41 results on '"Bech-Serra JJ"'
Search Results
2. RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells in DMD
- Author
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Jordi-Díaz-Manera, Xavier Suárez-Calvet, Patricia Piñol-Jurado, Eduard Gallardo, Bech Serra Jj, E. Fernández-Simón, S. López-Fernández, Ana Carrasco-Rozas, de la Torre C, and de Luna N
- Subjects
musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,RHOA ,biology ,Chemistry ,Duchenne muscular dystrophy ,Fasudil ,medicine.disease ,Hedgehog signaling pathway ,Cell biology ,Precursor cell ,biology.protein ,medicine ,ROCK2 ,Dystrophin ,Platelet-derived growth factor receptor - Abstract
The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) leads to muscle necrosis and replacement of muscle tissue by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been previously studied, the pathways that are activated by PDGF-AA in muscular dystrophies have not been described so far. Herein we report the effects of PDGF-AA on the fibrotic process in muscular dystrophies by performing a quantitative proteomic study in DMD isolated fibro-adipogenic precursor cells (FAPs) treated with PDGF-AA. In vitro studies showed that RhoA/ROCK2 pathway is activated by PDGF-AA and induces the activation of FAPs. The inhibition of RhoA/ROCK signalling pathway by C3-exoenzyme or fasudil attenuated the effects of PDGF-AA. The blocking effects of RhoA/ROCK pathway were analysed in the dba/2J-mdx murine model with fasudil. Grip strength test showed an improvement in the muscle function and histological studies demonstrated reduction of the fibrotic area. Our results suggest that blockade of RhoA/ROCK could attenuate the activation of FAPs and could be considered a potential therapeutic approach for muscular dystrophies. more...
- Published
- 2021
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Catalog
3. Antitumor Activity of the Novel BTK Inhibitor TG-1701 Is Associated with Disruption of Ikaros Signaling in Patients with B-cell Non-Hodgkin Lymphoma
- Author
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Ribeiro, ML, Reyes-Garau, D, Vinyoles, M, Peleja, NP, Santos, JC, Armengol, M, Fernandez-Serrano, M, Mor, AS, Bech-Serra, JJ, Blecua, P, Musulen, E, De La Torre, C, Miskin, H, Esteller, M, Bosch, F, Menendez, P, Normant, E, and Roue, G more...
- Abstract
Purpose: Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non-Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of phosphoproteomic profiling for the early identification of BTKi responders remains underexplored. Experimental Design: A set of six clinical samples from an ongoing phase I trial dosing patients with chronic lymphocytic leukemia (CLL) with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA sequencing (RNA-seq) analysis. The activity of TG-1701 was evaluated in a panel of 11 B-NHL cell lines and mouse xenografts, including two NF-kappa B-and BTKC481S-driven BTKi-resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, Western blot analysis, immunostaining, and gene knockout (KO) experiments. Results: A nonsupervised, phosphoproteomic-based clustering did match the early clinical outcomes of patients with CLL and separated a group of "early-responders" from a group of "late-responders." This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including YES1 and MYC, in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug in vitro and in vivo in late-responder patients and in BTKC481S, BTKKO, and noncanonical NF-kappa B models. Conclusions: These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action. more...
- Published
- 2021
4. Disulfide driven folding for a conditionally disordered protein
- Author
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Fraga, H, Pujols, J, Gil-Garcia, M, Roque, A, Bernardo-Seisdedos, G, Santambrogio, C, Bech-Serra, J, Canals, F, Bernadó, P, Grandori, R, Millet, O, Ventura, S, Bech-Serra, JJ, Fraga, H, Pujols, J, Gil-Garcia, M, Roque, A, Bernardo-Seisdedos, G, Santambrogio, C, Bech-Serra, J, Canals, F, Bernadó, P, Grandori, R, Millet, O, Ventura, S, and Bech-Serra, JJ more...
- Abstract
Conditionally disordered proteins are either ordered or disordered depending on the environmental context. The substrates of the mitochondrial intermembrane space (IMS) oxidoreductase Mia40 are synthesized on cytosolic ribosomes and diffuse as intrinsically disordered proteins to the IMS, where they fold into their functional conformations; behaving thus as conditionally disordered proteins. It is not clear how the sequences of these polypeptides encode at the same time for their ability to adopt a folded structure and to remain unfolded. Here we characterize the disorder-to-order transition of a Mia40 substrate, the human small copper chaperone Cox17. Using an integrated real-time approach, including chromatography, fluorescence, CD, FTIR, SAXS, NMR, and MS analysis, we demonstrate that in this mitochondrial protein, the conformational switch between disordered and folded states is controlled by the formation of a single disulfide bond, both in the presence and in the absence of Mia40. We provide molecular details on how the folding of a conditionally disordered protein is tightly regulated in time and space, in such a way that the same sequence is competent for protein translocation and activity. more...
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- 2017
5. Proteomics of circulating extracellular vesicles reveals diverse clinical presentations of COVID-19 but fails to identify viral peptides.
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Gualdrón-López M, Ayllon-Hermida A, Cortes-Serra N, Resa-Infante P, Bech-Serra JJ, Aparici-Herraiz I, Nicolau-Fernandez M, Erkizia I, Gutierrez-Chamorro L, Marfil S, Pradenas E, Ávila Nieto C, Cucurull B, Montaner-Tarbés S, Muelas M, Sotil R, Ballana E, Urrea V, Fraile L, Montoya M, Vergara J, Segales J, Carrillo J, Izquierdo-Useros N, Blanco J, Fernandez-Becerra C, de La Torre C, Pinazo MJ, Martinez-Picado J, and Del Portillo HA more...
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- Animals, Humans, Male, Antibodies, Neutralizing blood, Antibodies, Neutralizing immunology, Female, Peptides metabolism, Antibodies, Viral blood, Mesocricetus, Adult, Middle Aged, Cricetinae, Viral Proteins metabolism, Extracellular Vesicles metabolism, COVID-19 virology, COVID-19 immunology, Proteomics, SARS-CoV-2
- Abstract
Extracellular vesicles (EVs) released by virus-infected cells have the potential to encapsulate viral peptides, a characteristic that could facilitate vaccine development. Furthermore, plasma-derived EVs may elucidate pathological changes occurring in distal tissues during viral infections. We hypothesized that molecular characterization of EVs isolated from COVID-19 patients would reveal peptides suitable for vaccine development. Blood samples were collected from three cohorts: severe COVID-19 patients (G1), mild/asymptomatic cases (G2), and SARS-CoV-2-negative healthcare workers (G3). Samples were obtained at two time points: during the initial phase of the pandemic in early 2020 (m0) and eight months later (m8). Clinical data analysis revealed elevated inflammatory markers in G1. Notably, non-vaccinated individuals in G1 exhibited increased levels of neutralizing antibodies at m8, suggesting prolonged exposure to viral antigens. Proteomic profiling of EVs was performed using three distinct methods: immunocapture (targeting CD9), ganglioside-capture (utilizing Siglec-1) and size-exclusion chromatography (SEC). Contrary to our hypothesis, this analysis failed to identify viral peptides. These findings were subsequently validated through Western blot analysis targeting the RBD of the SARS-CoV-2 Spike protein's and comparative studies using samples from experimentally infected Syrian hamsters. Furthermore, analysis of the EV cargo revealed a diverse molecular profile, including components involved in the regulation of viral replication, systemic inflammation, antigen presentation, and stress responses. These findings underscore the potential significance of EVs in the pathogenesis and progression of COVID-19., Competing Interests: HP, MMo, and LF are shareholders of Innovex Therapeutics. SM-T was a former employee of Innovex Therapeutics. JM-P has received institutional grants and educational/consultancy fees from AbiVax; AstraZeneca; Gilead Sciences; Grifols; Janssen; Merck Sharp & Dohme; and ViiV Healthcare; all outside the submitted work. JC and JB are shareholders of Albajuna Therapeutics SL, NI-U reports institutional grants from Grifols, Dentaid, Hipra and Amassence. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gualdrón-López, Ayllon-Hermida, Cortes-Serra, Resa-Infante, Bech-Serra, Aparici-Herraiz, Nicolau-Fernandez, Erkizia, Gutierrez-Chamorro, Marfil, Pradenas, Ávila Nieto, Cucurull, Montaner-Tarbés, Muelas, Sotil, Ballana, Urrea, Fraile, Montoya, Vergara, Segales, Carrillo, Izquierdo-Useros, Blanco, Fernandez-Becerra, de La Torre, Pinazo, Martinez-Picado and del Portillo.) more...
