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1. Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

Author

Bertrand Fabre, Thomas Lambour, Luc Garrigues, François Amalric, Nathalie Vigneron, Thomas Menneteau, Alexandre Stella, Bernard Monsarrat, Benoît Van den Eynde, Odile Burlet‐Schiltz, and Marie‐Pierre Bousquet‐Dubouch

Subjects
affinity purification, correlation profiling, label‐free quantitative proteomics, mass spectrometry, Biology (General), QH301-705.5, Medicine (General), R5-920
Abstract

Abstract In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub‐complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high‐resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub‐complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon‐γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.

Published
2015
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2. Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

Author

Luc Garrigues, Florence Roux-Dalvai, Emmanuelle Mouton-Barbosa, Mathilde Beau, Luc Sensebé, Anne Gonzalez-de-Peredo, Dusan Zivkovic, Marie-Pierre Bousquet, Marie-Laure Renoud, Thomas Menneteau, Odile Burlet-Schiltz, Alexandre Stella, Bertrand Fabre, François Amalric, Isabelle Ader, Institut de pharmacologie et de biologie structurale (IPBS), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut de médecine moléculaire de Rangueil (I2MR), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-IFR150-Institut National de la Santé et de la Recherche Médicale (INSERM), Equipe 1, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), STROMALab, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement Français du Sang-Centre National de la Recherche Scientifique (CNRS), Université de Montréal [Montréal], Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Etablissement Français du Sang-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université de Montréal (UdeM)

Subjects
Proteasome Endopeptidase Complex, Stromal cell, [SDV]Life Sciences [q-bio], Cell, Protein degradation, SILAC, Biochemistry, Mass Spectrometry, Cell Line, Analytical Chemistry, Interferon-gamma, 03 medical and health sciences, Absolute quantification, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Stable isotope labeling by amino acids in cell culture, medicine, Humans, Stem cells, Molecular Biology, Protein complex analysis, Cell Proliferation, 030304 developmental biology, 0303 health sciences, Chemistry, Research, Human Adipose-derived Mesenchymal Stromal/Stem Cells, 030302 biochemistry & molecular biology, Mesenchymal stem cell, 20S Proteasome, Reproducibility of Results, Targeted mass spectrometry, Cell Differentiation, Mesenchymal Stem Cells, stoichiometry, 3. Good health, Cell biology, Oxygen, medicine.anatomical_structure, Proteasome, Selected reaction monitoring, Stem cell
Abstract

20S proteasomes are very heterogeneous protein complexes involved in many cellular processes. In the present study, we combined an MRM-based assay with the production and purification of entire SILAC labelled proteasome to monitor absolute quantities of the different 20S proteasome subtypes in various human cells and tissues. This method applied to adipocyte-derived stem cells (ADSCs) amplified under various conditions highlights an increased expression of immunoproteasome when this type of cell is primed with IFNγ or amplified in a 20% O2 environment., Graphical Abstract Highlights Design of an MRM assay to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes. Use of purified isotopically labelled 20S proteasome as internal standard for accurate quantification. Variation in the expression of immunoproteasome in adipocyte-derived stem cells (ADSCs) grown under different O2 levels might be causal for change in cells differentiation capacity. The status of 20S proteasome during ADSCs expansion might constitute an additional relevant quality control parameter to contribute to predict, among other quality markers, their therapeutic capacity., The proteasome controls a multitude of cellular processes through protein degradation and has been identified as a therapeutic target in oncology. However, our understanding of its function and the development of specific modulators are hampered by the lack of a straightforward method to determine the overall proteasome status in biological samples. Here, we present a method to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes based on a robust, absolute SILAC-based multiplexed LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sensitivity. The method was initially optimized and validated by comparison with a reference ELISA assay and by analyzing the dynamics of catalytic subunits in HeLa cells following IFNγ-treatment and in range of human tissues. It was then successfully applied to reveal IFNγ- and O2-dependent variations of proteasome status during primary culture of Adipose-derived-mesenchymal Stromal/Stem Cells (ADSCs). The results show the critical importance of controlling the culture conditions during cell expansion for future therapeutic use in humans. We hypothesize that a shift from the standard proteasome to the immunoproteasome could serve as a predictor of immunosuppressive and differentiation capacities of ADSCs and, consequently, that quality control should include proteasomal quantification in addition to examining other essential cell parameters. The method presented also provides a new powerful tool to conduct more individualized protocols in cancer or inflammatory diseases where selective inhibition of the immunoproteasome has been shown to reduce side effects.

