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1. ATP7B-Deficient Hepatocytes Reveal the Importance of Protein Misfolding Induced at Low Copper Concentration

Author: Peggy Charbonnier, Benoît Chovelon, Corinne Ravelet, Tuan Dung Ngo, Mireille Chevallet, and Aurélien Deniaud
Subjects: copper homeostasis, ATP7B, Wilson disease, hepatocytes, protein misfolding, CrispR/Cas9, Cytology, QH573-671
Abstract: Copper is a transition metal essential for human life. Its homeostasis is regulated in the liver, which delivers copper to the whole body and excretes its excess outside the organism in the feces through the bile. These functions are regulated within hepatocytes, and the ATP7B copper transporter is central to making the switch between copper use and excretion. In Wilson disease, the gene coding for ATP7B is mutated, leading to copper overload, firstly, in the liver and the brain. To better understand the role of ATP7B in hepatocytes and to provide a smart tool for the development of novel therapies against Wilson disease, we used the CrispR/Cas9 tool to generate hepatocyte cell lines with the abolished expression of ATP7B. These cell lines revealed that ATP7B plays a major role at low copper concentrations starting in the micromolar range. Moreover, metal stress markers are induced at lower copper concentrations compared to parental cells, while redox stress remains not activated. As shown recently, the main drawback induced by copper exposure is protein unfolding that is drastically exacerbated in ATP7B-deficient cells. Our data enabled us to propose that the zinc finger domain of DNAJ-A1 would serve as a sensor of Cu stress. Therefore, these Wilson-like hepatocytes are of high interest to explore in more detail the role of ATP7B.
Published: 2022
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2. Ligand-Promoted Surface Solubilization of TiO2 Nanoparticles by the Enterobactin Siderophore in Biological Medium

Author: Jérôme Laisney, Mireille Chevallet, Caroline Fauquant, Camille Sageot, Yohann Moreau, Daniela Predoi, Nathalie Herlin-Boime, Colette Lebrun, and Isabelle Michaud-Soret
Subjects: titanium dioxide, nanoparticle, siderophore, enterobactin, ligand-promoted dissolution, biological medium, Microbiology, QR1-502
Abstract: Titanium dioxide nanoparticles (TiO2-NPs) are increasingly used in consumer products for their particular properties. Even though TiO2 is considered chemically stable and insoluble, studying their behavior in biological environments is of great importance to figure their potential dissolution and transformation. The interaction between TiO2-NPs with different sizes and crystallographic forms (anatase and rutile) and the strong chelating enterobactin (ent) siderophore was investigated to look at a possible dissolution. For the first time, direct evidence of anatase TiO2-NP surface dissolution or solubilization (i.e., the removal of Ti atoms located at the surface) in a biological medium by this siderophore was shown and the progressive formation of a hexacoordinated titanium–enterobactin (Ti–ent) complex observed. This complex was characterized by UV–visible and Fourier transform infrared (FTIR) spectroscopy (both supported by Density Functional Theory calculations) as well as electrospray ionization mass spectrometry (ESI-MS) and X-ray photoelectron spectroscopy (XPS). A maximum of ca. 6.3% of Ti surface atoms were found to be solubilized after 24 h of incubation, releasing Ti–ent complexes in the micromolar range that could then be taken up by bacteria in an iron-depleted medium. From a health and environmental point of view, the effects associated to the solubilization of the E171 TiO2 food additive in the presence of enterobactin and the entrance of the Ti–enterobactin complex in bacteria were questioned.
Published: 2022
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3. Deciphering silver nanoparticle fate in liver up to biliary excretion using HepG2/C3A spheroids in scenarios mimicking different exposure pathways

Author: Yousr Rekik, Vanessa Tardillo Suárez, Vikas Raj Sharma, Mireille Chevallet, Benoit Gallet, Denis Falconet, Peggy Charbonnier, Isabelle Kieffer, Rémi Tucoulou, Pierre-Henri Jouneau, Giulia Veronesi, Aurélien Deniaud, Métaux et Organes (MET&OR), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), European Synchroton Radiation Facility [Grenoble] (ESRF), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), LIPID, Physiologie cellulaire et végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Observatoire des Sciences de l'Univers de Grenoble (OSUG ), Institut national des sciences de l'Univers (INSU - CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Météo-France, Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Chimie computationnelle, modélisation et rayons X (COMX), European Synchrotron Radiation Facility (project LS-2967), Auvergne Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), the fonds FEDER and the GIS-Infrastrutures en Biologie Santé et Agronomie (IBISA), CEA DRF-Impulsion grant FIB-Bio, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
Subjects: Materials Science (miscellaneous), [SDV.TOX]Life Sciences [q-bio]/Toxicology, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, [CHIM.MATE]Chemical Sciences/Material chemistry, General Environmental Science
Abstract: International audience; Silver nanoparticles (AgNP) are heavily used in consumer products and medical devices, including face masks to fight against COVID-19. AgNP effects on mammals have been studied both using cellular and animal models. The former elucidate molecular mechanisms occurring at the subcellular level, while the latter bring insight into the toxicity and accumulation at the organ level. It has not yet been possible to reconcile these two kinds of studies in a translational approach. In the current study, we use 3D HepG2/C3A hepatocyte cultures that partially reproduce liver architecture, leading to the formation of active bile canaliculi and exposure scenarios mimicking parenteral or oral exposure to AgNP. By using a combination of cutting edge imaging techniques, atomic spectroscopy, and cell biology, we reveal the formation of Ag sulphide or Ag-organothiol complexes in the different conditions, the storage of excess Ag into vacuoles, the excretion of Ag(i) ions into bile canaliculi through the ATP7B Cu transporter, and modifications of the mitochondrial network. These findings, made possible by the use of a pseudo-organ as a model, reveal exposure-dependent mechanisms that can inspire future studies on nanomaterial metabolism in mammals and risk-assessment, and thereof regulations. Moreover, they can help bridge the gap between cellular and animal studies, bringing significant advances into the understanding of AgNP fate in mammals.
Published: 2023
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4. Pharmacological characterization of the native store-operated calcium channels of cortical neuronal from embryonic mouse brain

Author: Sylvain Chauvet, Louis Jarvis, Mireille Chevallet, Niroj Shrestha, Klaus Groschner, and Alexandre Bouron
Subjects: Calcium, Neurons, Cortex, Store-operated channels, Orai channels, Therapeutics. Pharmacology, RM1-950
Abstract: In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of transient receptor potential channel of C type 3 (TRPC3 channel). However Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to precise the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2, Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, GSK-7975A) were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6, GSK-7975A, and also to EVP4593, zinc, copper and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ~10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing the mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2 (iPLA2). Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex.
Published: 2016
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5. Lectin recognition and hepatocyte endocytosis of GalNAc-decorated nanostructured lipid carriers

Author: Mireille Chevallet, Franck Fieschi, Francois Bulteau Bulteau, Christelle Gateau, Pascale Delangle, Aurélien Deniaud, Michel Thépaut, Isabelle Texier, Laura Gauthier, Corinne Vivès, Chimie Interface Biologie pour l’Environnement, la Santé et la Toxicologie (CIBEST ), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Département Microtechnologies pour la Biologie et la Santé (DTBS), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Métaux et Organes (MET&OR), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
Subjects: Acetylgalactosamine, surface functionalisation, N-acetylgalactosamine, nanostructured lipid carriers, Pharmaceutical Science, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 02 engineering and technology, Endocytosis, macrophage galactose-lectin, targeted drug delivery, N-Acetylgalactosamine, 03 medical and health sciences, chemistry.chemical_compound, asialoglycoprotein receptor, 0302 clinical medicine, Lectins, medicine, Humans, Drug Carriers, biology, Lectin, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Hep G2 Cells, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, 021001 nanoscience & nanotechnology, Lipids, Nanostructures, Cell biology, carbohydrates (lipids), medicine.anatomical_structure, chemistry, Targeted drug delivery, 030220 oncology & carcinogenesis, Hepatocyte, Drug delivery, biology.protein, hepatocytes, lipids (amino acids, peptides, and proteins), Asialoglycoprotein receptor, Nanocarriers, 0210 nano-technology
Abstract: International audience; Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimisation of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDA-approved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that a high density of GalNAc was required to favour hepatocyte internalisation via the ASGPR pathway. Interaction studies using surface plasmon resonance and the macrophage galactose-lectin as GalNAc-recognising lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is the first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.
Published: 2020
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6. Correlative transmission electron microscopy and high-resolution hard X-ray fluorescence microscopy of cell sections to measure trace element concentrations at the organelle level

Author: Mireille Chevallet, Vanessa Tardillo Suárez, Rémi Tucoulou, Pierre-Henri Jouneau, Benoit Gallet, Giulia Veronesi, Aurélien Deniaud, European Synchrotron Radiation Facility (ESRF), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Métaux et Organes (MET&OR), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Chimie computationnelle, modélisation et rayons X (COMX), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
Subjects: Silver, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, Metal ions in aqueous solution, X-ray fluorescence, Metal Nanoparticles, 02 engineering and technology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Silver nanoparticle, X-ray fluorescence microscopy, law.invention, Metal, 03 medical and health sciences, Microscopy, Electron, Transmission, Structural Biology, law, Organelle, Microscopy, 030304 developmental biology, Organelles, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, X-Rays, Resolution (electron density), Spectrometry, X-Ray Emission, Environmental exposure, 021001 nanoscience & nanotechnology, Trace Elements, Mitochondria, Microscopy, Fluorescence, Transmission electron microscopy, visual_art, visual_art.visual_art_medium, Biophysics, Hepatocytes, Electron microscope, 0210 nano-technology
Abstract: Metals are essential to all forms of life and their concentration and distribution in the organisms are tightly regulated. Indeed, in their free form, metal ions are toxic. Therefore, an excess of physiologic metal ions or the uptake of non-physiologic metal ions can be highly detrimental for the organisms. It is thus fundamental to understand metals distribution and dynamics in physiologic or disrupted conditions, for instance in metal-related pathologies or upon environmental exposure to metals. Elemental imaging techniques can serve this purpose, by allowing the visualization and the quantification of metal species in a tissue or down to the interior of a cell. Among these techniques, synchrotron radiation-based X-ray fluorescence (SR-XRF) microscopy is the most sensitive to date, and great progresses were made to reach spatial resolutions as low as 20×20 nm2. Until recently, 2D XRF mapping was used on whole cells, thus summing up the signal from the whole thickness of the cell. In the last two years, we have developed a methodology to work on thin cell sections, in order to analyze the metal content at the level of the organelle. Herein, we propose a correlative method to couple SR-XRF to electron microscopy, with the aim to quantify the elemental content in an organelle of interest. As a proof-of-concept, the technique was applied to the analysis of mitochondria from hepatocytes exposed to silver nanoparticles. It was thus possible to identify mitochondria with higher concentration of Ag(I) ions compared to the surrounding cytosol. The versatility of the method makes it suitable to answer a large panel of biological questions, for instance related to metal homeostasis in biological organisms.
Published: 2021
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7. Nuclear translocation of silver ions and hepatocyte nuclear receptor impairment upon exposure to silver nanoparticles