- Published
- 2024
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6. TOR complex 1 negatively regulates NDR kinase Cbk1 to control cell separation in budding yeast.
- Author
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Foltman M, Mendez I, Bech-Serra JJ, de la Torre C, Brace JL, Weiss EL, Lucas M, Queralt E, and Sanchez-Diaz A
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- Phosphorylation, Anaphase, Cell Separation, Mechanistic Target of Rapamycin Complex 1, Saccharomycetales
- Abstract
The target of rapamycin (TOR) signalling pathway plays a key role in the coordination between cellular growth and the cell cycle machinery in eukaryotes. The underlying molecular mechanisms by which TOR might regulate events after anaphase remain unknown. We show for the first time that one of the 2 TOR complexes in budding yeast, TORC1, blocks the separation of cells following cytokinesis by phosphorylation of a member of the NDR (nuclear Dbf2-related) protein-kinase family, the protein Cbk1. We observe that TORC1 alters the phosphorylation pattern of Cbk1 and we identify a residue within Cbk1 activation loop, T574, for which a phosphomimetic substitution makes Cbk1 catalytically inactive and, indeed, reproduces TORC1 control over cell separation. In addition, we identify the exocyst component Sec3 as a key substrate of Cbk1, since Sec3 activates the SNARE complex to promote membrane fusion. TORC1 activity ultimately compromises the interaction between Sec3 and a t-SNARE component. Our data indicate that TORC1 negatively regulates cell separation in budding yeast by participating in Cbk1 phosphorylation, which in turn controls the fusion of secretory vesicles transporting hydrolase at the site of division., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Foltman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.) more...
- Published
- 2023
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7. SIRT1 regulates DNA damage signaling through the PP4 phosphatase complex.
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Rasti G, Becker M, Vazquez BN, Espinosa-Alcantud M, Fernández-Duran I, Gámez-García A, Ianni A, Gonzalez J, Bosch-Presegué L, Marazuela-Duque A, Guitart-Solanes A, Segura-Bayona S, Bech-Serra JJ, Scher M, Serrano L, Shankavaram U, Erdjument-Bromage H, Tempst P, Reinberg D, Olivella M, Stracker TH, de la Torre C, and Vaquero A more...
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- Animals, Humans, Mammals metabolism, Phosphoric Monoester Hydrolases, Phosphorylation, Signal Transduction, DNA Damage, Sirtuin 1 metabolism
- Abstract
The Sirtuin family of NAD+-dependent enzymes plays an important role in maintaining genome stability upon stress. Several mammalian Sirtuins have been linked directly or indirectly to the regulation of DNA damage during replication through Homologous recombination (HR). The role of one of them, SIRT1, is intriguing as it seems to have a general regulatory role in the DNA damage response (DDR) that has not yet been addressed. SIRT1-deficient cells show impaired DDR reflected in a decrease in repair capacity, increased genome instability and decreased levels of γH2AX. Here we unveil a close functional antagonism between SIRT1 and the PP4 phosphatase multiprotein complex in the regulation of the DDR. Upon DNA damage, SIRT1 interacts specifically with the catalytical subunit PP4c and promotes its inhibition by deacetylating the WH1 domain of the regulatory subunits PP4R3α/β. This in turn regulates γH2AX and RPA2 phosphorylation, two key events in the signaling of DNA damage and repair by HR. We propose a mechanism whereby during stress, SIRT1 signaling ensures a global control of DNA damage signaling through PP4., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.) more...
- Published
- 2023
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8. Biomarkers Found in the Tumor Interstitial Fluid may Help Explain the Differential Behavior Among Keratinocyte Carcinomas.
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Matas-Nadal C, Bech-Serra JJ, Gatius S, Gomez X, Ribes-Santolaria M, Guasch-Vallés M, Pedraza N, Casanova JM, de la Torre Gómez C, Garí E, and Aguayo-Ortiz RS
- Subjects
- Humans, Extracellular Fluid metabolism, NF-kappa B metabolism, Proteomics, Keratinocytes metabolism, Biomarkers, Tumor metabolism, Tumor Microenvironment, Skin Neoplasms metabolism, Carcinoma, Basal Cell metabolism, Carcinoma, Basal Cell pathology, Carcinoma, Squamous Cell metabolism
- Abstract
Basal cell carcinomas (BCCs) and cutaneous squamous cell carcinomas (SCCs) are the most frequent types of cancer, and both originate from the keratinocyte transformation, giving rise to the group of tumors called keratinocyte carcinomas (KCs). The invasive behavior is different in each group of KC and may be influenced by their tumor microenvironment. The principal aim of the study is to characterize the protein profile of the tumor interstitial fluid (TIF) of KC to evaluate changes in the microenvironment that could be associated with their different invasive and metastatic capabilities. We obtained TIF from 27 skin biopsies and conducted a label-free quantitative proteomic analysis comparing seven BCCs, 16 SCCs, and four normal skins. A total of 2945 proteins were identified, 511 of them quantified in more than half of the samples of each tumoral type. The proteomic analysis revealed differentially expressed TIF proteins that could explain the different metastatic behavior in both KCs. In detail, the SCC samples disclosed an enrichment of proteins related to cytoskeleton, such as Stratafin and Ladinin-1. Previous studies found their upregulation positively correlated with tumor progression. Furthermore, the TIF of SCC samples was enriched with the cytokines S100A8/S100A9. These cytokines influence the metastatic output in other tumors through the activation of NF-kB signaling. According to this, we observed a significant increase in nuclear NF-kB subunit p65 in SCCs but not in BCCs. In addition, the TIF of both tumors was enriched with proteins involved in the immune response, highlighting the relevance of this process in the composition of the tumor environment. Thus, the comparison of the TIF composition of both KCs provides the discovery of a new set of differential biomarkers. Among them, secreted cytokines such as S100A9 may help explain the higher aggressiveness of SCCs, while Cornulin is a specific biomarker for BCCs. Finally, the proteomic landscape of TIF provides key information on tumor growth and metastasis, which can contribute to the identification of clinically applicable biomarkers that may be used in the diagnosis of KC, as well as therapeutic targets., Competing Interests: Conflict of interest The authors declare no conflict of interest to disclosure., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.) more...
- Published
- 2023
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9. Epigenetic inactivation of the 5-methylcytosine RNA methyltransferase NSUN7 is associated with clinical outcome and therapeutic vulnerability in liver cancer.
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Ortiz-Barahona V, Soler M, Davalos V, García-Prieto CA, Janin M, Setien F, Fernández-Rebollo I, Bech-Serra JJ, De La Torre C, Guil S, Villanueva A, Zhang PH, Yang L, Guarnacci M, Schumann U, Preiss T, Balaseviciute U, Montal R, Llovet JM, and Esteller M more...