Published
2019
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3. Connectivity mapping of glomerular proteins identifies dimethylaminoparthenolide as a new inhibitor of diabetic kidney disease

Author

Julie Klein, Cécile Caubet, Mylène Camus, Manousos Makridakis, Colette Denis, Marion Gilet, Guylène Feuillet, Simon Rascalou, Eric Neau, Luc Garrigues, Olivier Thillaye du Boullay, Harald Mischak, Bernard Monsarrat, Odile Burlet-Schiltz, Antonia Vlahou, Jean Sébastien Saulnier-Blache, Jean-Loup Bascands, Joost P. Schanstra, Equipe 7 Inserm U1048, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Evotec (France) SAS [Toulouse], Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Biomedical Research Foundation of the Academy of Athens (BRFAA), Laboratoire Hétérochimie Fondamentale et Appliquée (LHFA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Institut de Chimie de Toulouse (ICT-FR 2599), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD), Mosaiques Diagnostics GmbH [Hanovre, Allemagne], Diabète athérothrombose et thérapies Réunion Océan Indien (DéTROI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de La Réunion (UR), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Evotec [Toulouse], Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Toulouse (ICT-FR 2599), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Saulnier-Blache, Jean Sébastien, Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Toulouse (UT), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Toulouse (ICT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), and Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Proteomics, Male, [SDV]Life Sciences [q-bio], Kidney Glomerulus, lcsh:Medicine, Angiotensin-Converting Enzyme Inhibitors, Diabetic nephropathy, Article, Renin-Angiotensin System, Angiotensin Receptor Antagonists, Mice, Chronic kidney disease, Connectome, Animals, Diabetic Nephropathies, lcsh:Science, Kidney diseases, Drug discovery, lcsh:R, Diagnostic markers, Computational biology and bioinformatics, [SDV] Life Sciences [q-bio], Mice, Inbred C57BL, Diabetes Mellitus, Type 1, Gene Expression Regulation, Nephrology, lcsh:Q, Sesquiterpenes, Biomarkers, Glomerular Filtration Rate
Abstract

International audience; Abstract While blocking the renin angiotensin aldosterone system (RAAS) has been the main therapeutic strategy to control diabetic kidney disease (DKD) for many years, 25–30% of diabetic patients still develop the disease. In the present work we adopted a systems biology strategy to analyze glomerular protein signatures to identify drugs with potential therapeutic properties in DKD acting through a RAAS-independent mechanism. Glomeruli were isolated from wild type and type 1 diabetic (Ins2Akita) mice treated or not with the angiotensin-converting enzyme inhibitor (ACEi) ramipril. Ramipril efficiently reduced the urinary albumin/creatine ratio (ACR) of Ins2Akita mice without modifying DKD-associated renal-injuries. Large scale quantitative proteomics was used to identify the DKD-associated glomerular proteins (DKD-GPs) that were ramipril-insensitive (RI-DKD-GPs). The raw data are publicly available via ProteomeXchange with identifier PXD018728. We then applied an in silico drug repurposing approach using a pattern-matching algorithm (Connectivity Mapping) to compare the RI-DKD-GPs’s signature with a collection of thousands of transcriptional signatures of bioactive compounds. The sesquiterpene lactone parthenolide was identified as one of the top compounds predicted to reverse the RI-DKD-GPs’s signature. Oral treatment of 2 months old Ins2Akita mice with dimethylaminoparthenolide (DMAPT, a water-soluble analogue of parthenolide) for two months at 10 mg/kg/d by gavage significantly reduced urinary ACR. However, in contrast to ramipril, DMAPT also significantly reduced glomerulosclerosis and tubulointerstitial fibrosis. Using a system biology approach, we identified DMAPT, as a compound with a potential add-on value to standard-of-care ACEi-treatment in DKD.

