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1. Hamster Melatonin Receptors: Cloning and Binding Characterization of MT₁ and Attempt to Clone MT₂

Author

Gautier, Célia, Dufour, Emilie, Dupré, Clémence, Lizzo, Giulia, Caignard, Sarah, Riest-Fery, Isabelle, Brasseur, Chantal, Legros, Celine, Delagrange, Philippe, Nosjean, Olivier, Simonneaux, Valerie, Boutin, Jean, Guenin, Sophie-Pénélope, Département de Chimie Moléculaire (DCM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biotechnologies, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Institut de Recherches SERVIER (IRS), Département des Sciences Expérimentales, Biotechnologies, Pharamcologie Moléculaire et Cellulaire, Équipe 'Rythme, vie et mort de la rétine', Institut des Neurosciences Cellulaires et Intégratives (INCI), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche de Croissy, and Institut de Recherche Servier

Subjects
[SDV.BA]Life Sciences [q-bio]/Animal biology
Abstract

International audience; For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT₁ and MT₂) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT₁, MT₂, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT₁ receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (

Published
2018
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2. Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2

Author

Giulia Lizzo, Philippe Delagrange, Céline Legros, Jean A. Boutin, Sophie-Pénélope Guenin, Olivier Nosjean, Sarah Caignard, Clémence Dupré, Valérie Simonneaux, Célia Gautier, Isabelle Riest-Fery, Emilie Dufour, Chantal Brasseur, Département de Chimie Moléculaire (DCM), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biotechnologies, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Département des Sciences Expérimentales, Biotechnologies, Pharamcologie Moléculaire et Cellulaire, Équipe 'Rythme, vie et mort de la rétine', Institut des Neurosciences Cellulaires et Intégratives (INCI), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche de Croissy, Institut de Recherche Servier, Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), and Université de Strasbourg (UNISTRA)-Institut des Neurosciences Cellulaires et Intégratives (INCI)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], cloning, Hamster, melatonin, chimeric construction, Catalysis, European hamster, Inorganic Chemistry, lcsh:Chemistry, 03 medical and health sciences, Exon, 0302 clinical medicine, Consensus sequence, melatonin receptors, Physical and Theoretical Chemistry, Receptor, Molecular Biology, Gene, lcsh:QH301-705.5, Spectroscopy, ComputingMilieux_MISCELLANEOUS, Cloning, Genetics, biology, Organic Chemistry, molecular pharmacology, General Medicine, biology.organism_classification, European hamster (Cricetus cricetus), Stop codon, Computer Science Applications, 030104 developmental biology, lcsh:Biology (General), lcsh:QD1-999, 030217 neurology & neurosurgery
Abstract

For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT₁ and MT₂) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT₁, MT₂, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT₁ receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (Phodopus sungorus) expresses a non-functional MT₂. We observed that unlike this hamster, the European hamster (Cricetus cricetus) does not have a stop codon in the MT₂ sequence. Thus, we undertook the tedious task of cloning the MT₂ receptor. We partially succeeded, sequencing the complete exon 2 and a fragment of exon 1 (from putative amino acids 12 to 38 and 77 to 323), after several years of efforts. In order to show that the protein parts we cloned were capable to sustain some binding capacities, we designed a chimeric MT₂ receptor using a consensus sequence to replace the unknown amino acids, based on other small rodent MT₂ sequences. This chimeric construct could bind melatonin in the nanomolar range. This work is meant to be the basis for attempts from other laboratories of the community to determine the complete natural sequence of the European hamster MT₂ receptor. The present work is the first to show that, among the hamsters, if the Siberian is a natural knockout for MT₂, the European one is not.

Published
2018
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3. Gene expression profiling during hibernation in the European hamster