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- Humans, 5-Methylcytosine, CpG Islands, DNA Methylation, Epigenesis, Genetic, Gene Expression Regulation, Neoplastic, Proteomics, RNA, Messenger metabolism, RNA-Binding Proteins metabolism, Transcription Factors genetics, Liver Neoplasms genetics, Methyltransferases genetics, Methyltransferases metabolism
- Abstract
Background: RNA modifications are important regulators of transcript activity and an increasingly emerging body of data suggests that the epitranscriptome and its associated enzymes are altered in human tumors., Methods: Combining data mining and conventional experimental procedures, NSUN7 methylation and expression status was assessed in liver cancer cell lines and primary tumors. Loss-of-function and transfection-mediated recovery experiments coupled with RNA bisulfite sequencing and proteomics determined the activity of NSUN7 in downstream targets and drug sensitivity., Results: In this study, the initial screening for genetic and epigenetic defects of 5-methylcytosine RNA methyltransferases in transformed cell lines, identified that the NOL1/NOP2/Sun domain family member 7 (NSUN7) undergoes promoter CpG island hypermethylation-associated with transcriptional silencing in a cancer-specific manner. NSUN7 epigenetic inactivation was common in liver malignant cells and we coupled bisulfite conversion of cellular RNA with next-generation sequencing (bsRNA-seq) to find the RNA targets of this poorly characterized putative RNA methyltransferase. Using knock-out and restoration-of-function models, we observed that the mRNA of the coiled-coil domain containing 9B (CCDC9B) gene required NSUN7-mediated methylation for transcript stability. Most importantly, proteomic analyses determined that CCDC9B loss impaired protein levels of its partner, the MYC-regulator Influenza Virus NS1A Binding Protein (IVNS1ABP), creating sensitivity to bromodomain inhibitors in liver cancer cells exhibiting NSUN7 epigenetic silencing. The DNA methylation-associated loss of NSUN7 was also observed in primary liver tumors where it was associated with poor overall survival. Interestingly, NSUN7 unmethylated status was enriched in the immune active subclass of liver tumors., Conclusion: The 5-methylcytosine RNA methyltransferase NSUN7 undergoes epigenetic inactivation in liver cancer that prevents correct mRNA methylation. Furthermore, NSUN7 DNA methylation-associated silencing is associated with clinical outcome and distinct therapeutic vulnerability., (© 2023. The Author(s).) more...
- Published
- 2023
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10. RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells: implications for Duchenne muscular dystrophy.
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Fernández-Simón E, Suárez-Calvet X, Carrasco-Rozas A, Piñol-Jurado P, López-Fernández S, Pons G, Bech Serra JJ, de la Torre C, de Luna N, Gallardo E, and Díaz-Manera J
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- Animals, Humans, Mice, Mice, Inbred mdx, Muscle, Skeletal pathology, Platelet-Derived Growth Factor, Proteomics, Rho Guanine Nucleotide Exchange Factors metabolism, Stem Cells metabolism, Stem Cells pathology, rho-Associated Kinases metabolism, rho-Associated Kinases therapeutic use, rhoA GTP-Binding Protein metabolism, rhoA GTP-Binding Protein therapeutic use, Muscular Dystrophy, Duchenne pathology
- Abstract
Background: The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and replacement by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been studied, pathways activated by PDGF-AA have not been described so far. Our aim was to study the molecular role of PDGF-AA in the fibrotic process of DMD., Methods: Skeletal muscle fibro-adipogenic progenitor cells (FAPs) from three DMD treated with PDGF-AA at 50 ng/mL were analysed by quantitative mass spectrometry-based proteomics. Western-blot, immunofluorescence, and G-LISA were used to confirm the mass spectrometry results. We evaluated the effects of PDGF-AA on the activation of RhoA pathway using two inhibitors, C3-exoenzyme and fasudil. Cell proliferation and migration were determined by BrdU and migration assay. Actin reorganization and collagen synthesis were measured by phalloidin staining and Sircol assay, respectively. In an in vivo proof of concept study, we treated dba/2J-mdx mice with fasudil for 6 weeks. Muscle strength was assessed with the grip strength. Immunofluorescence and flow cytometry analyses were used to study fibrotic and inflammatory markers in muscle tissue., Results: Mass spectrometry revealed that RhoA pathway proteins were up-regulated in treated compared with non-treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expression was significantly increased in DMD muscles compared with healthy controls by a 7.7-fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF-AA (n = 3, 1.88-fold increase, P < 0.01) and both C3-exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF-AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3-exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, 1.76-fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen-I expression area (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, P < 0.01)., Conclusions: Our results suggest that PDGF-AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.) more...
- Published
- 2022
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11. Antitumor Activity of the Novel BTK Inhibitor TG-1701 Is Associated with Disruption of Ikaros Signaling in Patients with B-cell Non-Hodgkin Lymphoma.
- Author
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Ribeiro ML, Reyes-Garau D, Vinyoles M, Profitós Pelejà N, Santos JC, Armengol M, Fernández-Serrano M, Sedó Mor A, Bech-Serra JJ, Blecua P, Musulen E, De La Torre C, Miskin H, Esteller M, Bosch F, Menéndez P, Normant E, and Roué G more...
- Subjects
- Agammaglobulinaemia Tyrosine Kinase, Animals, Humans, Mice, Piperidines therapeutic use, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Pyrazoles pharmacology, Pyrimidines pharmacology, Signal Transduction, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Lymphoma, Non-Hodgkin drug therapy
- Abstract
Purpose: Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non-Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of phosphoproteomic profiling for the early identification of BTKi responders remains underexplored., Experimental Design: A set of six clinical samples from an ongoing phase I trial dosing patients with chronic lymphocytic leukemia (CLL) with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA sequencing (RNA-seq) analysis. The activity of TG-1701 was evaluated in a panel of 11 B-NHL cell lines and mouse xenografts, including two NF-κB- and BTK
C481S -driven BTKi-resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, Western blot analysis, immunostaining, and gene knockout (KO) experiments., Results: A nonsupervised, phosphoproteomic-based clustering did match the early clinical outcomes of patients with CLL and separated a group of "early-responders" from a group of "late-responders." This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including YES1 and MYC , in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug in vitro and in vivo in late-responder patients and in BTKC481S , BTKKO , and noncanonical NF-κB models., Conclusions: These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action., (©2021 The Authors; Published by the American Association for Cancer Research.) more...- Published
- 2021
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12. Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis.
- Author
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Diesch J, Le Pannérer MM, Winkler R, Casquero R, Muhar M, van der Garde M, Maher M, Herráez CM, Bech-Serra JJ, Fellner M, Rathert P, Brooks N, Zamora L, Gentilella A, de la Torre C, Zuber J, Götze KS, and Buschbeck M more...
- Subjects
- Antimetabolites, Antineoplastic pharmacology, Cell Line, Tumor, DNA Methylation drug effects, Humans, Leukemia, Myelomonocytic, Acute, Azacitidine pharmacology, CREB-Binding Protein antagonists & inhibitors, CREB-Binding Protein genetics, Protein Biosynthesis drug effects, RNA metabolism
- Abstract
The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis., (© 2021. The Author(s).) more...
- Published
- 2021
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13. Evaluation of Tumor Interstitial Fluid-Extraction Methods for Proteome Analysis: Comparison of Biopsy Elution versus Centrifugation.
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Matas-Nadal C, Bech-Serra JJ, Guasch-Vallés M, Fernández-Armenteros JM, Barceló C, Casanova JM, de la Torre Gómez C, Aguayo Ortiz R, and Garí E
- Subjects
- Biopsy, Centrifugation, Extracellular Fluid, Humans, Proteome, Proteomics, Carcinoma, Squamous Cell diagnosis, Skin Neoplasms
- Abstract
The analysis of tumor interstitial fluid (TIF) composition is a valuable procedure to identify antimetastatic 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 (10 000 g ) was required to obtain TIF from cSCC by centrifugation. Second, 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 there exists an elevated consistence of TIF composition independently of the method used. However, we observed a 3-fold increase of extracellular proteins in nonoverlapped proteome obtained by centrifugation. We therefore conclude that centrifugation is the method of choice to study the proteome of TIF from cutaneous biopsies. more...
- Published
- 2020
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14. Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.
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Janin M, Ortiz-Barahona V, de Moura MC, Martínez-Cardús A, Llinàs-Arias P, Soler M, Nachmani D, Pelletier J, Schumann U, Calleja-Cervantes ME, Moran S, Guil S, Bueno-Costa A, Piñeyro D, Perez-Salvia M, Rosselló-Tortella M, Piqué L, Bech-Serra JJ, De La Torre C, Vidal A, Martínez-Iniesta M, Martín-Tejera JF, Villanueva A, Arias A, Cuartas I, Aransay AM, La Madrid AM, Carcaboso AM, Santa-Maria V, Mora J, Fernandez AF, Fraga MF, Aldecoa I, Pedrosa L, Graus F, Vidal N, Martínez-Soler F, Tortosa A, Carrato C, Balañá C, Boudreau MW, Hergenrother PJ, Kötter P, Entian KD, Hench J, Frank S, Mansouri S, Zadeh G, Dans PD, Orozco M, Thomas G, Blanco S, Seoane J, Preiss T, Pandolfi PP, and Esteller M more...