Published
2020
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4. The Intestinal Glucose–Apelin Cycle Controls Carbohydrate Absorption in Mice

Author

Isabelle Castan-Laurell, Bernard Masri, Estelle Wanecq, Yassine Sakar, Cédric Dray, Sylvain Galvani, Philippe Valet, Larry S. Barak, Anne Nègre-Salvayre, Robert Ducroc, Luc Garrigues, Danièle Daviaud, Bernard Monsarrat, Odile Burlet-Schiltz, Claire Vinel, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de recherche biomédicale Bichat-Beaujon (CRB3), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherches en Cancérologie de Toulouse (CRCT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Department of Cell Biology, Duke University Medical Center, This work was funded by Inserm, Université Paris Diderot Paris 7 and Université Paul 19 Sabatier. Y.S. was a recipient of Fondation pour la Recherche Médicale (FRM)., Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Hormones, facteurs de croissance et physiopathologie vasculaire, IFR 31 Louis Bugnard (IFR 31), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-IFR 31 Louis Bugnard (IFR 31), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, [SDV]Life Sciences [q-bio], Mass Spectrometry, Intestinal absorption, Mice, Random Allocation, 0302 clinical medicine, AMP-activated protein kinase, Reference Values, Glucose homeostasis, Sodium-Glucose Transporter 1, ComputingMilieux_MISCELLANEOUS, Glucose Transporter Type 2, 0303 health sciences, Microscopy, Confocal, diabetes, biology, Gastroenterology, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Immunohistochemistry, Apelin, apelin, Intercellular Signaling Peptides and Proteins, medicine.medical_specialty, mouse model, Blotting, Western, Carbohydrates, 030209 endocrinology & metabolism, Carbohydrate metabolism, 03 medical and health sciences, Internal medicine, medicine, Animals, 030304 developmental biology, Analysis of Variance, adipokine, Hepatology, Glucose transporter, Biological Transport, Mice, Inbred C57BL, Disease Models, Animal, Glucose, Endocrinology, Intestinal Absorption, biology.protein, GLUT2, Calorie intake, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Chromatography, Liquid
Abstract

International audience; BACKGROUND & AIMS: Glucose is absorbed into intestine cells via the sodium glucose transporter 1 (SGLT-1) and glucose transporter 2 (GLUT2); various peptides and hormones control this process. Apelin is a peptide that regulates glucose homeostasis and is produced by proximal digestive cells; we studied whether glucose modulates apelin secretion by enterocytes and the effects of apelin on intestinal glucose absorption. METHODS: We characterized glucose-related luminal apelin secretion in vivo and ex vivo by mass spectroscopy and immunologic techniques. The effects of apelin on (14)C-labeled glucose transport were determined in jejunal loops and in mice following apelin gavage. We determined levels of GLUT2 and SGLT-1 proteins and phosphorylation of AMPKα2 by immunoblotting. The net effect of apelin on intestinal glucose transepithelial transport was determined in mice. RESULTS: Glucose stimulated luminal secretion of the pyroglutaminated apelin-13 isoform ([Pyr-1]-apelin-13) in the small intestine of mice. Apelin increased specific glucose flux through the gastric epithelial barrier in jejunal loops and in vivo following oral glucose administration. Conversely, pharmacologic apelin blockade in the intestine reduced the increased glycemia that occurs following oral glucose administration. Apelin activity was associated with phosphorylation of AMPKα2 and a rapid increase of the GLUT2/SGLT-1 protein ratio in the brush border membrane. CONCLUSIONS: Glucose amplifies its own transport from the intestinal lumen to the bloodstream by increasing luminal apelin secretion. In the lumen, active apelin regulates carbohydrate flux through enterocytes by promoting AMPKα2 phosphorylation and modifying the ratio of SGLT-1:GLUT2. The glucose-apelin cycle might be pharmacologically handled to regulate glucose absorption and assess better control of glucose homeostasis.