Author: Mireille Chevallet, Christine Moriscot, Isabelle Michaud-Soret, Benoit Gallet, Giulia Veronesi, Peggy Charbonnier, Wojciech Bal, Cécile Cottet-Rousselle, Aurélien Deniaud, Rémi Tucoulou, Vanessa Tardillo Suárez, Elizaveta Karepina, Alexandra Fuchs, Pierre-Henri Jouneau, European Synchrotron Radiation Facility (ESRF), Biologie des Métaux (BioMet ), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Groupe Microscopie Electronique et Méthodes (IBS-MEM), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Laboratory of Fundamental and Applied Bioenergetics = Laboratoire de bioénergétique fondamentale et appliquée (LBFA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Integrated Structural Biology Grenoble (ISBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, INSERM, U1160, Département de Gastroentérologie, Université Paris Diderot - Paris 7 (UPD7), Groupe modélisation et chimie théorique (MCT ), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and Integrated Structural Biology Grenoble (ISBG - UMS 3518 )
Subjects: [SDV.EE.SANT]Life Sciences [q-bio]/Ecology, environment/Health, 0303 health sciences, Chemistry, Nucleolus, Materials Science (miscellaneous), 02 engineering and technology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 021001 nanoscience & nanotechnology, Silver nanoparticle, Nuclear translocation, 03 medical and health sciences, Subcellular distribution, Human health, medicine.anatomical_structure, Nuclear receptor, Hepatocyte, [SDE]Environmental Sciences, medicine, Biophysics, 0210 nano-technology, Intracellular, 030304 developmental biology, General Environmental Science
Abstract: International audience; The impact on human health of long-term exposure to low doses of silver nanoparticles (AgNPs) remainsunderstudied. Cellular studies have shown the intracellular dissolution of AgNPs within endolysosomesfollowed by Ag(I) binding to biomolecular thiolate-containing molecules. However, the precise subcellulardistribution of Ag(I) and the nature of the disrupted physiological pathways remained unknown. Novelimaging approaches enabled us to visualize the trafficking of AgNP-containing lysosomes towards aperinuclear location and a nuclear transfer of Ag(I) species with accumulation in the nucleoli. These Ag(I)species impaired nuclear receptor activity, disrupting critical mechanisms of liver physiology in low doseexposure scenarios, thus justifying further research into defining a framework for the safe use of AgNPs.
Published: 2020
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8. Comparative proteomic analysis of the molecular responses of mouse macrophages to titanium dioxide and copper oxide nanoparticles unravels some toxic mechanisms for copper oxide nanoparticles in macrophages.

Author: Sarah Triboulet, Catherine Aude-Garcia, Lucie Armand, Véronique Collin-Faure, Mireille Chevallet, Hélène Diemer, Adèle Gerdil, Fabienne Proamer, Jean-Marc Strub, Aurélie Habert, Nathalie Herlin, Alain Van Dorsselaer, Marie Carrière, and Thierry Rabilloud
Subjects: Medicine, Science
Abstract: Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.
Published: 2015
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9. Safer-by-design biocides made of tri-thiol bridged silver nanoparticle assemblies

Author: Pascale Delangle, Marianne Marchioni, Pierre-Henri Jouneau, Aurélien Deniaud, Christelle Gateau, Wai Li Ling, Didier Leonard, Mireille Chevallet, Isabelle A.M. Worms, Thomas Gallon, Giulia Veronesi, Laura Carlini, Chiara Battocchio, Isabelle Michaud-Soret, Marchioni, Marianne, Veronesi, Giulia, Worms, Isabelle, Ling, Wai Li, Gallon, Thoma, Leonard, Didier, Gateau, Christelle, Chevallet, Mireille, Jouneau, Pierre-Henri, Carlini, Laura, Battocchio, Chiara, Delangle, Pascale, Michaud-Soret, Isabelle, Deniaud, Aurélien, Biologie des Métaux (BioMet ), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Département Interfaces pour l'énergie, la Santé et l'Environnement (DIESE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Groupe modélisation et chimie théorique (MCT ), European Synchrotron Radiation Facility (ESRF), Institut National de L'Energie Solaire (INES), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS), Département des Technologies des NanoMatériaux (DTNM), Laboratoire d'Innovation pour les Technologies des Energies Nouvelles et les nanomatériaux (LITEN), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)-Institut National de L'Energie Solaire (INES), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Chimie Interface Biologie pour l’Environnement, la Santé et la Toxicologie (CIBEST ), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Surfaces, Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA ), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Univ. Roma Tre, Dept. of Sciences, ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de L'Energie Solaire (INES), Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)
Subjects: chemistry.chemical_classification, Biocide, Silver, Metal Nanoparticles, Nanotechnology, 02 engineering and technology, [CHIM.MATE]Chemical Sciences/Material chemistry, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, Silver nanoparticle, 0104 chemical sciences, Highly sensitive, Nanomaterials, Organic molecules, chemistry, Drug Stability, Drug Design, Thiol, General Materials Science, [SDV.TOX.ECO]Life Sciences [q-bio]/Toxicology/Ecotoxicology, Sulfhydryl Compounds, 0210 nano-technology, No release, Disinfectants
Abstract: International audience; Silver nanoparticles (AgNPs) are efficient biocides increasingly used in consumer products and medical devices. Their activity is due to their capacity to release bioavailable Ag(I) ions making them long-lasting biocides but AgNPs themselves are usually easily released from the product. Besides, AgNPs are highly sensitive to various chemical environments that triggers their transformation, decreasing their activity. Altogether, widespread use of AgNPs leads to bacterial resistance and safety concerns for humans and the environment. There is thus a crucial need for improvement. Herein, a proof of concept for a novel biocide based on AgNP assemblies bridged together by a tri-thiol bioinspired ligand is presented. The final nanomaterial is stable and less sensitive to chemical environments with AgNPs completely covered by organic molecules tightly bound via their thiol functions. Therefore, these AgNP assemblies can be considered as safer-by-design and innovative biocides, since they deliver a sufficient amount of Ag(I) for biocidal activity with no release of AgNPs, which are insensitive to transformations in the nanomaterial.
Published: 2020
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10. Canalicular domain structure and function in matrix-free hepatic spheroids

Author: Ananya Shrivastava, Aurélien Deniaud, Elizaveta Karepina, Vikas Raj Sharma, Mireille Chevallet, Peggy Charbonnier, Benoit Gallet, Pierre-Henri Jouneau, Biologie des Métaux (BioMet ), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Groupe Microscopie Electronique et Méthodes (IBS-MEM), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
Subjects: Cell Culture Techniques, Biomedical Engineering, Context (language use), [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Bone canaliculus, digestive system, 03 medical and health sciences, Live cell imaging, Spheroids, Cellular, Cell polarity, medicine, Humans, General Materials Science, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, Actin, 030304 developmental biology, 0303 health sciences, Chemistry, Bile Canaliculi, 030302 biochemistry & molecular biology, Spheroid, Cell Polarity, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Hep G2 Cells, Cell Dedifferentiation, Cell biology, medicine.anatomical_structure, Microscopy, Fluorescence, Cell culture, Hepatocyte, Hepatocytes
Abstract: International audience; Liver is pivotal in organism metabolism. This organ is receiving nutriments from the portal vein and then storing, metabolizing, distributing in the circulation or excreting excess and xenobiotics in bile. Liver architecture and hepatocyte polarization are crucial to achieve these functions. To study these mechanisms in details, relevant cell culture systems are required, which is not the case with standard 2D cell culture. Besides, primary hepatocytes rapidly de-differenciate making them inefficient in forming physiological system. Herein, we used an hepatoma-derived cell line to produce matrix-free hepatic spheroids and developed an integrated structural cell biology methodology by combining light sheet fluorescence microscopy and 3D electron microscopy to study their function and structure. Within these spheroids, hepatocytes polarize and organize to form bile canaliculi active for both organics and inorganics excretion. Besides, live imaging revealed the high dynamic of actin networks in basal membranes compared to their high stability in the apical pole that constitutes bile canaliculi. Finally, the first structure of active bile canaliculi was solved at nm resolution and showed the very high density of microvilli coming from all cells constituting the canaliculus. Therefore, this study is the first comprehensive and in-depth functional and structural study of bile canaliculi in a physiological-relevant context.
Published: 2020
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11. Silver nanoparticle fate in mammals: Bridging in vitro and in vivo studies

Author: Mireille Chevallet, Aurélien Deniaud, Marianne Marchioni, Pierre-Henri Jouneau, Isabelle Michaud-Soret, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA ), Modélisation et Exploration des Matériaux (MEM), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-11-IDEX-0001,Amidex,INITIATIVE D'EXCELLENCE AIX MARSEILLE UNIVERSITE(2011), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Biocide, Biodistribution, Future studies, Silver sulfide, 02 engineering and technology, Cellular imaging, 010402 general chemistry, 01 natural sciences, Chloride, Silver nanoparticle, Inorganic Chemistry, chemistry.chemical_compound, In vivo, Materials Chemistry, Electron microscopy, [CHIM.COOR]Chemical Sciences/Coordination chemistry, Physical and Theoretical Chemistry, Silver speciation, Chemistry, [CHIM.MATE]Chemical Sciences/Material chemistry, 021001 nanoscience & nanotechnology, In vitro, 0104 chemical sciences, Human exposure, Biophysics, Silver nanoparticles, 0210 nano-technology, Sulfur
Abstract: International audience; Silver nanoparticles (AgNPs) are exponentially used in various consumer products including medical devices. This production leads to an increasing human exposure to silver in different forms. Indeed, AgNPs are subject to various transformations in aqueous aerobic conditions that trigger the production of Ag(I) species. The main environmental transformation produces the non-toxic species silver sulfide. Transformations occurring in mammals are more diverse and mainly depend on the interaction of AgNPs with thiol, chloride and proteins. Any of these species have a different impact on AgNPs and induces AgNP dissolution into Ag(I) species, aggregation and/or stabilization. The transformations occurring also depend on the exposure route. The main one is dietary but medical exposure is also growing with the massive use of nanosilver as biocide in medical devices. For the former, AgNP modifications and Ag distribution has been extensively studied using in vitro and in vivo models, while data related to medical use of nanosilver are scarce. However, most of the in vitro and in vivo data often remain inconsistent. In this review, we describe both in vitro, in cellulo and in vivo data about AgNP transformations, silver speciation and biodistribution. We try to reconcile all these data and describe the latest methods for the future studies of AgNP fate in mammals.
Published: 2018
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12. Corrigendum: Pharmacological Characterization of the Native Store-Operated Calcium Channels of Cortical Neurons from Embryonic Mouse Brain

Author: Louis Jarvis, Mireille Chevallet, Niroj Shrestha, Alexandre Bouron, Klaus Groschner, and Sylvain Chauvet
Subjects: inorganic chemicals, Gene isoform, Pharmacology, 0303 health sciences, calcium, Voltage-dependent calcium channel, ORAI1, neurons, STIM2, Biology, store-operated channels, 3. Good health, Cell biology, 03 medical and health sciences, Transient receptor potential channel, 0302 clinical medicine, TRPC3, cortex, Biochemistry, Pharmacology (medical), Channel blocker, 030217 neurology & neurosurgery, TRPC, Orai channels, 030304 developmental biology
Abstract: In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of transient receptor potential channel of C type 3 (TRPC3 channel). However Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to precise the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2, Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, GSK-7975A) were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6, GSK-7975A, and also to EVP4593, zinc, copper and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ~10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing the mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2 (iPLA2). Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex.
Published: 2017
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13. Textural, Structural and Biological Evaluation of Hydroxyapatite Doped with Zinc at Low Concentrations