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- Animals, Biomarkers, Tumor, Cell Line, Tumor, DNA Methylation, Humans, Methyltransferases genetics, Mice, Nude, Muscle Proteins genetics, Neoplasm Transplantation, RNA, Ribosomal, 28S, Brain Neoplasms metabolism, Epigenesis, Genetic, Glioma metabolism, Methyltransferases metabolism, Muscle Proteins metabolism, Protein Biosynthesis physiology, Ribosomes metabolism
- Abstract
Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease. more...
- Published
- 2019
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15. Aging-related tau astrogliopathy (ARTAG): not only tau phosphorylation in astrocytes.
- Author
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Ferrer I, García MA, González IL, Lucena DD, Villalonga AR, Tech MC, Llorens F, Garcia-Esparcia P, Martinez-Maldonado A, Mendez MF, Escribano BT, Bech-Serra JJ, Sabido E, de la Torre Gómez C, and Del Rio JA more...
- Subjects
- Aged, Aged, 80 and over, Aging metabolism, Aging pathology, Animals, Astrocytes classification, Corpus Callosum metabolism, Female, Fornix, Brain metabolism, Glial Fibrillary Acidic Protein metabolism, Hippocampus metabolism, Humans, Male, Mice, Mice, Inbred C57BL, Middle Aged, Oligodendroglia metabolism, Phosphorylation, Superoxide Dismutase metabolism, White Matter metabolism, tau Proteins chemistry, tau Proteins classification, Astrocytes metabolism, Astrocytes pathology, Tauopathies metabolism, Tauopathies pathology, tau Proteins metabolism
- Abstract
Aging-related tau astrogliopathy (ARTAG) is defined by the presence of two types of tau-bearing astrocytes: thorn-shaped astrocytes (TSAs) and granular/fuzzy astrocytes in the brain of old-aged individuals. The present study is focused on TSAs in rare forms of ARTAG with no neuronal tau pathology or restricted to entorhinal and transentorhinal cortices, to avoid bias from associated tauopathies. TSAs show 4Rtau phosphorylation at several specific sites and abnormal tau conformation, but they lack ubiquitin and they are not immunostained with tau-C3 antibodies which recognize truncated tau at Asp421. Astrocytes in ARTAG have atrophic processes, reduced glial fibrillary acidic protein (GFAP) and increased superoxide dismutase 2 (SOD2) immunoreactivity. Gel electrophoresis and western blotting of sarkosyl-insoluble fractions reveal a pattern of phospho-tau in ARTAG characterized by two bands of 68 and 64 kDa, and several middle bands between 35 and 50 kDa which differ from what is seen in AD. Phosphoproteomics of dissected vulnerable regions identifies an increase of phosphorylation marks in a large number of proteins in ARTAG compared with controls. GFAP, aquaporin 4, several serine-threonine kinases, microtubule associated proteins and other neuronal proteins are among the differentially phosphorylated proteins in ARTAG thus suggesting a hyper-phosphorylation background that affects several molecules, including many kinases and proteins from several cell compartments and various cell types. Finally, present results show for the first time that tau seeding is produced in neurons of the hippocampal complex, astrocytes, oligodendroglia and along fibers of the corpus callosum, fimbria and fornix following inoculation into the hippocampus of wild type mice of sarkosyl-insoluble fractions enriched in hyper-phosphorylated tau from selected ARTAG cases. These findings show astrocytes as crucial players of tau seeding in tauopathies., (© 2018 International Society of Neuropathology.) more...
- Published
- 2018
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16. SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.
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Baro B, Játiva S, Calabria I, Vinaixa J, Bech-Serra JJ, de LaTorre C, Rodrigues J, Hernáez ML, Gil C, Barceló-Batllori S, Larsen MR, and Queralt E
- Subjects
- Amino Acid Sequence, Cell Cycle Proteins chemistry, Cytokinesis, Endocytosis, Gene Ontology, Metaphase, Molecular Docking Simulation, Phosphorylation, Protein Binding, Protein Interaction Maps, Protein Phosphatase 2 chemistry, Proteome metabolism, Reproducibility of Results, Saccharomyces cerevisiae Proteins chemistry, Substrate Specificity, Cell Cycle Proteins metabolism, Isotope Labeling methods, Phosphoproteins metabolism, Protein Phosphatase 2 metabolism, Proteomics methods, Saccharomyces cerevisiae Proteins metabolism, Saccharomycetales metabolism
- Abstract
Background: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells., Results: We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction., Conclusions: We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome. more...
- Published
- 2018
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17. "Exosomics"-A Review of Biophysics, Biology and Biochemistry of Exosomes With a Focus on Human Breast Milk.
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de la Torre Gomez C, Goreham RV, Bech Serra JJ, Nann T, and Kussmann M
- Abstract
Exosomes are biomolecular nanostructures released from cells. They carry specific biomolecular information and are mainly researched for their exquisite properties as a biomarker source and delivery system. We introduce exosomes in the context of other extracellular vesicles, describe their biophysical isolation and characterisation and discuss their biochemical profiling. Motivated by our interest in early-life nutrition and health, and corresponding studies enrolling lactating mothers and their infants, we zoom into exosomes derived from human breast milk. We argue that these should be more extensively studied at proteomic and micronutrient profiling level, because breast milk exosomes provide a more specific window into breast milk quality from an immunological (proteomics) and nutritional (micronutrient) perspective. Such enhanced breast milk exosome profiling would thereby complement and enrich the more classical whole breast milk analysis and is expected to deliver more functional insights than the rather descriptive analysis of human milk, or larger fractions thereof, such as milk fat globule membrane. We substantiate our arguments by a bioinformatic analysis of two published proteomic data sets of human breast milk exosomes. more...
- Published
- 2018
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18. Disulfide driven folding for a conditionally disordered protein.
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Fraga H, Pujols J, Gil-Garcia M, Roque A, Bernardo-Seisdedos G, Santambrogio C, Bech-Serra JJ, Canals F, Bernadó P, Grandori R, Millet O, and Ventura S
- Subjects
- Amino Acid Sequence, Carrier Proteins metabolism, Copper Transport Proteins, Disulfides metabolism, Humans, Intrinsically Disordered Proteins metabolism, Mitochondrial Membrane Transport Proteins metabolism, Mitochondrial Precursor Protein Import Complex Proteins, Models, Molecular, Protein Conformation, Scattering, Small Angle, Sequence Homology, X-Ray Diffraction, Carrier Proteins chemistry, Disulfides chemistry, Intrinsically Disordered Proteins chemistry, Mitochondrial Membrane Transport Proteins chemistry, Protein Folding
- Abstract
Conditionally disordered proteins are either ordered or disordered depending on the environmental context. The substrates of the mitochondrial intermembrane space (IMS) oxidoreductase Mia40 are synthesized on cytosolic ribosomes and diffuse as intrinsically disordered proteins to the IMS, where they fold into their functional conformations; behaving thus as conditionally disordered proteins. It is not clear how the sequences of these polypeptides encode at the same time for their ability to adopt a folded structure and to remain unfolded. Here we characterize the disorder-to-order transition of a Mia40 substrate, the human small copper chaperone Cox17. Using an integrated real-time approach, including chromatography, fluorescence, CD, FTIR, SAXS, NMR, and MS analysis, we demonstrate that in this mitochondrial protein, the conformational switch between disordered and folded states is controlled by the formation of a single disulfide bond, both in the presence and in the absence of Mia40. We provide molecular details on how the folding of a conditionally disordered protein is tightly regulated in time and space, in such a way that the same sequence is competent for protein translocation and activity. more...
- Published
- 2017
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19. Characterization of secretomes from a human blood brain barrier endothelial cells in-vitro model after ischemia by stable isotope labeling with aminoacids in cell culture (SILAC).