Author

Giulia Lizzo, Olivier Nosjean, Sophie-Pénélope Guenin, Clémence Dupré, Chantal Brasseur, Valérie Simonneaux, Célia Gautier, Jean-Philippe Stephan, Damien Valour, Béatrice Bothorel, Albane Gaudeau, Dominique Ciocca, Jean A. Boutin, Isabelle Riest-Fery, PEX Biotechnologie Chimie & Biologie, Institut des Neurosciences Cellulaires et Intégratives (INCI), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), PEX Méthodologie et Valorisation des Données, Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biotechnologies, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Centre de Recherche de Croissy, Institut de Recherche Servier, Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Biologie du Développement et Reproduction (BDR), Institut National de la Recherche Agronomique (INRA), Biotechnologies, Pharamcologie Moléculaire et Cellulaire, Centre de Recherches de Croissy, Équipe 'Rythme, vie et mort de la rétine', Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), and Université de Strasbourg (UNISTRA)-Institut des Neurosciences Cellulaires et Intégratives (INCI)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Hibernation, Male, [SDV]Life Sciences [q-bio], Adipose tissue, lcsh:Medicine, THYROID-HORMONE, CIRCADIAN CLOCK, METABOLIC REGULATOR, Energy homeostasis, Adipose Tissue, Brown, Brown adipose tissue, lcsh:Science, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, 13-LINED GROUND-SQUIRRELS, BROWN ADIPOSE-TISSUE, Period Circadian Proteins, SUPRACHIASMATIC NUCLEUS, Circadian Rhythm, CLOCK, Europe, medicine.anatomical_structure, BODY-WEIGHT, Triiodothyronine, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Arousal, PER1, medicine.medical_specialty, Period (gene), Hypothalamus, Biology, Iodide Peroxidase, Article, DAILY TORPOR, 03 medical and health sciences, Cricetulus, Internal medicine, medicine, Animals, Gene Expression Profiling, lcsh:R, Molecular Sequence Annotation, Torpor, PARS TUBERALIS, 030104 developmental biology, Endocrinology, Gene Ontology, Gene Expression Regulation, lcsh:Q, Energy Metabolism, MAMMALIAN HIBERNATION
Abstract

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body’s clocks at the beginning of the active period.

Published
2018
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4. Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6): Chemical Synthesis of Catalytically Active Calstabin and Mutants

Author

Leonard M. G. Chavas, Magali Jullian, Gilles Ferry, Mathias Antoine, Serena Sirigu, Lisa Bruyand, Olivier Nosjean, Karine Puget, Benjamin Fould, Tiphaine Huet, Laurent Vuillard, Luisa Ronga, Marine Bacchi, Jean A. Boutin, Institut de Recherches Internationales Servier [Suresnes] (IRIS), Institut Curie [Paris], Pharmacologie Moléculaire et Cellulaire, Institut de Recherche Servier, Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Institut de Recherches Servier, Centre de Recherches de Croissy, Régulations Naturelles et Artificielles (ARNA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Bordeaux Ségalen [Bordeaux 2], Institut des sciences analytiques et de physico-chimie pour l'environnement et les materiaux (IPREM), Université de Pau et des Pays de l'Adour (UPPA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Biotechnologies, Pharmacologie Moléculaire et Cellulaire, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Biotechnologies, Pharamcologie Moléculaire et Cellulaire, and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)

Subjects
chemistry.chemical_classification, 010405 organic chemistry, calstabin, Chemical biology, Total synthesis, catalytic activity, Isomerase, 010402 general chemistry, Native chemical ligation, 01 natural sciences, Biochemistry, Chemical synthesis, 0104 chemical sciences, Amino acid, Enzyme, FKBP, chemistry, [CHIM]Chemical Sciences, synthetic biology, total synthesis, crystallography, Molecular Biology
Abstract

International audience; Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.

Published
2016
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5. Piceatannol and resveratrol share inhibitory effects on hydrogen peroxide release, monoamine oxidase and lipogenic activities in adipose tissue, but differ in their antilipolytic properties

Author

Pascale Mauriège, Simon Deleruyelle, Picard Marceau, Francisco Les, Jean A. Boutin, Christian Carpéné, Balázs Balogh, Françoise Nepveu, Denis Richard, Simon Biron, Laure-Estelle Cassagnes, Jose M. Arbones-Mainar, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Universidad San Jorge, Hôpital de Rangueil, CHU Toulouse [Toulouse], Pharmacochimie et Biologie pour le Développement (PHARMA-DEV), Institut de Recherche pour le Développement (IRD)-Institut de Chimie de Toulouse (ICT-FR 2599), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC), Institut de Recherches SERVIER (IRS), Semmelweis University [Budapest], Instituto Aragonés de Ciencias de la Salud [Zaragoza] (IACS), Faculté de médecine de l'Université Laval [Québec] (ULaval), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Institut de Recherche pour le Développement (IRD)-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), Université de Toulouse (UT), Université Laval [Québec] (ULaval), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Biotechnologies, Pharamcologie Moléculaire et Cellulaire, Institut de Recherches Servier, Laboratoire de Logique, Algorithmique et Informatique (LLAIC1), Université d'Auvergne - Clermont-Ferrand I (UdA), Institut de médecine moléculaire de Rangueil (I2MR), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-IFR150-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Adult, 0301 basic medicine, Benzylamines, Amine oxidase, Monoamine oxidase, Lipolysis, [SDV]Life Sciences [q-bio], Amine oxidases, Subcutaneous Fat, Tyramine, Adipose tissue, Resveratrol, Toxicology, Dietary stilbenes, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Adipocyte, Stilbenes, Adipocytes, Animals, Humans, Obesity, Monoamine Oxidase, ComputingMilieux_MISCELLANEOUS, Piceatannol, biology, Lipogenesis, Electron Spin Resonance Spectroscopy, food and beverages, General Medicine, Catalase, Oxidants, Hydrogen peroxide, Mice, Inbred C57BL, Molecular Docking Simulation, 030104 developmental biology, chemistry, Biochemistry, Oxidative stress, 030220 oncology & carcinogenesis, Adipocyte lipolysis, Biocatalysis, biology.protein, Female
Abstract