Author: Daniela Predoi, Aurélien Deniaud, Mireille Chevallet, Isabelle Michaud-Soret, Simona Liliana Iconaru, Alina Mihaela Prodan, Nicolas Buton, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), National Institute of Materials Physics, Magurele-Ilfov, Romania, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), HORIBA Jobin Yvon SAS, HORIBA Jobin Yvon S.A.S., 6-18, rue du Canal, 91165 Longjumeau Cédex, Emergency Hospital Floreasca Bucharest, Emergency Hospital Floreasca Bucharest, 8 Calea Floresca, Sector 1, 014461 Bucharest, Romania, Surgery Department, Carol Davila University of Medicine and Pharmacy, and Surgery Department, Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari, Sector 5, 050474 Bucharest, Romania.
Subjects: Staphylococcus aureus, Materials science, Scanning electron microscope, chemistry.chemical_element, 02 engineering and technology, Zinc, 010402 general chemistry, 01 natural sciences, lcsh:Technology, Article, Ion, X-ray photoelectron spectroscopy, Escherichia coli, General Materials Science, HepG2 cell viability, Fourier transform infrared spectroscopy, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], lcsh:Microscopy, Volume concentration, lcsh:QC120-168.85, lcsh:QH201-278.5, lcsh:T, Doping, zinc, technology, industry, and agriculture, hydroxyapatite, [CHIM.MATE]Chemical Sciences/Material chemistry, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Crystallography, chemistry, lcsh:TA1-2040, lcsh:Descriptive and experimental mechanics, Crystallite, lcsh:Electrical engineering. Electronics. Nuclear engineering, 0210 nano-technology, lcsh:Engineering (General). Civil engineering (General), lcsh:TK1-9971, Nuclear chemistry
Abstract: International audience; The present work was focused on the synthesis and characterization of hydroxyapatite doped with low concentrations of zinc (Zn:HAp) (0.01 < x Zn < 0.05). The incorporation of low concentrations of Zn 2+ ions in the hydroxyapatite (HAp) structure was achieved by co-precipitation method. The physico-chemical properties of the samples were characterized by X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), Scanning Electron Microscopy (SEM), zeta-potential, and DLS and N 2-BET measurements. The results obtained by XRD and FTIR studies demonstrated that doping hydroxyapatite with low concentrations of zinc leads to the formation of a hexagonal structure with lattice parameters characteristic to hydroxyapatite. The XRD studies have also shown that the crystallite size and lattice parameters of the unit cell depend on the substitutions of Ca 2+ with Zn 2+ in the apatitic structure. Moreover, the FTIR analysis revealed that the water content increases with the increase of zinc concentration. Furthermore, the Energy Dispersive X-ray Analysis (EDAX) and XPS analyses showed that the elements Ca, P, O, and Zn were found in all the Zn:HAp samples suggesting that the synthesized materials were zinc doped hydroxyapatite, Ca 10−x Zn x (PO 4) 6 (OH), with 0.01 ≤ x Zn ≤ 0.05. Antimicrobial assays on Staphylococcus aureus and Escherichia coli bacterial strains and HepG2 cell viability assay were carried out.
Published: 2017
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14. Impact of labile metal nanoparticles on cellular homeostasis. Current developments in imaging, synthesis and applications

Author: Mireille Chevallet, Alexandra Fuchs, Isabelle Michaud-Soret, Elisabeth Mintz, Giulia Veronesi, Aurélien Deniaud, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), European Synchrotron Radiation Facility (ESRF), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: Elemental imaging, Silver, Biophysics, Nanoparticle, Cellular homeostasis, Metal Nanoparticles, Nanotechnology, Context (language use), 02 engineering and technology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010402 general chemistry, 01 natural sciences, Biochemistry, Risk Assessment, Labile metal nanoparticles, X-ray fluorescence microscopy, Cell Line, Toxicity Tests, Animals, Homeostasis, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Particle Size, Metal nanoparticles, Molecular Biology, chemistry.chemical_classification, Inflammation, Biomolecule, Silver Compounds, Oxidation reduction, Cell Biology, Metal homeostasis, 021001 nanoscience & nanotechnology, 0104 chemical sciences, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Zinc, chemistry, Microscopy, Fluorescence, [SDV.TOX]Life Sciences [q-bio]/Toxicology, Biological Assay, Zinc Oxide, 0210 nano-technology, Oxidation-Reduction, Copper
Abstract: Background The use of nanomaterials is constantly increasing in electronics, cosmetics, food additives, and is emerging in advanced biomedical applications such as theranostics, bio-imaging and therapeutics. However their safety raises concerns and requires appropriate methods to analyze their fate in vivo. Scope of review In this review, we describe the current knowledge about the toxicity of labile metal (ZnO, CuO and Ag) nanoparticles (NPs) both at the organism and cellular levels, and describe the pathways that are triggered to maintain cellular homeostasis. We also describe advanced elemental imaging approaches to analyze intracellular NP fate. Finally, we open the discussion by presenting recent developments in terms of synthesis and applications of Ag and CuO NPs. Major conclusions Labile metal nanoparticles (MeNPs) release metal ions that trigger a cellular response involving biomolecules binding to the ions followed by regulation of the redox balance. In addition, specific mechanisms are set up by the cell in response to physiological ions such as Cu(I) and Zn(II). Among all types of NPs, labile MeNPs induce the strongest inflammatory responses which are most probably due to the combined effects of the NPs and of its released ions. Interestingly, recent developments in imaging technologies enable the intracellular visualization of both the NPs and their ions and promise new insights into nanoparticle fate and toxicity. General significance The exponential use of nanotechnologies associated with the difficulties of assessing their impact on health and the environment has prompted scientists to develop novel methodologies to characterize these nanoobjects in a biological context.
Published: 2017
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15. From Secretome Analysis to Immunology

Author: Patrice N. Marche, Rachel Couderc, Mireille Chevallet, Heidi Freitas, Thierry Rabilloud, Christian L. Villiers, Hélène Diemer, and Alain Van Dorsselaer
Subjects: CD86, 0303 health sciences, CD40, CLEC7A, Antigen presentation, Biology, Natural killer T cell, Biochemistry, Analytical Chemistry, 03 medical and health sciences, 0302 clinical medicine, Immunology, Interleukin 12, biology.protein, Antigen-presenting cell, Molecular Biology, CD80, 030304 developmental biology, 030215 immunology
Abstract: Dendritic cells are known to be activated by a wide range of microbial products, leading to cytokine production and increased levels of membrane markers such as major histocompatibility complex class II molecules. Such activated dendritic cells possess the capacity to activate naive T cells. In the present study we demonstrated that immature dendritic cells secrete both the YM1 lectin and lipocalin-2. By testing the ligands of these two proteins, chitosan and siderophores, respectively, we also demonstrated that chitosan, a degradation product of various fungal and protozoal cell walls, induces an activation of dendritic cells at the membrane level, as shown by the up-regulation of membrane proteins such as class II molecules, CD80 and CD86 via a TLR4-dependent mechanism, but is not able to induce cytokine production. This led to the production of activated dendritic cells unable to stimulate T cells. However, costimulation with other microbial products overcame this partial activation and restored the capacity of these activated dendritic cells to stimulate T cells. In addition, successive stimulation with chitosan and then by lipopolysaccharide induced a dose-dependent change in the cytokinic IL-12/IL-10 balance produced by the dendritic cells.
Published: 2009
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16. Power and limitations of electrophoretic separations in proteomics strategies

Author: Emmanuelle Leize-Wagner, Mireille Chevallet, Ali R. Vaezzadeh, Noelle Potier, Cécile Lelong, and Thierry Rabilloud
Subjects: chemistry.chemical_classification, Gel electrophoresis, 0303 health sciences, Chromatography, 010401 analytical chemistry, Peptide, Condensed Matter Physics, Mass spectrometry, Proteomics, 01 natural sciences, General Biochemistry, Genetics and Molecular Biology, Protein detection, 0104 chemical sciences, Analytical Chemistry, 03 medical and health sciences, Electrophoresis, chemistry, Spectroscopy, 030304 developmental biology
Abstract: Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed.
Published: 2008
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17. Metal homeostasis disruption and mitochondrial dysfunction in hepatocytes exposed to sub-toxic doses of zinc oxide nanoparticles

Author: Benoit Gallet, Elisabeth Mintz, Mireille Chevallet, Alexandra Fuchs, Pierre-Henri Jouneau, Isabelle Michaud-Soret, K. Um, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA ), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de biologie structurale (IBS - UMR 5075), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA), Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-11-LABX-0003/11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), and ANR-10-LABX-0049/10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010)
Subjects: 0301 basic medicine, HMOX1, chemistry.chemical_element, Metal Nanoparticles, 02 engineering and technology, Zinc, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Mitochondrion, medicine.disease_cause, Superoxide dismutase, 03 medical and health sciences, medicine, Homeostasis, Humans, General Materials Science, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, biology, GCLM, Hep G2 Cells, 021001 nanoscience & nanotechnology, 3. Good health, Mitochondria, Heme oxygenase, 030104 developmental biology, Biochemistry, chemistry, [SDV.TOX]Life Sciences [q-bio]/Toxicology, biology.protein, Hepatocytes, Zinc Oxide, 0210 nano-technology, Metallothionein 1X, Oxidation-Reduction, Oxidative stress
Abstract: International audience; Increased production and use of zinc oxide nanoparticles (ZnO-NPs) in consumer products has prompted the scientific community to investigate their potential toxicity, and understand their impact on the environment and organisms. Molecular mechanisms involved in ZnO-NP toxicity are still under debate and focus essentially on high dose expositions. In our study, we chose to evaluate the effect of sub-toxic doses of ZnO-NPs on human hepatocytes (HepG2) with a focus on metal homeostasis and redox balance disruptions. We showed massive dissolution of ZnO-NPs outside the cell, transport and accumulation of zinc ions inside the cell but no evidence of nanoparticle entry, even when analysed by high resolution TEM microscopy coupled with EDX. Gene expression analysis highlighted zinc homeostasis disruptions as shown by metallothionein 1X and zinc transporter 1 and 2 (ZnT1, ZnT2) over-expression. Major oxidative stress response genes, such as superoxide dismutase 1, 2 and catalase were not induced. Phase 2 enzymes in term of antioxidant response, such as heme oxygenase 1 (HMOX1) and the regulating subunit of the glutamate-cysteine ligase (GCLM) were slightly upregulated, but these observations may be linked solely to metal homeostasis disruptions, as these actors are involved in both metal and ROS responses. Finally, we observed abnormal mitochondria morphologies and autophagy vesicles in response to ZnO-NPs, indicating a potential role of mitochondria in storing and protecting cells from zinc excess but ultimately causing cell death at higher doses.
Published: 2016
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18. The Na+/K+-ATPase and the amyloid-beta peptide aβ1-40 control the cellular distribution, abundance and activity of TRPC6 channels