- Author
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Llombart V, García-Berrocoso T, Bech-Serra JJ, Simats A, Bustamante A, Giralt D, Reverter-Branchat G, Canals F, Hernández-Guillamon M, and Montaner J
- Subjects
- Amino Acids chemistry, Biomarkers metabolism, Blood-Brain Barrier pathology, Brain Ischemia pathology, Cell Line, Transformed, Endothelial Cells pathology, Humans, Isotope Labeling methods, Blood-Brain Barrier metabolism, Brain Ischemia metabolism, Endothelial Cells metabolism, Models, Biological, Proteome metabolism
- Abstract
Unlabelled: The human immortalized brain endothelial cell line hCMEC/D3 is considered a simple in-vitro model of the blood-brain-barrier. Our aim was to characterize changes in the secretome of hCMEC/D3 subjected to oxygen and glucose deprivation (OGD) to identify new proteins altered after ischemia and that might trigger blood-brain-barrier disruption and test their potential as blood biomarkers for ischemic stroke. Using a quantitative proteomic approach based on SILAC, 19 proteins were found differentially secreted between OGD and normoxia/normoglycemia conditions. Among the OGD-secreted proteins, protein folding was the main molecular function identified and for the main canonical pathways there was an enrichment in epithelial adherens junctions and aldosterone signaling. Western blot was used to verify the MS results in a set of 9 differentially secreted proteins and 5 of these were analyzed in serum samples of 38 ischemic stroke patients, 18 stroke-mimicking conditions and 18 healthy controls., Significance: "We characterized changes in the secretome of hCMEC/D3 cells after an ischemic insult by SILAC and identified proteins associated with ischemia that might be involved in the disruption of the blood-brain barrier. Besides we analyzed the putative potential of the candidate proteins to become biomarkers for the diagnosis of ischemic stroke., (Copyright © 2015 Elsevier B.V. All rights reserved.) more...
- Published
- 2016
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20. Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines.
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Rosa M, Bech-Serra JJ, Canals F, Zajac JM, Talmont F, Arsequell G, and Valencia G
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- Amino Acid Sequence, Blotting, Western, Chymotrypsin metabolism, Humans, Models, Molecular, Molecular Sequence Data, Peptides metabolism, Pichia genetics, Protein Structure, Secondary, Proteomics instrumentation, Proteomics methods, Receptors, Opioid, mu genetics, Receptors, Opioid, mu metabolism, Recombinant Fusion Proteins metabolism, Trypsin metabolism, Chromatography, Liquid methods, Receptors, Opioid, mu chemistry, Recombinant Fusion Proteins chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Tandem Mass Spectrometry methods
- Abstract
Human μ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR. more...
- Published
- 2015
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21. A mouse model uncovers LKB1 as an UVB-induced DNA damage sensor mediating CDKN1A (p21WAF1/CIP1) degradation.
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Esteve-Puig R, Gil R, González-Sánchez E, Bech-Serra JJ, Grueso J, Hernández-Losa J, Moliné T, Canals F, Ferrer B, Cortés J, Bastian B, Ramón Y Cajal S, Martín-Caballero J, Flores JM, Vivancos A, García-Patos V, and Recio JÁ more...
- Subjects
- AMP-Activated Protein Kinases, Animals, Animals, Newborn, Apoptosis genetics, Apoptosis radiation effects, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p21 genetics, Disease Models, Animal, Hepatocyte Growth Factor genetics, Humans, Keratinocytes metabolism, Keratinocytes pathology, Keratinocytes radiation effects, Mice, Transgenic, Neoplasms, Squamous Cell etiology, Neoplasms, Squamous Cell pathology, Phosphorylation, Protein Kinases metabolism, Protein Serine-Threonine Kinases genetics, Repressor Proteins metabolism, Skin Neoplasms etiology, Skin Neoplasms genetics, Skin Neoplasms pathology, Cyclin-Dependent Kinase Inhibitor p21 metabolism, DNA Damage radiation effects, Protein Serine-Threonine Kinases metabolism, Ultraviolet Rays adverse effects
- Abstract
Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response. more...
- Published
- 2014
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22. Relevance of IGFBP2 proteolysis in glioma and contribution of the extracellular protease ADAMTS1.
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Martino-Echarri E, Fernández-Rodríguez R, Bech-Serra JJ, Plaza-Calonge Mdel C, Vidal N, Casal C, Colomé N, Seoane J, Canals F, and Rodríguez-Manzaneque JC
- Subjects
- ADAMTS1 Protein, Cell Line, Tumor, Disease Progression, Gene Expression Regulation, Neoplastic, HEK293 Cells, Humans, Phosphorylation, Proteolysis, Signal Transduction, Transfection, ADAM Proteins genetics, ADAM Proteins metabolism, Brain Neoplasms genetics, Glioma genetics, Insulin-Like Growth Factor Binding Protein 2 genetics, Insulin-Like Growth Factor Binding Protein 2 metabolism
- Abstract
Expression of IGFBP2 (Insulin-like Growth Factor Binding Protein 2) has been positively correlated with glioma progression. Although the proteolysis of IGFBP2 has been widely recognized, with consequences as a major modulator of IGFII signaling, the relevance of this post-translational modification has not been well studied in tumors. Using an in vivo proteomic approach by Isotope-Coded Protein Label (ICPL), we identified IGFBP2 as a target of the extracellular protease ADAMTS1 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 1). Notably, the proteolytic pattern of IGFBP2 was also detected in human glioma culture cells and, more importantly, in all glioma samples evaluated. In addition, high expression of ADAMTS1 correlates with higher levels of cleaved IGFBP2 in glioblastoma multiforme cases. Using gene expression public databases, we confirmed that IGFBP2 is a poor prognosis marker for gliomas, and we also observed an important contribution of ADAMTS1.Finally, we showed the impact of ADAMTS1 on IGFII-mediated IGF1R phosphorylation and cellular migration. Our results support a functional interaction between IGFBP2 and ADAMTS1 and suggest the need to evaluate post-translational modifications of IGFBP2 in glioma, in order to approach new therapies. more...
- Published
- 2014
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23. The mitochondrial intermembrane space oxireductase Mia40 funnels the oxidative folding pathway of the cytochrome c oxidase assembly protein Cox19.
- Author
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Fraga H, Bech-Serra JJ, Canals F, Ortega G, Millet O, and Ventura S
- Subjects
- Amino Acid Motifs, Biological Transport, Active physiology, Disulfides chemistry, Disulfides metabolism, Kinetics, Mitochondria chemistry, Mitochondria genetics, Mitochondrial Membrane Transport Proteins chemistry, Mitochondrial Membrane Transport Proteins genetics, Mitochondrial Precursor Protein Import Complex Proteins, Molecular Chaperones chemistry, Molecular Chaperones genetics, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Mitochondria metabolism, Mitochondrial Membrane Transport Proteins metabolism, Molecular Chaperones metabolism, Protein Folding, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40. more...
- Published
- 2014
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24. Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome.
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Segura V, Medina-Aunon JA, Mora MI, Martínez-Bartolomé S, Abian J, Aloria K, Antúnez O, Arizmendi JM, Azkargorta M, Barceló-Batllori S, Beaskoetxea J, Bech-Serra JJ, Blanco F, Monteiro MB, Cáceres D, Canals F, Carrascal M, Casal JI, Clemente F, Colomé N, Dasilva N, Díaz P, Elortza F, Fernández-Puente P, Fuentes M, Gallardo O, Gharbi SI, Gil C, González-Tejedo C, Hernáez ML, Lombardía M, Lopez-Lucendo M, Marcilla M, Mato JM, Mendes M, Oliveira E, Orera I, Pascual-Montano A, Prieto G, Ruiz-Romero C, Sánchez del Pino MM, Tabas-Madrid D, Valero ML, Vialas V, Villanueva J, Albar JP, and Corrales FJ more...
- Subjects
- Chromatography, Liquid, Humans, Mass Spectrometry, Sequence Analysis, RNA, Chromosomes, Human, Pair 16, Proteome, Transcriptome
- Abstract
The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study. more...
- Published
- 2014
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25. Guidelines for reporting quantitative mass spectrometry based experiments in proteomics.
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Martínez-Bartolomé S, Deutsch EW, Binz PA, Jones AR, Eisenacher M, Mayer G, Campos A, Canals F, Bech-Serra JJ, Carrascal M, Gay M, Paradela A, Navajas R, Marcilla M, Hernáez ML, Gutiérrez-Blázquez MD, Velarde LF, Aloria K, Beaskoetxea J, Medina-Aunon JA, and Albar JP more...
