrometry (MS). MS-based proteomics is starting to mature and to deliver through a combination of developments in instrumentation, sample preparation and computational analysis. Here we describe this emerging next generation of proteomics and highlight recent applications. © 2013 Macmillan Publishers Limited.

Wurm S.,Spanish National Cancer Research Center | Zhang J.,Qingdao University | Guinea-Viniegra J.,Spanish National Cancer Research Center | Garcia F.,Proteomics Unit | And 4 more authors.
Genes and Development | Year: 2015

Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation. © 2015 Wurm et al.

Munoz J.,Proteomics Unit | Heck A.J.R.,University Utrecht
Angewandte Chemie - International Edition | Year: 2014

A herculean task: Determining the human proteome sets the ultimate challenge in cell biology as it is thought to consist of more than 1000000 proteoforms in contrast to "only" 20000 protein-coding genes. Two teams of researchers have now proved the translation of 18000 proteins (and more than 27000 isoforms) by mass spectrometry. They obtained important information on the extent of protein translation and alternative splicing. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA Weinheim.

Schonthaler H.B.,BBVA Foundation CNIO Cancer Cell Biology Programme | Guinea-Viniegra J.,BBVA Foundation CNIO Cancer Cell Biology Programme | Wculek S.K.,BBVA Foundation CNIO Cancer Cell Biology Programme | Ruppen I.,Proteomics Unit | And 6 more authors.
Immunity | Year: 2013

Psoriasis is a common heterogeneous inflammatory skin disease with a complex pathophysiology and limited treatment options. Here we performed proteomic analyses of human psoriatic epidermis and found S100A8-S100A9, also called calprotectin, as the most upregulated proteins, followed by the complement component C3. Both S100A8-S100A9 and C3 are specifically expressed in lesional psoriatic skin. S100A9 is shown here to function as a chromatin component modulating C3 expression in mouse and human cells by binding to a region upstream of the C3 start site. When S100A9 was genetically deleted in mouse models of skin inflammation, the psoriasis-like skin disease and inflammation were strongly attenuated, with a mild immune infiltrate and decreased amounts of C3. In addition, inhibition of C3 in the mouse model strongly reduced the inflammatory skin disease. Thus, S100A8-S100A9 can regulate C3 at the nuclear level and present potential new therapeutic targets for psoriasis. © 2013 Elsevier Inc.

Chiva C.,Proteomics Unit | Chiva C.,University Pompeu Fabra | Sabido E.,Proteomics Unit | Sabido E.,University Pompeu Fabra
Journal of Proteomics | Year: 2014

Protein quantitation based on the generation of reporter ions from chemical labels is a widely used quantitative proteomics approach that enables measuring changes in protein abundance in response to biological perturbations. Isobaric labeling strategies at the MS2 level allow simultaneous measurements of different samples but it requires a fine-tuning of the collision energy used in HCD fragmentation to simultaneously obtain confident peptide identifications and highly sensitive and accurate quantitation. Although the recent development of dual CID/HCD fragmentation methods to circumvent these limitations, the fact is that many laboratories still use HCD-only methods for routine TMT protein quantitation experiments. Here, we have explored the effect of the collision energy on peptide identification and quantitation using HCD-only fragmentation methods on a linear ion trap/orbitrap mass spectrometer bearing an axial field HCD fragmentation cell. Our results using the HCD-only method show that a balance between the increase in the number of peptide identifications and the decrease in the precision of peptide quantitation is attained at a normalized collision energy of 40%. The HCD-only method at 40% does not only yield better results than those obtained using a higher collision energies, but it also outperforms the results obtained using the available CID/HCD dual method. Biological significance: In this work we have explored the effect of the collision energy on peptide identification and quantitation using HCD-only fragmentation methods on an Orbitrap Velos Pro mass spectrometer. Our results show that when using a HCD-only method, a balance between the number of peptide identifications and the precision of peptide quantitation is attained at a normalized collision energy (NCE) of 40%. This contrast with the parameters routinely used in many laboratories, which are set at NCE 45%. The single HCD method at 40% does not only yield better results than those obtained using a collision energy of 45% but it also outperforms the results obtained using the available CID/HCD dual method. Therefore, we suggest that the single HCD method using the optimal NCE of 40% can therefore become the method of choice in routinely TMT protein quantitation experiments. © 2013 Elsevier B.V.

Fernandez-Irigoyen J.,Proteomics Unit | Corrales F.J.,University of Navarra | Santamaria E.,Proteomics Unit
Journal of Proteomics | Year: 2012

The olfactory bulb (OB) is the first site for the processing of olfactory information in the brain and its deregulation is associated with neurodegenerative disorders. Although different efforts have been made to characterize the human brain proteome in depth, the protein composition of the human OB remains largely unexplored. We have performed a comprehensive analysis of the human OB proteome employing protein and peptide fractionation methods followed by LC-MS/MS, identifying 1529 protein species, corresponding to 1466 unique proteins, which represents a 7-fold increase in proteome coverage with respect to previous OB proteome descriptions from translational models. Bioinformatic analyses revealed that protein components of the OB participated in a plethora of biological process highlighting hydrolase and phosphatase activities and nucleotide and RNA binding activities. Interestingly, 631 OB proteins identified were not previously described in protein datasets derived from large-scale Human Brain Proteome Project (HBPP) studies. In particular, a subset of these differential proteins was mainly involved in axon guidance, opioid signaling, neurotransmitter receptor binding, and synaptic plasticity. Taken together, these results increase our knowledge about the molecular composition of the human OB and may be useful to understand the molecular basis of the olfactory system and the etiology of its disorders. © 2012 Elsevier B.V.

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