Author: Mohamed Benharouga, Alexandre Bouron, Sylvain Chauvet, Michel Jadot, Mireille Chevallet, Peter Faller, Addis Abebe, Marielle Boonen, Louis Jarvis, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Department of Physiological Chemistry, Facultés Universitaires Notre Dame de la Paix (FUNDP), Laboratoire de chimie de coordination (LCC), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie de Toulouse (ICT-FR 2599), 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)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Toulouse (ICT), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), 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: Amyloid beta, TRPC6, Peptide, Aβ peptides, Ouabain, chemistry.chemical_compound, Mice, Aβpeptides, BAPTA, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, TRPC6 Cation Channel, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Calcium Signaling, Na+/K+-ATPase, Molecular Biology, TRPC Cation Channels, Neurons, chemistry.chemical_classification, Trafficking, Amyloid beta-Peptides, biology, Na/K pump, Depolarization, Cell Biology, Na/K, Peptide Fragments, Protein Transport, HEK293 Cells, Biochemistry, chemistry, pump, biology.protein, Biophysics, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Sodium-Potassium-Exchanging ATPase, Lysosomes, Veratridine, medicine.drug
Abstract: The Na(+)/K(+)-ATPase interacts with the non-selective cation channels TRPC6 but the functional consequences of this association are unknown. Experiments performed with HEK cells over-expressing TRPC6 channels showed that inhibiting the activity of the Na(+)/K(+)-ATPase with ouabain reduced the amount of TRPC6 proteins and depressed Ca(2+) entry through TRPC6. This effect, not mimicked by membrane depolarization with KCl, was abolished by sucrose and bafilomycin-A, and was partially sensitive to the intracellular Ca(2+) chelator BAPTA/AM. Biotinylation and subcellular fractionation experiments showed that ouabain caused a multifaceted redistribution of TRPC6 to the plasma membrane and to an endo/lysosomal compartment where they were degraded. The amyloid beta peptide Aβ(1-40), another inhibitor of the Na(+)/K(+)-ATPase, but not the shorter peptide Aβ1-16, reduced TRPC6 protein levels and depressed TRPC6-mediated responses. In cortical neurons from embryonic mice, ouabain, veratridine (an opener of voltage-gated Na(+) channel), and Aβ(1-40) reduced TRPC6-mediated Ca(2+) responses whereas Aβ(1-16) was ineffective. Furthermore, when Aβ(1-40) was co-added together with zinc acetate it could no longer control TRPC6 activity. Altogether, this work shows the existence of a functional coupling between the Na(+)/K(+)-ATPase and TRPC6. It also suggests that the abundance, distribution and activity of TRPC6 can be regulated by cardiotonic steroids like ouabain and the naturally occurring peptide Aβ(1-40) which underlines the pathophysiological significance of these processes.
Published: 2015
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19. Improved mass spectrometry compatibility is afforded by ammoniacal silver staining

Author: Mireille Chevallet, Thierry Rabilloud, Emmanuelle Leize-Wagner, Hélène Diemer, Alain Van Dorsselaer, Sylvie Luche, Contrôle moléculaire de la réponse immune specifique, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pluridisciplinaire Hubert Curien (IPHC), and Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)
Subjects: Proteomics, Silver Staining, Low protein, Mass spectrometry, 01 natural sciences, Biochemistry, MESH: Silver Staining, Mass Spectrometry, Article, Adduct, Silver stain, 03 medical and health sciences, chemistry.chemical_compound, Ammonia, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Humans, Quantitative Biology - Genomics, Electrophoresis, Gel, Two-Dimensional, MESH: Ammonia, Molecular Biology, 030304 developmental biology, Genomics (q-bio.GN), 0303 health sciences, MESH: Humans, Chromatography, MESH: Peptides, Chemistry, MESH: Proteomics, 010401 analytical chemistry, Reproducibility of Results, MESH: Electrophoresis, Gel, Two-Dimensional, 0104 chemical sciences, 3. Good health, Staining, MESH: Reproducibility of Results, Silver nitrate, FOS: Biological sciences, electrophoresis, MESH: Isoelectric Focusing, Mass spectrum, Isoelectric Focusing, Peptides, MESH: Spectrum Analysis, Mass
Abstract: Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer., Comment: website publisher http://www.interscience.wiley.com
Published: 2006
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20. Oxidative stress response: a proteomic view

Author: Thierry Rabilloud, Sylvie Luche, Emmanuelle Leize-Wagner, Mireille Chevallet, Contrôle moléculaire de la réponse immune specifique, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biochimie et biophysique des systèmes intégrés (BBSI), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Substances naturelles/chimie moléculaire, Université Louis Pasteur - Strasbourg I-Ecole européenne de chimie, polymères et matériaux [Strasbourg]-Centre National de la Recherche Scientifique (CNRS), and Rabilloud, Thierry
Subjects: Proteomics, Proteomics methods, Proteome, medicine.disease_cause, Biochemistry, Protein expression, Superoxide dismutase, 03 medical and health sciences, 0302 clinical medicine, MESH: Biology, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Animals, Humans, Quantitative Biology - Genomics, MESH: Animals, Recent onset, Biology, Molecular Biology, 030304 developmental biology, Genomics (q-bio.GN), 0303 health sciences, MESH: Humans, MESH: Oxidative Stress, biology, Chemistry, MESH: Proteomics, Protein level, MESH: Gene Expression Regulation, Cell biology, MESH: Proteome, Oxidative Stress, Gene Expression Regulation, FOS: Biological sciences, biology.protein, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Peroxiredoxin, 030217 neurology & neurosurgery, Oxidative stress
Abstract: The oxidative stress response is characterized by various effects on a range of biologic molecules. When examined at the protein level, both expression levels and protein modifications are altered by oxidative stress. While these effects have been studied in the past by classic biochemical methods, the recent onset of proteomics methods has allowed the oxidative stress response to be studied on a much wider scale. The input of proteomics in the study of oxidative stress response and in the evidence of an oxidative stress component in biologic phenomena is reviewed in this paper.
Published: 2005
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21. Regeneration of Peroxiredoxins during Recovery after Oxidative Stress

Author: Elsa Wagner, Mireille Chevallet, Alain Van Dorsselaer, Sylvie Luche, Emmanuelle Leize-Wagner, and Thierry Rabilloud
Subjects: biology, Regeneration (biology), Active site, Cell Biology, respiratory system, Cycloheximide, Proteomics, medicine.disease_cause, Biochemistry, chemistry.chemical_compound, Redox enzymes, chemistry, biology.protein, medicine, Cysteine sulfinic acid, Molecular Biology, Oxidative stress, Cysteine
Abstract: Peroxiredoxins (prx) are redox enzymes using an activated cysteine as their active site. This activated cysteine can be easily overoxidized to cysteine sulfinic acid or cysteine sulfonic acid, especially under oxidative stress conditions. The regeneration of peroxiredoxins after a short, intense oxidative stress was studied, using a proteomics approach. Important differences in regeneration speed were found, prx2 being the fastest regenerated protein, followed by prx1, whereas prx3 and prx6 were regenerated very slowly. Further study of the mechanism of this regeneration by pulse-chase experiments using stable isotope labeling and cycloheximide demonstrated that the fast-regenerating peroxiredoxins are regenerated at least in part by a retroreduction mechanism. This demonstrates that the overoxidation can be reversible under certain conditions. The pathway of this retroreduction and the reasons explaining the various regeneration speeds of the peroxiredoxins remain to be elucidated.
Published: 2003
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22. Two EPR-detectable [4Fe–4S] clusters, N2a and N2b, are bound to the NuoI (TYKY) subunit of NADH:ubiquinone oxidoreductase (Complex I) from Rhodobacter capsulatus

Author: Mireille Chevallet, Joël Lunardi, Ronald van Belzen, Jean-Paul Issartel, Alain Dupuis, and Simon P. J. Albracht
Subjects: Iron-Sulfur Proteins, Models, Molecular, Pyridines, TYKY, Protein subunit, Mutant, Biophysics, Biochemistry, Rhodobacter capsulatus, NADH:ubiquinone oxidoreductase, Iron–sulfur cluster N2, Complex I, Point Mutation, NADH, NADPH Oxidoreductases, NuoI, Peptide sequence, Ferredoxin, R. capsulatus, chemistry.chemical_classification, Electron Transport Complex I, Rhodobacter, biology, Immunochemistry, Cell Membrane, Electron Spin Resonance Spectroscopy, Cell Biology, NAD, biology.organism_classification, Anti-Bacterial Agents, Amino acid, chemistry, Oxidation-Reduction, Cysteine
Abstract: NADH:ubiquinone oxidoreductases (Complex I) contain a subunit, TYKY in the bovine enzyme and NuoI in the enzyme from Rhodobacter capsulatus, which is assumed to bind two [4Fe–4S] clusters because it contains two sets of conserved cysteine motifs similar to those found in the 2[4Fe–4S] ferredoxins. It was recently shown that the TYKY subunit is not an ordinary 2[4Fe–4S] ferredoxin, but has a unique amino acid sequence, which is only found in NAD(P)H:quinone oxidoreductases and certain membrane-bound [NiFe]-hydrogenases expected to be involved in redox-linked proton translocation [FEBS Lett. 485 (2000) 1]. We have generated a set of R. capsulatus mutants in which five out of the eight conserved cysteine residues in NuoI were replaced by other amino acids. The resulting mutants fell into three categories with virtually no, intermediate or quite normal Complex I activities. EPR-spectroscopic analysis of the membranes of the C67S and C106S mutants, two mutants belonging to the second and third group, respectively, showed a specific 50% decrease of the EPR signal attributed to cluster N2. It is concluded that the NuoI (TYKY) subunit binds two clusters N2, called N2a and N2b, which exhibit very similar spectral features when analyzed by X-band EPR spectroscopy.
Published: 2003
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23. Structure-efficiency relationships of zwitterionic detergents as protein solubilizers in two-dimensional electrophoresis

Author: Sylvie Luche, Christophe Tastet, Thierry Rabilloud, Mireille Chevallet, and Stéphane Charmont
Subjects: chemistry.chemical_classification, Two-dimensional gel electrophoresis, Chemistry, cvg.computer_videogame, Detergents, Erythrocyte Membrane, Hydrophobia, Biochemistry, Combinatorial chemistry, Quaternary Ammonium Compounds, Structure-Activity Relationship, chemistry.chemical_compound, Electrophoresis, Two dimensional electrophoresis, Zwitterion, Humans, Structure–activity relationship, Organic chemistry, Electrophoresis, Gel, Two-Dimensional, cvg, Molecular Biology, Linker, Alkyl
Abstract: Several zwitterionic detergents differing in their polar heads, linker parts and hydrophobia tail were synthesized and evaluated for their efficiency in protein solubilizers for two-dimensional electrophoresis. A model system consisting of human red blood cell ghosts was used for this purpose. This study leads to the description of several new efficient detergents and allowed us to derive structural constraints for the design and synthesis of efficient detergents for two-dimensional electrophoresis. These constraints apply to the hydrophilic head (sulfobetaine but not carboxybetaine), to the hydrophobic tail (12 to 16 alkyl carbons long, linear alkyl or alkylaryl) and to the presence and nature of the linker between the hydrophilic head and hydrophobic tail.
Published: 2003
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24. A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress

Author: Emmanuelle Leize-Wagner, Sylvie Luche, Thierry Rabilloud, Alain Van Dorsselaer, Elsa Wagner, Lucia Penna, and Mireille Chevallet
Subjects: Gene isoform, Spectrometry, Mass, Electrospray Ionization, Molecular Sequence Data, Sulfinic acid, medicine.disease_cause, Biochemistry, Mass Spectrometry, medicine, Humans, Protein Isoforms, Electrophoresis, Gel, Two-Dimensional, Amino Acid Sequence, Cysteine, Binding site, Molecular Biology, Peptide sequence, chemistry.chemical_classification, Binding Sites, Sequence Homology, Amino Acid, biology, Peroxiredoxins, Cell Biology, respiratory system, Sulfinic Acids, Oxygen, Oxidative Stress, Peroxidases, chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, biology.protein, Sulfonic Acids, Peptides, Peroxiredoxin, Oxidative stress, HeLa Cells, Research Article, Peroxidase
Abstract: Peroxiredoxins are often encountered as double spots when analysed by two-dimensional electrophoresis. The quantitative balance between these two spots depends on the physiological conditions, and is altered in favour of the acidic variant by oxidative stress for all the peroxiredoxins we could analyse. Using HeLa cells as a model system, we have further analysed the two protein isoforms represented by the two spots for each peroxiredoxin. The use of selected enzyme digestion and MS demonstrated that the acidic variant of all the peroxiredoxins analysed is irreversibly oxidized at the active-site cysteine into cysteine sulphinic or sulphonic acid. Thus, this acidic variant represents an inactivation form of the peroxiredoxins, and provides a useful marker of oxidative damage to the cells.
Published: 2002
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25. Functional consequences of the over-expression of TRPC6 channels in HEK cells: impact on the homeostasis of zinc

Author: Amélie Harel, Thierry Rabilloud, Sylvie Luche, Louis Jarvis, Sébastien Degot, Alexandre Bouron, Guylain Boulay, Mireille Chevallet, Violaine Chapuis, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Protéomique, Métaux et Différenciation (ProMD ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Cytoo cell architecture, cytoo, Department of Pharmacology, University of Sherbrook, Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Biophysics, Ionophore, chemistry.chemical_element, Zinc, Biology, Biochemistry, TRPC6, Biomaterials, 03 medical and health sciences, 0302 clinical medicine, Ezrin, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], TRPC6 Cation Channel, Homeostasis, Humans, Polycyclic Compounds, Cation Transport Proteins, Actin, TRPC Cation Channels, 030304 developmental biology, 0303 health sciences, HEK 293 cells, Metals and Alloys, Proton Pump Inhibitors, Actin cytoskeleton, Cell biology, Cytoskeletal Proteins, HEK293 Cells, Actin Depolymerizing Factors, chemistry, Chemistry (miscellaneous), 030220 oncology & carcinogenesis, Intracellular
Abstract: International audience; The canonical transient receptor potential 6 (TRPC6) protein is a non-selective cation channel able to transport essential trace elements like iron (Fe) and zinc (Zn) through the plasma membrane. Its over-expression in HEK-293 cells causes an intracellular accumulation of Zn, indicating that it could be involved in Zn transport. This finding prompted us to better understand the role played by TRPC6 in Zn homeostasis. Experiments done using the fluorescent probe FluoZin-3 showed that HEK cells possess an intracellular pool of mobilisable Zn present in compartments sensitive to the vesicular proton pump inhibitor Baf-A, which affects endo/lysosomes. TRPC6 over-expression facilitates the basal uptake of Zn and enhances the size of the pool of Zn sensitive to Baf-A. Quantitative RT-PCR experiments showed that TRPC6 over-expression does not affect the mRNA expression of Zn transporters (ZnT-1, ZnT-5, ZnT-6, ZnT-7, ZnT-9, Zip1, Zip6, Zip7, and Zip14); however it up-regulates the mRNA expression of metallothionein-I and -II. This alters the Zn buffering capacities of the cells as illustrated by the experiments done using the Zn ionophore Na pyrithione. In addition, HEK cells over-expressing TRPC6 grow slower than their parental HEK cells. This feature can be mimicked by growing HEK cells in a culture medium supplemented with 5 μM of Zn acetate. Finally, a proteomic analysis revealed that TRPC6 up-regulates the expression of the actin-associated proteins ezrin and cofilin-1, and changes the organisation of the actin cytoskeleton without changing the cellular actin content. Altogether, these data indicate that TRPC6 is participating in the transport of Zn and influences the Zn storage and buffering capacities of the cells.
Published: 2014
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26. The Complex I from Rhodobacter capsulatus

Author: H. Duborjal, Jean-Paul Issartel, Elisabeth Darrouzet, Joël Lunardi, Alain Dupuis, and Mireille Chevallet
Subjects: Iron–sulfur cluster, Operon, NADH-ubiquinone oxidoreductase, Protein subunit, Molecular Sequence Data, Piericidin, Biophysics, nuo Operon, Biochemistry, Rhodobacter capsulatus, chemistry.chemical_compound, Quinone binding, Oxidoreductase, Rotenone, Complex I, NAD(P)H Dehydrogenase (Quinone), Amino Acid Sequence, Binding site, chemistry.chemical_classification, Rhodobacter, biology, Quinone binding site, Cell Biology, biology.organism_classification, chemistry, Homologous recombination
Abstract: The NADH-ubiquinone oxidoreductase (type I NDH) of Rhodobacter capsulatus is a multisubunit enzyme encoded by the 14 genes of the nuo operon. This bacterial enzyme constitutes a valuable model for the characterization of the mitochondrial Complex I structure and enzymatic mechanism for the following reasons. (i) The mitochondria-encoded ND subunits are not readily accessible to genetic manipulation. In contrast, the equivalents of the mitochondrial ND1, ND2, ND4, ND4L, ND5 and ND6 genes can be easily mutated in R. capsulatus by homologous recombination. (ii) As illustrated in the case of ND1 gene, point mutations associated with human cytopathies can be reproduced and studied in this model system. (iii) The R. capsulatus model also allows the recombinant manipulations of iron–sulfur (Fe–S) subunits and the assignment of Fe–S clusters as illustrated in the case of the NUOI subunit (the equivalent of the mitochondrial TYKY subunit). (iv) Finally, like mitochondrial Complex I, the NADH-ubiquinone oxidoreductase of R. capsulatus is highly sensitive to the inhibitor piericidin-A which is considered to bind to or close to the quinone binding site(s) of Complex I. Therefore, isolation of R. capsulatus mutants resistant to piericidin-A represents a straightforward way to map the inhibitor binding sites and to try and define the location of quinone binding site(s) in the enzyme. These illustrations that describe the interest in the R. capsulatus NADH-ubiquinone oxidoreductase model for the general study of Complex I will be critically developed in the present review.
Published: 1998
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27. Distal genes of thenuooperon ofRhodobacter capsulatusequivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH–ubiquinone oxidoreductase

Author: Béatrice Pierrard, Alain Dupuis, Mireille Chevallet, Joël Lunardi, Ronald van Belzen, Elisabeth Darrouzet, Simon P. J. Albracht, and Hervé Duborjal
Subjects: chemistry.chemical_classification, Rhodobacter, Operon, Genetic Complementation Test, Mutant, Electron Spin Resonance Spectroscopy, Biology, biology.organism_classification, Microbiology, Rhodobacter capsulatus, NADH dehydrogenase activity, Bacterial Proteins, Biochemistry, chemistry, Genes, Bacterial, Oxidoreductase, Mutation, NADH, NADPH Oxidoreductases, ORFS, Molecular Biology, Gene, Biogenesis, Plasmids
Abstract: Seven out of the 13 proteins encoded by the mitochondrial genome of mammals (peptides ND1 to ND6 plus ND4L) are subunits of the respiratory NADH–ubiquinone oxidoreductase (complex I). The function of these ND subunits is still poorly understood. We have used the NADH–ubiquinone oxidoreductase of Rhodobacter capsulatus as a model for the study of the function of these proteins. In this bacterium, the 14 genes encoding the NADH–ubiquinone oxidoreductase are clustered in the nuo operon. We report here on the biochemical and spectroscopic characterization of mutants individually disrupted in five nuo genes, equivalent to mitochondrial genes nd1, nd2, nd5, nd6 and nd4L. Disruption of any of these genes in R. capsulatus leads to the suppression of NADH dehydrogenase activity at the level of the bacterial membranes and to the disappearance of complex I-associated iron–sulphur clusters. Individual NUO subunits can still be immunodetected in the membranes of these mutants, but they do not form a functional subcomplex. In contrast to these observations, disruption of two ORFs (orf6 and orf7 ), also present in the distal part of the nuo operon, does not suppress NADH dehydrogenase activity or complex I-associated EPR signals, thus demonstrating that these ORFs are not essential for the biosynthesis of complex I.
Published: 1998
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28. Interference of CuO nanoparticles with metal homeostasis in hepatocytes under sub-toxic conditions

Author: Isabelle Michaud-Soret, Isabelle Pignot-Paintrand, Josiane Arnaud, Mireille Chevallet, Doris Cassio, Peggy Charbonnier, Martine Cuillel, Caroline Fauquant, Elisabeth Mintz, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biochimie Hormonale et Nutritionnelle, Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble, Signalisation calcique et interactions cellulaires dans le foie, and Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Cell Survival, Endosome, Golgi Apparatus, Metal Nanoparticles, Oxidative phosphorylation, 010501 environmental sciences, Biology, Real-Time Polymerase Chain Reaction, Endocytosis, Polymerase Chain Reaction, 01 natural sciences, Mass Spectrometry, Superoxide dismutase, Bile canaliculus, 03 medical and health sciences, homeostasis, hepatocyte, Humans, Nanotechnology, General Materials Science, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Cation Transport Proteins, 030304 developmental biology, 0105 earth and related environmental sciences, Adenosine Triphosphatases, 0303 health sciences, nanoparticle, Hep G2 Cells, copper oxide, Hsp70, Cell biology, Heme oxygenase, Oxidative Stress, Gene Expression Regulation, Microscopy, Fluorescence, Biochemistry, Copper-Transporting ATPases, copper, Hepatocytes, Microscopy, Electron, Scanning, biology.protein, Metallothionein 1X
Abstract: International audience; Copper oxide nanoparticles (CuO-NP) were studied for their toxicity and mechanism of action on hepatocytes (HepG2), in relation to Cu homeostasis disruption. Indeed, hepatocytes, in the liver, are responsible for the whole body Cu balance and should be a major line of defence in the case of exposure to CuO-NP. We investigated the early responses to sub-toxic doses of CuO-NP and compared them to equivalent doses of Cu added as salt to see if there is a specific nano-effect related to Cu homeostasis in hepatocytes. The expression of the genes encoding the Cu-ATPase ATP7B, metallothionein 1X, heme oxygenase 1, heat shock protein 70, superoxide dismutase 1, glutamate cysteine ligase modifier subunit, metal responsive element-binding transcription factor 1 and zinc transporter 1 was analyzed by qRT-PCR. These genes are known to be involved in response to Cu, Zn and/or oxidative stresses. Except for MTF1, ATP7B and SOD1, we clearly observed an up regulation of these genes expression in CuO-NP treated cells, as compared to CuCl2. In addition, ATP7B trafficking from the Golgi network to the bile canaliculus membrane was observed in WIF-B9 cells, showing a need for Cu detoxification. This shows an increase in the intracellular Cu concentration, probably due to Cu release from endosomal CuO-NP solubilisation. Our data show that CuO-NP enter hepatic cells, most probably by endocytosis, bypassing the cellular defence mechanism against Cu, thus acting as a Trojan horse. Altogether, this study suggests that sub-toxic CuO-NP treatments induce successively a Cu overload, a Cu-Zn exchange on metallothioneins and MTF1 regulation on both Cu and Zn homeostasis.
Published: 2014
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29. Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses

Author: Thierry Rabilloud, Catherine Aude-Garcia, Hélène Diemer, Aurélie Habert, Mireille Chevallet, Nathalie Herlin-Boime, Alain Van Dorsselaer, Véronique Collin-Faure, Fabienne Proamer, Marie Carrière, Sarah Triboulet, Daniel Hanau, Jean-Marc Strub, Protéomique, Métaux et Différenciation (ProMD ), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Biologie des Cellules Dendritiques Humaines, EFS-Cancéropôle du Grand Est-Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Francis PERRIN (LFP - URA 2453), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Grenoble Alpes (UGA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Université Grenoble Alpes (UGA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Spectrométrie de Masse BioOrganique, Département des Sciences Analytiques, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France (LSMBO-DSA-IPHC), Centre National de la Recherche Scientifique (CNRS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Proteomics, Metal Nanoparticles, chemistry.chemical_element, 02 engineering and technology, Oxidative phosphorylation, Nitric Oxide, medicine.disease_cause, Biochemistry, Cell Line, Analytical Chemistry, Nitric oxide, Mitochondrial Proteins, Superoxide dismutase, Mice, 03 medical and health sciences, chemistry.chemical_compound, Phagocytosis, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Animals, Quantitative Biology - Genomics, Molecular Biology, Cells, Cultured, 030304 developmental biology, Membrane Potential, Mitochondrial, Genomics (q-bio.GN), 0303 health sciences, biology, Macrophages, Research, Copper toxicity, Glutathione, 021001 nanoscience & nanotechnology, medicine.disease, Copper, Oxidative Stress, chemistry, FOS: Biological sciences, biology.protein, Signal transduction, 0210 nano-technology, Oxidative stress, Signal Transduction
Abstract: International audience; The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents.
Published: 2013
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30. Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myelo��d cell lines nuclear proteomes

Author: Cécile Lelong, Thierry Rabilloud, Hélène Diemer, Alain Van Dorsselaer, Sylvie Luche, Mireille Chevallet, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), and Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Proteomics, Proteome, Biophysics, 01 natural sciences, Biochemistry, Mass Spectrometry, Mice, 03 medical and health sciences, Cell Line, Tumor, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Transcriptional regulation, Animals, Quantitative Biology - Genomics, Electrophoresis, Gel, Two-Dimensional, Nuclear protein, 030304 developmental biology, Cell Nucleus, Genomics (q-bio.GN), 0303 health sciences, Two-dimensional gel electrophoresis, Chemistry, Macrophages, 010401 analytical chemistry, Nuclear Proteins, Dendritic Cells, 0104 chemical sciences, Chromatin, Cell nucleus, medicine.anatomical_structure, FOS: Biological sciences, Heterochromatin protein 1, Protein Processing, Post-Translational, Software
Abstract: International audience; One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance proteins, particularly those localized in the organelles. An additional problem with nuclear proteins lies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of nuclear proteins compared to contaminant proteins, and also to obtain reproducible conditions compatible with 2D-gel electrophoresis. The NaCl procedure has been chosen. To test the interest of this procedure, the nuclear protein expression profiles of macrophages and dendritic cells have been compared with a proteomic approach by 2D-gel electrophoresis. Delta2D software and mass spectrometry analyses have allowed pointing out some proteins of interest. We have chosen some of them, involved in transcriptional regulation and/or chromatin structure for further validations. The immunoblotting experiments have shown that most of the observed changes are due to post-translational modifications, thereby exemplifying the interest of the 2D gel approach. Finally, this approach allowed us to reach not only high abundance nuclear proteins but also lower abundance proteins, such as the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics because of its ability to visualize intact proteins with their modifications.
Published: 2012
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31. Interference between nanoparticles and metal homeostasis

Author: Jean-Marc Moulis, Thierry Rabilloud, Patrice Catty, Jean-Luc Ravanat, Véronique Collin-Faure, Serge M. Candéias, A. Petit, P Charbonnier, Martine Cuillel, Sylvie Luche, Elisabeth Mintz, Nathalie Herlin-Boime, Isabelle Michaud-Soret, Marie Carrière, Axelle Casanova, S Ollagnier de Choudens, Cécile Lelong, C Aude Garcia, V Nivière, Thierry Douki, Sylvie Sauvaigo, Mireille Chevallet, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biochimie et biophysique des systèmes intégrés (BBSI), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF), Laboratoire Francis PERRIN (LFP - URA 2453), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Laboratoire Structure et Dynamique par Résonance Magnétique (LCF) (LSDRM), Nanosciences et Innovation pour les Matériaux, la Biomédecine et l'Energie (ex SIS2M) (NIMBE UMR 3685), Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), and Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: History, Materials science, DNA damage, Nanoparticle, 010501 environmental sciences, medicine.disease_cause, 01 natural sciences, Education, Lipid peroxidation, 03 medical and health sciences, chemistry.chemical_compound, medicine, 030304 developmental biology, 0105 earth and related environmental sciences, chemistry.chemical_classification, 0303 health sciences, Reactive oxygen species, 89.60.Gg, # 87.85.Qr, # 87.14.Cc, 87.14.G, 89.60.Ec, 61.46.Df, technology, industry, and agriculture, Computer Science Applications, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, Biochemistry, chemistry, Toxicity, Oxidative stress, Genotoxicity, Homeostasis
Abstract: International audience; The TiO2 nanoparticles (NPs) are now produced abundantly and widely used in a variety of consumer products. Due to the important increase in the production of TiO2-NPs, potential widespread exposure of humans and environment may occur during both the manufacturing process and final use. Therefore, the potential toxicity of TiO2-NPs on human health and environment has attracted particular attention. Unfortunately, the results of the large number of studies on the toxicity of TiO2-NPs differ significantly, mainly due to an incomplete characterization of the used nanomaterials in terms of size, shape and crystalline structure and to their unknown state of agglomeration/aggregation. The purpose of our project entitled NanoBioMet is to investigate if interferences between nanoparticles and metal homeostasis could be observed and to study the toxicity mechanisms of TiO2-NPs with well-characterized physicochemical parameters, using proteomic and molecular approaches. A perturbation of metal homeostasis will be evaluated upon TiO2-NPs exposure which could generate reactive oxygen species (ROS) production. Moreover, oxidative stress consequences such as DNA damage and lipid peroxidation will be studied. The toxicity of TiO2-NPs of different sizes and crystalline structures will be evaluated both in prokaryotic (E. coli) and eukaryotic cells (A549 human pneumocytes, macrophages, and hepatocytes). First results of the project will be presented concerning the dispersion of TiO2-NPs in bacterial medium, proteomic studies on total extracts of macrophages and genotoxicity on pneumocytes.
Published: 2010
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32. Two-dimensional gel electrophoresis in proteomics: Past, present and future

Author: Thierry Rabilloud, Cécile Lelong, Mireille Chevallet, Sylvie Luche, Biochimie et biophysique des systèmes intégrés (BBSI), and Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Genomics (q-bio.GN), Proteomics, Gel electrophoresis, 0303 health sciences, Two-dimensional gel electrophoresis, Proteome, Computer science, 010401 analytical chemistry, Biophysics, Computational biology, History, 20th Century, History, 21st Century, 01 natural sciences, Biochemistry, 0104 chemical sciences, 03 medical and health sciences, FOS: Biological sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Quantitative Biology - Genomics, Electrophoresis, Gel, Two-Dimensional, 030304 developmental biology
Abstract: International audience; Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned.
Published: 2010
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33. Silver Staining of Proteins in 2DE Gels

Author: Mireille Chevallet, Thierry Rabilloud, Cécile Lelong, and Sylvie Luche
Subjects: 0303 health sciences, Chromatography, Chemistry, 010401 analytical chemistry, Polyacrylamide, 01 natural sciences, Stain, 0104 chemical sciences, Silver stain, 03 medical and health sciences, chemistry.chemical_compound, Electrophoresis, Silver impregnation, Time range, 030304 developmental biology
Abstract: Silver staining detects proteins after electrophoretic separation on polyacrylamide gels. Its main positive features are its excellent sensitivity (in the low nanogram range) and the use of very simple and cheap equipment and chemicals. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation, and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 day after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.
Published: 2009
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34. Separation of Proteins by Gel Electrophoresis in the Tris-Taurine-HCl System

Author: Mireille Chevallet, Cécile Lelong, Thierry Rabilloud, and Sylvie Luche
Subjects: Free-flow electrophoresis, Gel electrophoresis, Chromatography, Two-dimensional gel electrophoresis, Electrochromatography, Gel electrophoresis of nucleic acids, Isoelectric focusing, Chemistry, Affinity electrophoresis, Gel electrophoresis of proteins
Published: 2009
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35. Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry

Author: Jean-Marc Strub, Sylvie Luche, Thierry Rabilloud, Hélène Diemer, Mireille Chevallet, Alain Van Dorsselaer, Biochimie et biophysique des systèmes intégrés (BBSI), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut Pluridisciplinaire Hubert Curien (IPHC), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)
Subjects: Mass spectrometry, 01 natural sciences, Biochemistry, Mass Spectrometry, Formaldehyde free, Cell Line, Silver stain, gel electrophoresis, Mice, 03 medical and health sciences, Malate Dehydrogenase, Formaldehyde, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Animals, Quantitative Biology - Genomics, Molecular Biology, silver staining, 030304 developmental biology, Genomics (q-bio.GN), Aldehydes, 0303 health sciences, Chromatography, Chemistry, 010401 analytical chemistry, Compatibility (geochemistry), New variant, Vinculin, proteins, 0104 chemical sciences, Electrophoresis, BORATE BUFFER, Homogeneous, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, FOS: Biological sciences
Abstract: International audience; Protein detection methods after electrophoresis have to be sensitive, homogeneous, and not to impair downstream analysis of proteins by MS. Speed, low cost, and user friendliness are also favored features. Silver staining combines many of these features, but its compatibility with MS is limited. We describe here, a new variant of silver staining that is completely formaldehyde-free. Reducing sugars in alkaline borate buffer are used as developers. While keeping the benefits of silver staining, this method is shown to afford a much better performance in terms of compatibility with MS, both in PMF by MALDI and in LC/ESI/MS/MS.
Published: 2008
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36. Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update