- Subjects
- Guidelines as Topic, Mass Spectrometry methods, Proteomics methods, Mass Spectrometry standards, Proteomics standards
- 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 labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM)., Biological 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 analyzed 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. This article is part of a Special Issue entitled: Standardization and Quality Control., (Copyright © 2013 Elsevier B.V. All rights reserved.) more...
- Published
- 2013
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26. A dominant-negative N-terminal fragment of HER2 frequently expressed in breast cancers.
- Author
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Morancho B, Parra-Palau JL, Ibrahim YH, Bernadó Morales C, Peg V, Bech-Serra JJ, Pandiella A, Canals F, Baselga J, Rubio I, and Arribas J
- Subjects
- Amino Acid Sequence, Animals, Breast Neoplasms epidemiology, Carcinoma epidemiology, Female, Gene Expression Regulation, Neoplastic, Gene Frequency, Genes, Dominant, HEK293 Cells, Humans, Mice, Mice, Inbred BALB C, Mice, Inbred NOD, Mice, Nude, Mice, SCID, Models, Biological, Molecular Sequence Data, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Structure, Tertiary genetics, Protein Structure, Tertiary physiology, Receptor, ErbB-2 metabolism, Tumor Cells, Cultured, Breast Neoplasms genetics, Carcinoma genetics, Receptor, ErbB-2 chemistry, Receptor, ErbB-2 genetics
- Abstract
The transmembrane tyrosine kinase HER2 (ErbB2, neu) is a prototypical biomarker for breast cancers and a therapeutic target. Although anti-HER2 therapies are remarkably effective, HER2-positive tumors are heterogeneous and some subtypes do not respond or develop resistance to these therapies. Here we show that H2NTF, a novel N-terminal fragment of HER2, is expressed at variable levels in 60% of the breast cancer samples analyzed. Characterization of H2NTF shows that it is devoid of the tyrosine kinase domain but it readily interacts with full-length HER2 and other HER receptors. As a consequence, H2NTF acts as a dominant-negative, attenuating the signaling triggered by full-length HER receptors. Expression of H2NTF results in resistance to the treatment with low concentrations of trastuzumab in vitro. However, cells expressing H2NTF and non-expressing cells have similar sensitivity to trastuzumab in vivo, indicating that H2NTF/trastuzumab complexes trigger antibody-dependent cell-mediated cytotoxicity. more...
- Published
- 2013
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27. Proteomic analysis of cerebrospinal fluid from obese women with idiopathic intracranial hypertension: a new approach for identifying new candidates in the pathogenesis of obesity.
- Author
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Lecube A, Poca MA, Colomé N, Bech-Serra JJ, Hernández C, García-Ramírez M, Gándara D, Canals F, and Simó R
- Subjects
- Adult, Case-Control Studies, Female, Humans, Middle Aged, Obesity cerebrospinal fluid, Obesity physiopathology, Prospective Studies, Obesity etiology, Proteomics methods, Pseudotumor Cerebri cerebrospinal fluid
- Abstract
Body weight control is tightly regulated in the hypothalamus. The inaccessibility of human brain tissue can be partially solved by using cerebrospinal fluid (CSF) as a tool for assessing the central nervous system's production of orexigen and anorexigen factors. Using proteomic analysis, the present study investigated the differentially displayed proteins in human CSF from obese and non-obese subjects. We designed a case-control study conducted in a reference hospital where eight obese (cases) and eight non-obese (controls) women with idiopathic intracranial hypertension were included. Intracranial hypertension was normalised through the placement of a ventriculo- or lumboperitoneal shunt in the 12 months before their inclusion in the study. Isotope-coded protein label (for proteins > 10 kDa) and label-free liquid chromatography (for proteins < 10 kDa) associated with mass spectrometry analysis were used. Eighteen differentially expressed proteins were identified. Many of them fall into three main groups: inflammation (osteopontin, fibrinogen γ and β chain, α1 acid glycoprotein 2 and haptoglobin), neuroendocrine mediators (neurosecretory protein VGF, neuroendocrine protein 7B2, chromogranin-A and chromogranin B), and brain plasticity (testican-1, isoform 10 of fibronectin, galectin-3 binding protein and metalloproteinase inhibitor type 2). The differential production of osteopontin, neurosecretory protein VGF, chromogranin-A and fibrinogen γ chain was further confirmed by either enzyme-linked immunosorbent assay or western blotting. In conclusion, we have identified potential candidates that could be involved in the pathogenesis of obesity. Further studies aiming to investigating the precise role of these proteins in the pathogenesis of obesity and their potential therapeutic implications are needed., (© 2012 The Authors. Journal of Neuroendocrinology © 2012 Blackwell Publishing Ltd.) more...
- Published
- 2012
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28. Protein arginine methyltransferase 5 regulates ERK1/2 signal transduction amplitude and cell fate through CRAF.
- Author
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Andreu-Pérez P, Esteve-Puig R, de Torre-Minguela C, López-Fauqued M, Bech-Serra JJ, Tenbaum S, García-Trevijano ER, Canals F, Merlino G, Avila MA, and Recio JA
- Subjects
- Animals, COS Cells, Cell Differentiation drug effects, Chlorocebus aethiops, Epidermal Growth Factor metabolism, Epidermal Growth Factor pharmacology, HEK293 Cells, Humans, MAP Kinase Signaling System drug effects, Mitogen-Activated Protein Kinase 1 genetics, Mitogen-Activated Protein Kinase 3 genetics, Nerve Growth Factor metabolism, Nerve Growth Factor pharmacology, PC12 Cells, Phosphorylation drug effects, Phosphorylation physiology, Protein Methyltransferases genetics, Protein-Arginine N-Methyltransferases, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins B-raf metabolism, Proto-Oncogene Proteins c-raf genetics, Rats, Cell Differentiation physiology, Cell Proliferation, MAP Kinase Signaling System physiology, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Protein Methyltransferases metabolism, Proto-Oncogene Proteins c-raf metabolism
- Abstract
The RAS to extracellular signal-regulated kinase (ERK) signal transduction cascade is crucial to cell proliferation, differentiation, and survival. Although numerous growth factors activate the RAS-ERK pathway, they can have different effects on the amplitude and duration of the ERK signal and, therefore, on the biological consequences. For instance, nerve growth factor, which elicits a larger and more sustained increase in ERK phosphorylation in PC12 cells than does epidermal growth factor (EGF), stimulates PC12 cell differentiation, whereas EGF stimulates PC12 cell proliferation. Here, we show that protein arginine methylation limits the ERK1/2 signal elicited by particular growth factors in different cell types from various species. We found that this restriction in ERK1/2 phosphorylation depended on methylation of RAF proteins by protein arginine methyltransferase 5 (PRMT5). PRMT5-dependent methylation enhanced the degradation of activated CRAF and BRAF, thereby reducing their catalytic activity. Inhibition of PRMT5 activity or expression of RAF mutants that could not be methylated not only affected the amplitude and duration of ERK phosphorylation in response to growth factors but also redirected the response of PC12 cells to EGF from proliferation to differentiation. This additional level of regulation within the RAS pathway may lead to the identification of new targets for therapeutic intervention. more...
- Published
- 2011
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29. ADAM17 (TACE) regulates TGFβ signaling through the cleavage of vasorin.
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Malapeira J, Esselens C, Bech-Serra JJ, Canals F, and Arribas J
- Subjects
- ADAM17 Protein, Cell Line, Tumor, Epithelial-Mesenchymal Transition, Humans, Hydrolysis, ADAM Proteins physiology, Carrier Proteins metabolism, Membrane Proteins metabolism, Signal Transduction physiology, Transforming Growth Factor beta physiology
- Abstract
The activity of a variety of extracellular signaling factors is tightly regulated by proteins containing A Disintegrin And a Metalloprotease domain (ADAM) metalloproteases through limited proteolysis. Thus, the identification of ADAM substrates may unveil novel components and mechanisms of cell signaling pathways. We report the identification of the transmembrane protein vasorin (VASN), a transforming growth factor-β (TGFβ) trap, as a substrate of ADAM17. The metalloprotease efficiently generates a soluble fragment encompassing the extracellular domain of VASN. Despite the importance of TGFβ in normal development and tumor progression, the regulation of VASN is completely unknown. Here, we show that only the soluble form of VASN inhibits TGFβ and that the secretion of VASN is tightly controlled by ADAM17. Hence, inhibition of ADAM17 leads to the upregulation of TGFβ signaling. Adding a new level of complexity to the function of ADAM17, we finally show that, through the cleavage of VASN, the metalloprotease controls TGFβ-mediated epithelial-to-mesenchymal transition. more...