Published
2013
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5. Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

Author

Bernard Monsarrat, Nathalie Vigneron, Benoît Van den Eynde, Thomas Lambour, Alexandre Stella, Marie-Pierre Bousquet-Dubouch, Bertrand Fabre, Thomas Menneteau, Luc Garrigues, Odile Burlet-Schiltz, François Amalric, and UCL - SSS/DDUV - Institut de Duve

Subjects
Proteomics, Proteasome Endopeptidase Complex, Supramolecular chemistry, Biology, Bioinformatics, 20s proteasome, General Biochemistry, Genetics and Molecular Biology, Chromatography, Affinity, Mass Spectrometry, label-free quantitative proteomics, Affinity chromatography, Tandem Mass Spectrometry, Correlation profiling, Cell Line, Tumor, correlation profiling, Humans, Label‐free quantitative proteomics, Protein Interaction Domains and Motifs, General Immunology and Microbiology, Mass spectrometry, Intracellular protein, Applied Mathematics, Gene Expression Profiling, affinity purification, Articles, U937 Cells, Affinity purification, Human cell, Cell biology, HEK293 Cells, Computational Theory and Mathematics, Proteasome, General Agricultural and Biological Sciences, Information Systems, Chromatography, Liquid
Abstract

In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.

Published
2015

6. Label-free Quantitative Urinary Proteomics Identifies the Arginase Pathway as a New Player in Congenital Obstructive Nephropathy

Author

Anthony Raevel, Bernard Monsarrat, Angelique Massoubre, David Bouyssié, Frédérique Sabourdy, J.P. Schanstra, Cécile Caubet, Caroline Le Gall, Alexandre Stella, Odile Burlet-Schiltz, Anne Gonzalez-de-Peredo, Stéphane Decramer, Julie Klein, Luc Garrigues, Chrystelle Lacroix, Flavio Bandin, Benjamin Breuil, Jean-Loup Bascands, Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées

Subjects
Male, Proteomics, Proteome, Urinary system, [SDV]Life Sciences [q-bio], Hydronephrosis, Biology, Kidney, Bioinformatics, Biochemistry, Analytical Chemistry, 03 medical and health sciences, 0302 clinical medicine, medicine, Animals, Humans, Renal Insufficiency, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Arginase, Research, Infant, Newborn, Infant, Kidney metabolism, Pathophysiology, Obstructive Nephropathy, 3. Good health, Mice, Inbred C57BL, Disease Models, Animal, medicine.anatomical_structure, Gene Expression Regulation, 030220 oncology & carcinogenesis, Signal Transduction
Abstract

Obstructive nephropathy is a frequently encountered situation in newborns. In previous studies, the urinary peptidome has been analyzed for the identification of clinically useful biomarkers of obstructive nephropathy. However, the urinary proteome has not been explored yet and should allow additional insight into the pathophysiology of the disease. We have analyzed the urinary proteome of newborns (n = 5/group) with obstructive nephropathy using label free quantitative nanoLC-MS/MS allowing the identification and quantification of 970 urinary proteins. We next focused on proteins exclusively regulated in severe obstructive nephropathy and identified Arginase 1 as a potential candidate molecule involved in the development of obstructive nephropathy, located at the crossroad of pro- and antifibrotic pathways. The reduced urinary abundance of Arginase 1 in obstructive nephropathy was verified in independent clinical samples using both Western blot and MRM analysis. These data were confirmed in situ in kidneys obtained from a mouse obstructive nephropathy model. In addition, we also observed increased expression of Arginase 2 and increased total arginase activity in obstructed mouse kidneys. mRNA expression analysis of the related arginase pathways indicated that the pro-fibrotic arginase-related pathway is activated during obstructive nephropathy. Taken together we have identified a new actor in the development of obstructive nephropathy in newborns using quantitative urinary proteomics and shown its involvement in an in vivo model of disease. The present study demonstrates the relevance of such a quantitative urinary proteomics approach with clinical samples for a better understanding of the pathophysiology and for the discovery of potential therapeutic targets.

Published
2014
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