Author: Cécile Lelong, Mireille Chevallet, Sylvie Luche, Thierry Rabilloud, Biochimie et biophysique des systèmes intégrés (BBSI), and Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Protein Denaturation, Proteomics, 01 natural sciences, Biochemistry, 03 medical and health sciences, proteomics, Two dimensional electrophoresis, mmebrane proteins, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Protein purification, Animals, Humans, Quantitative Biology - Genomics, Electrophoresis, Gel, Two-Dimensional, Lipid bilayer, Molecular Biology, 030304 developmental biology, Genomics (q-bio.GN), 0303 health sciences, Chromatography, Chemistry, 010401 analytical chemistry, Membrane Proteins, Reproducibility of Results, two-dimensional electrophoresis, 0104 chemical sciences, Electrophoresis, Membrane protein, Complex protein, FOS: Biological sciences
Abstract: International audience; The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field.
Published: 2008
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37. Zinc adaptation and resistance to cadmium toxicity in mammalian cells: molecular insight by proteomic analysis

Author: Jean-Marc Moulis, Hélène Diemer, Alain Martelli, Thierry Rabilloud, Estelle Rousselet, Alain Van Dorsselaer, Mireille Chevallet, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biochimie et biophysique des systèmes intégrés (BBSI), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF), Institut Pluridisciplinaire Hubert Curien (IPHC), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Proteomics, Cadmium Poisoning, Thapsigargin, Proteome, Cell Survival, Lactacystin, Tetrazolium Salts, Biochemistry, 4-Hydroxyphenylpyruvate Dioxygenase, Calcium in biology, HeLa, 03 medical and health sciences, chemistry.chemical_compound, Ubiquitin, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Humans, Quantitative Biology - Genomics, Electrophoresis, Gel, Two-Dimensional, Molecular Biology, 4-hydroxyphenylpyruvate, 030304 developmental biology, Carbonic Anhydrases, Genomics (q-bio.GN), 0303 health sciences, calcium, biology, Dose-Response Relationship, Drug, 030302 biochemistry & molecular biology, Tunicamycin, biology.organism_classification, epithelial cells, Thiazoles, Zinc, Proteasome, chemistry, Cell culture, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, FOS: Biological sciences, biology.protein, 2D electrophoresis, tyrosine, Cadmium, HeLa Cells
Abstract: To identify proteins involved in cellular adaptive responses to zinc, a comparative proteome analysis between a previously developed high zinc- and cadmium- resistant human epithelial cell line (HZR) and the parental HeLa cells has been carried out. Differentially produced proteins included co-chaperones, proteins associated with oxido-reductase activities, and ubiquitin. Biochemical pathways to which these proteins belong were probed for their involvement in the resistance of both cell lines against cadmium toxicity. Among endoplasmic reticulum stressors, thapsigargin sensitized HZR cells, but not HeLa cells, to cadmium toxicity more acutely than tunicamycin, implying that these cells heavily relied on proper intracellular calcium distribution. The similar sensitivity of both HeLa and HZR cells to inhibitors of the proteasome, such as MG-132 or lactacystin, excluded improved proteasome activity as a mechanism associated with zinc adaptation of HZR cells. The enzyme 4-hydroxyphenylpyruvate dioxygenase was overproduced in HZR cells as compared to HeLa cells. It transforms 4-hydroxyphenylpyruvate to homogentisate in the second step of tyrosine catabolism. Inhibition of 4-hydroxyphenylpyruvate dioxygenase decreased the resistance of HZR cells against cadmium, but not that of HeLa cells, suggesting that adaptation to zinc overload and increased 4-hydroxyphenylpyruvate removal are linked in HZR cells, Comment: in press in Proteomics
Published: 2008
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38. Preparing protein extracts for quantitative two-dimensional gel comparison

Author: Mireille Chevallet, Sylvie Luche, Thierry Rabilloud, and Christophe Tastet
Subjects: Cell Nucleus, Organelles, Chromatography, Chemistry, Tissue Extracts, Extraction (chemistry), Proteins, Buffers, Proteomics, Plant cell, Biochemistry, Electrophoresis, Structural Biology, Solubilization, Protein purification, Biological fluids, Animals, Humans, Sample preparation, Electrophoresis, Gel, Two-Dimensional, Isoelectric Focusing, Plant Proteins
Abstract: This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids. The optimized extraction process is strongly sample-dependent and cannot be described for every type of sample. Instead, typical protocols are provided as general guidelines and illustrate good starting points for sample-preparation optimization. These solubilization procedures take into account the constraints brought by two-dimensional electrophoresis and are thus well suited for proteomic approaches. Keywords: proteomics; protein extraction; sample preparation; two-dimensional electrophoresis; protein solubilization
Published: 2008

39. Toward a better analysis of secreted proteins: the example of the myeloid cells secretome

Author: Mireille Chevallet, Thierry Rabilloud, Hélène Diemer, Christian L. Villiers, Alain Van Dorssealer, Biochimie et biophysique des systèmes intégrés (BBSI), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut Pluridisciplinaire Hubert Curien (IPHC), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)
Subjects: Low protein, Lysis, Biology, Proteomics, 01 natural sciences, Biochemistry, Article, 03 medical and health sciences, Mice, proteomics, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Macrophage, Animals, Secretion, Quantitative Biology - Genomics, Molecular Biology, Cells, Cultured, 030304 developmental biology, interleukines, Genomics (q-bio.GN), 0303 health sciences, Two-dimensional gel electrophoresis, 010401 analytical chemistry, Proteins, Dendritic cell, 0104 chemical sciences, Cell biology, secretome, Secretory protein, myeloid cells, FOS: Biological sciences
Abstract: The analysis of secreted proteins represents a challenge for current proteomics techniques. Proteins are usually secreted at low concentrations in the culture media, which makes their recovery difficult. In addition, culture media are rich in salts and other compounds interfering with most proteomics techniques, which makes selective precipitation of proteins almost mandatory for a correct subsequent proteomics analysis. Last but not least, the non-secreted proteins liberated in the culture medium upon lysis of a few dead cells heavily contaminate the so-called secreted proteins preparations. Several techniques have been used in the past for concentration of proteins secreted in culture media. These techniques present several drawbacks, such as coprecipitation of salts or poor yields at low protein concentrations. Improved techniques based on carrier-assisted trichloroacetic acid precipitation are described and discussed in this paper. These techniques have been used to analyse the secretome of myeloid cells (macrophages, dendritic cells) and enabled to analyze proteins secreted at concentrations close to 1 ng/ml, thereby allowing to detect some of the cytokines (TNF, IL-12) secreted by the myeloid cells upon activation by bacterial products., Comment: sous presse sans Proteomics
Published: 2007
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40. Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view

Author: Alain Van Dorsselaer, Thierry Rabilloud, Emmanuelle Leize-Wagner, Pierre Lescuyer, Mireille Chevallet, Hélène Diemer, Contrôle moléculaire de la réponse immune specifique, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Electrochimie et physicochimie des complexes et systèmes interfaciaux (EPCSI), and Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)
Subjects: Proteomics, Mitochondrial DNA, MESH: Protein Transport, Nuclear gene, Proteome, Mitochondrial translation, MESH: Mitochondria, Clinical Biochemistry, Mitochondrion, Biochemistry, DNA, Mitochondrial, Article, Analytical Chemistry, Mitochondrial Proteins, 03 medical and health sciences, Ribosomal protein, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Tumor Cells, Cultured, Humans, Electrophoresis, Gel, Two-Dimensional, Quantitative Biology - Genomics, 030304 developmental biology, Genomics (q-bio.GN), 0303 health sciences, MESH: Humans, Chemistry, MESH: Proteomics, 030302 biochemistry & molecular biology, MESH: DNA, Mitochondrial, MESH: Mitochondrial Proteins, MESH: Electrophoresis, Gel, Two-Dimensional, Cell biology, Mitochondria, MESH: Proteome, Protein Transport, Membrane protein, Electron Transport Chain Complex Proteins, MESH: Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Cell culture, Apoptosis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, FOS: Biological sciences, MESH: T, MESH: Electron Transport Chain Complex Proteins
Abstract: The proper functioning of mitochondria requires that both the mitochondrial and the nuclear genome are functional. To investigate the importance of the mitochondrial genome, which encodes only 13 subunits of the respiratory complexes, the mitochondrial rRNAs and a few tRNAs, we performed a comparative study on the 143B cell line and on its Rho-0 counterpart, i.e., devoid of mitochondrial DNA. Quantitative differences were found, of course in the respiratory complexes subunits, but also in the mitochondrial translation apparatus, mainly mitochondrial ribosomal proteins, and in the ion and protein import system, i.e., including membrane proteins. Various mitochondrial metabolic processes were also altered, especially electron transfer proteins and some dehydrogenases, but quite often on a few proteins for each pathway. This study also showed variations in some hypothetical or poorly characterized proteins, suggesting a mitochondrial localization for these proteins. Examples include a stomatin-like protein and a protein sharing homologies with bacterial proteins implicated in tyrosine catabolism. Proteins involved in apoptosis control are also found modulated in Rho-0 mitochondria., Comment: website publisher: http://www3.interscience.wiley.com/
Published: 2006
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41. About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis

Author: Thierry Rabilloud, Hélène Diemer, Mireille Chevallet, Alain Van Dorsselaer, Chistophe Tastet, Sylvie Luche, Emmanuelle Leize-Wagner, Contrôle moléculaire de la réponse immune specifique, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Substances naturelles/chimie moléculaire, and Université Louis Pasteur - Strasbourg I-Ecole européenne de chimie, polymères et matériaux [Strasbourg]-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Hydrogen-Ion Concentration, Time Factors, Alkylation, Biochemistry, Iodoacetamide, Maleimides, chemistry.chemical_compound, Electrophoresis, Gel, Two-Dimensional, MESH: Animals, Disulfides, Maleimide, cysteine, mass spectrometry, MESH: Maleimides, chemistry.chemical_classification, MESH: Alkylation, 0303 health sciences, biology, MESH: Peptides, 030302 biochemistry & molecular biology, Temperature, Hydrogen-Ion Concentration, MESH: Temperature, 3. Good health, Mitochondria, MESH: Cattle, Major basic protein, Thiol, Cystine, MESH: Hydrogen Peroxide, MESH: Spectrum Analysis, Mass, basic proteins, Resolution (mass spectrometry), MESH: Mitochondria, MESH: Gels, thiol-disulfide exchange, Article, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], MESH: Water, Animals, Quantitative Biology - Genomics, MESH: Disulfides, Sulfhydryl Compounds, Derivatization, MESH: Sulfhydryl Compounds, Molecular Biology, 030304 developmental biology, MESH: Iodoacetamide, Genomics (q-bio.GN), MESH: Time Factors, Water, two-dimensional electrophoresis, MESH: Cystine, Hydrogen Peroxide, MESH: Cysteine, MESH: Electrophoresis, Gel, Two-Dimensional, Combinatorial chemistry, Electrophoresis, Dithiothreitol, chemistry, MESH: Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, FOS: Biological sciences, biology.protein, Cattle, Peptides, MESH: Dithiothreitol, Gels, Cysteine
Abstract: The influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels., Comment: web publisher http://www.interscience.wiley.com
Published: 2006
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42. [Concepts and therapeutic perspectives of proteomics]