- Published
- 2011
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30. Increased apoptosis after autoimmune regulator expression in epithelial cells revealed by a combined quantitative proteomics approach.
- Author
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Colomé N, Collado J, Bech-Serra JJ, Liiv I, Antón LC, Peterson P, Canals F, Jaraquemada D, and Alvarez I
- Subjects
- Calmodulin-Binding Proteins analysis, Cell Line, Tumor, Electrophoresis, Gel, Two-Dimensional, Flow Cytometry, HSP70 Heat-Shock Proteins analysis, Humans, Isotope Labeling, Reproducibility of Results, Signal Transduction, Transcription Factors genetics, Transcription Factors metabolism, Transfection, AIRE Protein, Apoptosis physiology, Epithelial Cells metabolism, Proteome analysis, Proteomics methods, Transcription Factors biosynthesis
- Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive autoimmune disease, affecting many endocrine tissues. APECED is associated to the lack of function of a single gene called AutoImmune REgulator (AIRE). Aire knockout mice develop various autoimmune disorders affecting different organs, indicating that Aire is a key gene in the control of organ-specific autoimmune diseases. AIRE is mainly expressed by medullary thymic epithelial cells (mTECs), and its absence results in the loss of tolerance against tissue restricted antigens (TRAs). Aire induces the transcription of genes encoding for TRAs in mTECs. In this report, the analysis of AIRE's effect on the cellular proteome was approached by the combination of two quantitative proteomics techniques, 2D-DIGE and ICPL, using an AIRE-transfected and nontransfected epithelial cell line. The results showed increased levels of several chaperones, (HSC70, HSP27 and tubulin-specific chaperone A) in AIRE-expressing cells, while various cytoskeleton interacting proteins, that is, transgelin, caldesmon, tropomyosin alpha-1 chain, myosin regulatory light polypeptide 9, and myosin-9, were decreased. Furthermore, some apoptosis-related proteins were differentially expressed. Data were confirmed by Western blot and flow cytometry analysis. Apoptosis assays with annexin V and etoposide demonstrated that AIRE-positive cells suffer more spontaneous apoptosis and are less resistant to apoptosis induction. more...
- Published
- 2010
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31. Proteomic analysis in cerebrospinal fluid of patients with atypical nonketotic hyperglycinemia and pulmonary hypertension - A pilot study.
- Author
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Rodríguez CE, Arranz JA, Colomé N, Bech-Serra JJ, Canals F, Del Toro M, and Riudor E
- Abstract
A variant phenotype of nonketotic hyperglycinemia has been described by our group associated with pulmonary hypertension. The aim of this study is to investigate the cerebrospinal fluid proteomes to get an insight into this neurodegenerative process producing leukoencephalopathy with white matter spongiform degeneration. DIGE and MALDI-TOF-TOF analyses were performed to carry out the proteomic study of four patients against three normal controls and one additional control of a classical nonketotic hyperglycinemia. The differential proteomic analysis showed a displacement of some series of spots toward the acidic side. The shifted proteins showed a high degree of carbonylation and increased methionine sulfoxidation was found in cystatin C and in vitamin-D-binding protein. These findings in addition to the increase of serum malondialdehyde concentration provide evidence of an oxidative stress in the patients under study, which is probably systemic rather than mainly confined to the CNS. The similarities of our findings with those found in other neurodegenerative diseases suggest that oxidative damage is commonly involved in these pathologies. DIGE technology improves the 2-D PAGE differential analysis and it is suitable in proteomic studies with a small number of cases., (Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...
- Published
- 2009
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32. A multi-laboratory study assessing reproducibility of a 2D-DIGE differential proteomic experiment.
- Author
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Bech-Serra JJ, Borthwick A, Colomé N, Albar JP, Wells M, Sánchez del Pino M, and Canals F
- Subjects
- Biochemistry methods, Cell Line, Tumor, Epidermal Growth Factor chemistry, Humans, Mass Spectrometry methods, Principal Component Analysis, Proteins chemistry, Reproducibility of Results, Spain, Time Factors, Electrophoresis, Gel, Two-Dimensional methods, Proteomics methods
- Published
- 2009
33. Very low-molecular-mass fragments of albumin in the plasma of patients with focal segmental glomerulosclerosis.
- Author
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Hellin JL, Bech-Serra JJ, Moctezuma EL, Chocron S, Santin S, Madrid A, Vilalta R, Canals F, Torra R, Meseguer A, and Nieto JL
- Subjects
- Adolescent, Child, Child, Preschool, Female, Glomerulosclerosis, Focal Segmental genetics, Humans, Male, Molecular Weight, Mutation, Proteomics, Glomerulosclerosis, Focal Segmental blood, Serum Albumin analysis
- Abstract
Background: Primary focal segmental glomerulosclerosis (FSGS) is a glomerular disease that frequently does not respond to treatment and progresses to kidney failure. FSGS can be of either genetic origin, caused by mutations in slit diaphragm proteins, such as podocin, or idiopathic origin of unknown cause., Study Design: Case series., Setting & Participants: Children with FSGS (aged 3-18 years); 15 with idiopathic and 11 with genetic forms of FSGS., Predictor: Genetic versus idiopathic forms., Outcomes & Measurements: Differentially expressed proteins in the plasma proteome, detected using 2-dimensional electrophoresis and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Western blot, and liquid chromatography electron spray ionization tandem mass spectrometry for fragmentation and identification of the peptides., Results: We found 3 very low-molecular-mass (9.2, 6.9, and 4.7 kDa; isoelectric point, 5.7) spots that were present in pooled samples from patients with genetic FSGS, but missing in patients with idiopathic FSGS and healthy individuals. Spots were identified using mass spectrometry as fragments of albumin, 2 of them apparently containing peptides from both C- and N-terminal parts of the whole protein. Proteomic analyses were carried out on all genetic patients individually; of these, 10 of 11 patients had > or =1 albumin fragment detected in the pool. We did not find an evident relationship between type of mutation or clinical status of patients and albumin fragments observed., Limitations: Very low-molecular-weight albumin fragments also can be produced by other diseases., Conclusions: We describe for the first time the presence of very low-molecular-mass albumin fragments in plasma of patients with FSGS with podocyte protein mutations that are absent in patients with idiopathic FSGS or healthy individuals. Additional studies are necessary to determine whether these fragments could be potential biomarkers to distinguish between genetic and idiopathic forms of FSGS. more...
- Published
- 2009
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34. HER2 carboxyl-terminal fragments regulate cell migration and cortactin phosphorylation.
- Author
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García-Castillo J, Pedersen K, Angelini PD, Bech-Serra JJ, Colomé N, Cunningham MP, Parra-Palau JL, Canals F, Baselga J, and Arribas J
- Subjects
- Breast Neoplasms metabolism, Breast Neoplasms pathology, Cell Line, Tumor, Cell Movement, Humans, Microscopy, Fluorescence, Models, Biological, Peptides chemistry, Phosphoproteins chemistry, Phosphorylation, Protein Structure, Tertiary, Signal Transduction, Cortactin chemistry, Gene Expression Regulation, Neoplastic, Receptor, ErbB-2 chemistry
- Abstract
A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF. more...
- Published
- 2009
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35. Quick two-dimensional differential in gel electrophoresis-based method to determine length and secondary structures of telomeric DNA.
- Author
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Lyakhovich A, Bech-Serra JJ, Canals F, and Surralles J
- Subjects
- Nucleic Acid Conformation, Structure-Activity Relationship, DNA chemistry, Electrophoresis, Gel, Two-Dimensional methods, Telomere chemistry
- Abstract
A novel differential in gel electrophoresis (DIGE)-based method has been developed and applied to measure telomere length and appearance of two-dimensional structural DNA changes. It can be applied to any area requiring quick and evident measurement of structural DNA changes. more...