Author: Pierre, Lescuyer, Mireille, Chevallet, and Thierry, Rabilloud
Subjects: Proteomics, Proteome, Genetic Diseases, Inborn, Animals, Humans
Abstract: The present paper aims at clarifying some important aspects of proteomics, i.e. the large scale analysis of proteins. To this purpose, the main types of proteomic analyses are presented, i.e. those aiming at determining expression levels and those aiming at unravelling protein-protein interactions networks. Their performances and limitations are outlined, as well as their potential applications in biomedicine, to give an reasoned view of the current state of the art.
Published: 2004

43. About the mechanism of interference of silver staining with peptide mass spectrometry

Author: Alain Van Dorsselaer, Emmanuelle Leize-Wagner, Mireille Chevallet, Thierry Rabilloud, Sylvie Luche, Sophie Richert, Institut Pluridisciplinaire Hubert Curien (IPHC), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Bioénergétique Cellulaire et Pathologique (BECP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)
Subjects: Silver Staining, Proteolysis, Polyacrylamide, Formaldehyde, Peptide, Carbohydrazide, Mass spectrometry, MESH: Formaldehyde, Peptide Mapping, Sensitivity and Specificity, 01 natural sciences, Biochemistry, MESH: Peptide Mapping, MESH: Silver Staining, Silver stain, 03 medical and health sciences, chemistry.chemical_compound, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Quantitative Biology - Genomics, Electrophoresis, Gel, Two-Dimensional, MESH: Proteins, Molecular Biology, 030304 developmental biology, Genomics (q-bio.GN), chemistry.chemical_classification, 0303 health sciences, Chromatography, medicine.diagnostic_test, Chemistry, MESH: Peptides, 010401 analytical chemistry, Proteins, MESH: Electrophoresis, Gel, Two-Dimensional, MESH: Sensitivity and Specificity, 0104 chemical sciences, Staining, Hydrazines, MESH: Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, FOS: Biological sciences, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, MESH: Hydrazines, Peptides
Abstract: International audience; The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.
Published: 2004
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44. Regeneration of peroxiredoxins during recovery after oxidative stress: only some overoxidized peroxiredoxins can be reduced during recovery after oxidative stress

Author: Mireille, Chevallet, Elsa, Wagner, Sylvie, Luche, Alain, van Dorsselaer, Emmanuelle, Leize-Wagner, and Thierry, Rabilloud
Subjects: Oxidative Stress, Binding Sites, Peroxidases, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Humans, Peroxiredoxins, Oxidation-Reduction, HeLa Cells
Abstract: Peroxiredoxins (prx) are redox enzymes using an activated cysteine as their active site. This activated cysteine can be easily overoxidized to cysteine sulfinic acid or cysteine sulfonic acid, especially under oxidative stress conditions. The regeneration of peroxiredoxins after a short, intense oxidative stress was studied, using a proteomics approach. Important differences in regeneration speed were found, prx2 being the fastest regenerated protein, followed by prx1, whereas prx3 and prx6 were regenerated very slowly. Further study of the mechanism of this regeneration by pulse-chase experiments using stable isotope labeling and cycloheximide demonstrated that the fast-regenerating peroxiredoxins are regenerated at least in part by a retroreduction mechanism. This demonstrates that the overoxidation can be reversible under certain conditions. The pathway of this retroreduction and the reasons explaining the various regeneration speeds of the peroxiredoxins remain to be elucidated.
Published: 2003

45. Solubilization of Proteins in Two-Dimensional Electrophoresis

Author: Thierry Rabilloud and Mireille Chevallet
Subjects: Gel electrophoresis, chemistry.chemical_classification, Two-dimensional gel electrophoresis, Enzyme, Biochemistry, chemistry, Nucleic acid, Affinity electrophoresis, Gel electrophoresis of proteins, Deamidation, Macromolecule
Abstract: The solubilization process for 2-D electrophoresis has to achieve several parallel goals: 1. Breaking Macromolecular Interactions in Order to Yield Separate Polypeptide Chains. This includes breaking disulfide bonds and disrupting all non-covalent interactions, both between proteins and between proteins and non-proteinaceous compounds such as lipids or nucleic acids. 2. Preventing Any Artefactual Modification of Polypeptides in the Solubilization Medium. Ideally, the perfect solubilization medium should freeze all the extracted polypeptides in their exact state prior to solubilization, both in terms of amino acid composition and in terms of post-translational modifications. This means that all the enzymes able to modify the proteins must be quickly and irreversibly inactivated. Such enzymes include of course proteases, which are the most difficult to inactivate, but also phosphatases, glycosidases, etc. In parallel, the solubilization protocol should not expose the polypeptides to conditions in which chemical modifications (e.g. deamidation of Asn and Gin, cleavage of Asp-Pro bonds) may occur. 3. Allowing Removal of Substances that May Interfere with Two-Dimensional Electrophoresis. In 2-D, proteins are the analytes. Thus, anything in the cell but proteins can be considered as an interfering substance. Many cellular compounds (e.g. coenzymes, hormones, simple sugars) do not interact with proteins or do not interfere with the electrophoretic process. However, many compounds bind to proteins and/or interfere with 2-D, and must be eliminated prior to electrophoresis if their amount exceeds a critical interference threshold. Such compounds mainly include salts, lipids, polysaccharides (including cell walls) and nucleic acids.
Published: 2000
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46. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis

Author: Mireille Chevallet, Véronique Santoni, Alexandra Poinas, David Rouquié, Alexandra Fuchs, Sylvie Kieffer, Joël Lunardi, Jérome Garin, Thierry Rabilloud, Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)
Subjects: Resolution (mass spectrometry), Neutrophils, [SDV]Life Sciences [q-bio], Clinical Biochemistry, Blotting, Western, Detergents, Arabidopsis, 01 natural sciences, Biochemistry, Analytical Chemistry, 03 medical and health sciences, chemistry.chemical_compound, Animals, Electrophoresis, Gel, Two-Dimensional, Solubility, ComputingMilieux_MISCELLANEOUS, Alkyl, 030304 developmental biology, Plant Proteins, chemistry.chemical_classification, 0303 health sciences, Chromatography, Molecular Structure, 010401 analytical chemistry, Membrane Proteins, 0104 chemical sciences, Electrophoresis, Chaotropic agent, chemistry, Membrane protein, Thiourea, Urea, Cattle
Abstract: Severe quantitative loss of protein is often observed in high-resolution two-dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14–16 carbons proved most efficient, solubilizing previously undetected membrane proteins.
Published: 1998
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47. The NuoI subunit of the Rhodobacter capsulatus respiratory Complex I (equivalent to the bovine TYKY subunit) is required for proper assembly of the membraneous and peripheral domains of the enzyme

Author: J.P. Issartel, Mireille Chevallet, A. Dupuis, R. van Belzen, Simon P. J. Albracht, J. Lunardi, and SILS Other Research (FNWI)
Subjects: Iron-Sulfur Proteins, Genotype, Protein subunit, Mutant, Molecular Sequence Data, Iron–sulfur cluster, Biology, Biochemistry, Rhodobacter capsulatus, Serine, chemistry.chemical_compound, Oxygen Consumption, NAD(P)H Dehydrogenase (Quinone), Animals, Humans, Amino Acid Sequence, Gene, Rhodobacter, Electron Spin Resonance Spectroscopy, biology.organism_classification, Phenotype, chemistry, Cytoplasm, Mutation, Cattle, sense organs, Homologous recombination
Abstract: The nuoI gene that encodes a ferredoxin-like subunit of the Rhodobacter capsulatus Complex I (a subunit equivalent to the bovine TYKY subunit) was mutated by homologous recombination. Both a nuoI-deleted mutant (delta nuoI mutant) and a point mutant in which Cys74 was replaced by a serine (C74S mutant) proved to be completely deficient in Complex I activity. These strains were unable to grow under anaerobic photosynthetic conditions. Their cytoplasmic membranes were also characterized by the absence of specific EPR signals assigned to FeS clusters N1 and N2. Immunochemical analysis of the mutant membranes with subunit-specific antibodies showed that the peripheral subunits were not assembled. Trans-complementation of the mutant strains by a native nuoI gene restored the wild-type phenotypes. In the C74S mutant, a limited amount of NuoI subunit still bound to the membraneous domain of Complex I, which is an indication that NuoI directly interacts with this domain. All these results clearly show that NuoI plays a critical role in the connection between the membraneous domain and the peripheral domain of Complex I.
Published: 1998

48. A nonradioactive double detection method for the assignment of spots in two-dimensional blots

Author: Vincent Procaccio, Thierry Rabilloud, and Mireille Chevallet
Subjects: Silver, Immunoblotting, Biophysics, Gold Colloid, Biochemistry, Stain, Rhodobacter capsulatus, law.invention, Bacterial Proteins, law, General pattern, Molecular Biology, Chemiluminescence, Chromatography, Spots, Staining and Labeling, Chemistry, Membrane Proteins, Proteins, Cell Biology, Molecular biology, Staining, Blot, Electrophoresis, Colloidal gold, Evaluation Studies as Topic, Luminescent Measurements
Abstract: A method for identification of spots on two-dimensional electrophoresis gels by means of immunoblotting is described. This method does not require radioactive-labeled proteins and is based on the dual use of colloidal gold staining to detect the general 2D pattern on the blot and on chemiluminescence to detect the antibody-reactive spot(s). Profit is taken from the fact that the general gold stain produces a background pattern on strong ECL exposures, which allows alignment of the antibody-reactive spot(s) on the general pattern and therefore easy identification of the spot(s) of interest on the two-dimensional maps.
Published: 1997

49. Identification of five Rhodobacter capsulatus genes encoding the equivalent of ND subunits of the mitochondrial NADH-ubiquinone oxidoreductase

Author: Joël Lunardi, Mireille Chevallet, Jean-Paul Issartel, Vincent Procaccio, Alain Dupuis, Béatrice Pierrard, Elisabeth Darrouzet, and André Peinncquin
Subjects: Molecular Sequence Data, Restriction Mapping, Rhodobacter capsulatus, Oxidoreductase, Terminology as Topic, Gene cluster, Operon, Genetics, NADH, NADPH Oxidoreductases, Amino Acid Sequence, Gene, Cloning, chemistry.chemical_classification, Rhodobacter, Electron Transport Complex I, biology, Base Sequence, General Medicine, biology.organism_classification, Open reading frame, Enzyme, Biochemistry, chemistry, Genes, Bacterial, Paracoccus denitrificans
Abstract: We previously reported the sequencing of two genes (ndhA and ndhI) encoding two of the subunits of the type-I NADH-ubiquinone oxidoreductase from Rhodobacter capsulatus (Rc). The present paper deals with the cloning and characterization of a chromosomal fragment clustering five new Rc genes which encode subunits of this enzyme. This gene cluster is located immediately downstream from ndhA and ndhI, and also contains two unidentified open reading frames (urf2, urf3). The five genes, nuoJ, nuoK, nuoL, nuoM and nuoN, encode proteins related, respectively, to mitochondrial (mt) subunits ND6, ND4L, ND5, ND4 and ND2. The overall organization of the nuo genes identified in Rc shows similarity to that of the Paracoccus denitrificans (Pd) nqo gene cluster.
Published: 1995

50. Erratum: Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry

Author: Mireille Chevallet, Sylvie Luche, Hélène Diemer, Jean-Marc Strub, Alain Van Dorsselaer, and Thierry Rabilloud
Subjects: Molecular Biology, Biochemistry
Published: 2010
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