- Published
- 2009
- Full Text
- View/download PDF
36. Post-transcriptional up-regulation of ADAM17 upon epidermal growth factor receptor activation and in breast tumors.
- Author
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Santiago-Josefat B, Esselens C, Bech-Serra JJ, and Arribas J
- Subjects
- ADAM17 Protein, Biotinylation, Cell Line, Tumor, Culture Media, Serum-Free pharmacology, Desmoglein 2 metabolism, Disease Progression, Humans, Recombinant Proteins, Time Factors, ADAM Proteins metabolism, Breast Neoplasms metabolism, Epidermal Growth Factor metabolism, ErbB Receptors metabolism, Gene Expression Regulation, Neoplastic, RNA Processing, Post-Transcriptional, Up-Regulation
- Abstract
ADAM17 is a transmembrane metalloprotease involved in the proteolytic release of the extracellular domain of many cell surface molecules, a process known as ectodomain shedding. Despite its likely participation in tumor progression and its current consideration as a therapeutic target, very little is known about the regulation of the expression of ADAM17. Here we show that long term treatment with epidermal growth factor (EGF) leads to a marked increase in the levels of ADAM17. EGF receptor activation does not affect the levels of the mRNA that encodes for, or the rate of synthesis of, ADAM17 but increases its half-life. The effect of EGF is biologically relevant because it increases the shedding of several substrates of ADAM17, including the desmosomal cadherin Dsg-2. Analysis of protein and mRNA levels in mammary tumor samples shows that in vivo the levels of ADAM17 can also be controlled post-transcriptionally. Finally, we show that both the shed extracellular domains of Dsg-2 and ADAM17 are frequently expressed in tumors, further supporting the participation of the metalloprotease in malignant progression. more...
- Published
- 2007
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37. Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation.
- Author
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Anido J, Scaltriti M, Bech Serra JJ, Santiago Josefat B, Todo FR, Baselga J, and Arribas J
- Subjects
- Amino Acid Sequence, Animals, Breast Neoplasms metabolism, Cell Line, Cell Nucleus metabolism, Codon, Initiator, Cytoplasm metabolism, Enzyme Activation, Female, Humans, Molecular Sequence Data, Mutation, Phosphorylation, Protein Structure, Tertiary, Receptor, ErbB-2 genetics, Cell Transformation, Neoplastic metabolism, Receptor, ErbB-2 biosynthesis, Translations
- Abstract
The overactivation of the HERs, a family of tyrosine kinase receptors, leads to the development of cancer. Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, full-length HER1, HER2 and HER3 have been detected in the nucleoplasm. Furthermore, limited proteolysis of HER4 generates nuclear C-terminal fragments (CTFs). Using cells expressing a panel of deletion and point mutants, here we show that HER2 CTFs are generated by alternative initiation of translation from methionines located near the transmembrane domain of the full-length molecule. In vitro and in vivo, HER2 CTFs are found in the cytoplasm and nucleus. Expression of HER2 CTFs to levels similar to those found in human tumors induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies. more...
- Published
- 2006
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38. Proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of ADAM17 and ADAM10 by difference gel electrophoresis.
- Author
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Bech-Serra JJ, Santiago-Josefat B, Esselens C, Saftig P, Baselga J, Arribas J, and Canals F
- Subjects
- ADAM Proteins genetics, ADAM17 Protein, Amyloid Precursor Protein Secretases, Animals, Aspartic Acid Endopeptidases, CHO Cells, Cell Line, Tumor, Cricetinae, Cricetulus, Electrophoresis, Gel, Two-Dimensional, Endopeptidases genetics, Epidermal Growth Factor pharmacology, Humans, Mice, Mutation, Neoplasms enzymology, Protein Structure, Tertiary genetics, Proteomics, Substrate Specificity, Up-Regulation, ADAM Proteins metabolism, Activated-Leukocyte Cell Adhesion Molecule metabolism, Desmoglein 2 metabolism, Endopeptidases metabolism, Neoplasms metabolism
- Abstract
In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy. more...
- Published
- 2006
- Full Text
- View/download PDF
39. ADAMs, cell migration and cancer.
- Author
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Arribas J, Bech-Serra JJ, and Santiago-Josefat B
- Subjects
- Animals, Caenorhabditis elegans genetics, Extracellular Matrix metabolism, Humans, Integrins metabolism, Metalloproteases metabolism, Models, Biological, Neoplasm Metastasis, Neoplasms pathology, Protein Binding, ADAM Proteins physiology, Cell Movement, Neoplasms metabolism
- Published
- 2006
- Full Text
- View/download PDF
40. Recycling of cell surface pro-transforming growth factor-{alpha} regulates epidermal growth factor receptor activation.
- Author
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Martínez-Arca S, Bech-Serra JJ, Hurtado-Küttner M, Borroto A, and Arribas J
- Subjects
- Animals, CHO Cells, Cell Proliferation, Clathrin metabolism, Cricetinae, Dogs, Endosomes metabolism, Flow Cytometry, Fluorescent Antibody Technique, HeLa Cells, Humans, Lysosomes metabolism, Sequence Deletion, Cell Membrane metabolism, Endocytosis physiology, ErbB Receptors metabolism, Protein Precursors metabolism, Protein Transport physiology, Signal Transduction, Transforming Growth Factor alpha metabolism
- Abstract
Impairments in signal transduction, leading to the regulation of cell proliferation, differentiation, or migration are frequently the cause of cancer. Since the accurate spatial and temporal location of their components is crucial to ensure the correct regulation of these signaling pathways, it could be anticipated that defects in intracellular trafficking are at the base of certain neoplasias. However, the trafficking of many components of pathways frequently up-regulated in cancers, such as the epidermal growth factor receptor (EGFR) pathway, are largely unknown. Here, we show that the pro-transforming growth factor-alpha (pro-TGF-alpha), a prototypical EGFR ligand, is endocytosed from the cell surface via a clathrin-dependent pathway. Internalized pro-TGF-alpha does not progress to the lysosome; instead, it is delivered to the cell surface via recycling endosomes. To analyze the functional meaning of the internalization of pro-TGF-alpha, we used a deletion construct that is normally transported to the cell surface but is deficiently endocytosed. Due to this impairment, the levels of this construct at the cell surface are dramatically augmented. Consequently, the deletion construct displays a higher EGFR-activating ability, revealing a link between the trafficking of pro-TGF-alpha and the signaling by the EGFR and opening the possibility that defects in the trafficking of the growth factor may contribute to the development of tumors. more...
- Published
- 2005
- Full Text
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41. Inactivating mutations block the tumor necrosis factor-alpha-converting enzyme in the early secretory pathway.
- Author
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Villanueva de la Torre T, Bech-Serra JJ, Ruiz-Paz S, Baselga J, and Arribas J
- Subjects
- ADAM Proteins, ADAM17 Protein, Amino Acid Sequence, Animals, CHO Cells, Cricetinae, Metalloendopeptidases chemistry, Metalloendopeptidases metabolism, Molecular Sequence Data, Phenotype, Sequence Homology, Amino Acid, Transfection, Metalloendopeptidases genetics, Mutation
- Abstract
The ectodomain of different transmembrane molecules is released by a proteolytic event known as shedding. The metalloprotease disintegrin proTNF-alpha converting enzyme (TACE) is responsible for the shedding of various proteins, including protransforming growth factor-alpha (proTGF-alpha) and amyloid-beta precursor protein (APP). Inactive TACE accumulates in the early secretory pathway of cell mutants (M1 and M2) defective in proTGF-alpha and APP shedding. Although previous evidences indicated that the component mutated in M1 and M2 cells is different from TACE, recent results show the existence of two heterozygous point mutations in TACE from M2 cells. Here, we show that wild-type TACE stably transfected in M2 cells is processed, transported to the cell surface, and rescues the proTGF-alpha and APP shedding-defective phenotype. Furthermore, M1 cells also express mutant TACE and transfection with wild-type TACE restores the wild-type phenotype. Therefore, different inactivating mutations result in the accumulation of TACE in the early secretory pathway, emphasizing the importance of the initial steps in the biosynthesis of TACE. more...
- Published
- 2004
- Full Text
- View/download PDF
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