International audience; Piceatannol is a hydroxylated derivative of resveratrol. While both dietary polyphenols coexist in edible plants and fruits, and share equivalent concentrations in several wines, the influence of piceatannol on adiposity has been less studied than that of resveratrol. Though resveratrol is now recognized to limit fat deposition in various obesity models, the benefit of its dietary supplementation remains under debate regarding human obesity treatment or prevention. The research for more potent resveratrol analogs is therefore still undergoing. This prompted us to compare various effects of piceatannol and resveratrol directly on human adipose tissue (hAT). Hydrogen peroxide release was measured by Amplex Red-based fluorescence in subcutaneous hAT samples from obese patients. Interactions of stilbenes with human amine oxidases and quinone reductase were assessed by radiometric methods, computational docking and electron paramagnetic resonance. Influences on lipogenic and lipolytic activities were compared in mouse adipocytes. Resveratrol and piceatannol inhibited monoamine oxidase (MAO) with respective IC50 of 18.5 and 133.7 μM, but not semicarbazide-sensitive amine oxidase (SSAO) in hAT. For both stilbenes, the docking scores were better for MAO than for SSAO. Piceatannol and resveratrol similarly hampered hydrogen peroxide detection in assays with and without hAT, while they shared pro-oxidant activities when incubated with purified quinone reductase. They exhibited similar dose-dependent inhibition of adipocyte lipogenic activity. Only piceatannol inhibited basal and stimulated lipolysis when incubated at a dose ≥100 μM. Thus, piceatannol exerted on fat cells dose-dependent effects similar to those of resveratrol, except for a stronger antilipolytic action. In this regard, piceatannol should be useful in limiting the lipotoxicity related to obesity when ingested or administered alone - or might hamper the fat mobilization induced by resveratrol when simultaneously administered with it.

Published
2016
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6. Characterization of the Mel1c melatoninergic receptor in platypus (Ornithorhynchus anatinus)

Author

Frank Grützner, Olivier Nosjean, Valérie Simonneaux, Jean A. Boutin, Tahlia Perry, Céline Legros, Célia Gautier, Sophie-Pénélope Guenin, Isabelle Riest-Fery, Institut de Recherches SERVIER (IRS), Institut des Neurosciences Cellulaires et Intégratives (INCI), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), University of Adelaide, Équipe 'Rythme, vie et mort de la rétine', Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Biotechnologies, Pharamcologie Moléculaire et Cellulaire, Institut de Recherches Servier, Centre de Recherche de Croissy, Institut de Recherche Servier, and Université de Strasbourg (UNISTRA)-Institut des Neurosciences Cellulaires et Intégratives (INCI)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Xenopus, [SDV]Life Sciences [q-bio], Receptors, Melatonin, lcsh:Medicine, Plasma protein binding, Biochemistry, Poultry, Chlorocebus aethiops, Gamefowl, Monotremes, Cloning, Molecular, lcsh:Science, Receptor, Phylogeny, ComputingMilieux_MISCELLANEOUS, Melatonin, Data Management, Mammals, Multidisciplinary, biology, Eukaryota, Phylogenetic Analysis, Animal Models, Molecular Pharmacology, Cell biology, Phylogenetics, Experimental Organism Systems, Vertebrates, COS Cells, Frogs, hormones, hormone substitutes, and hormone antagonists, Research Article, Protein Binding, medicine.drug, Computer and Information Sciences, Research and Analysis Methods, Melatonin receptor, Amphibians, Birds, 03 medical and health sciences, Model Organisms, biology.animal, [SDV.BA.ZV]Life Sciences [q-bio]/Animal biology/Vertebrate Zoology, medicine, Animals, Evolutionary Systematics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Platypus, Taxonomy, Evolutionary Biology, Base Sequence, Receptor, Melatonin, MT2, Receptor, Melatonin, MT1, lcsh:R, Organisms, Biology and Life Sciences, Hormones, 030104 developmental biology, Fowl, GPR50, Amniotes, lcsh:Q, Chickens
Abstract

International audience; Melatonin is a neurohormone produced in both animals and plants. It binds at least three G-protein-coupled receptors: MT1 and MT2, and Mel1cGPR. Mammalian GPR50 evolved from the reptilian/avian Mel1c and lost its capacity to bind melatonin in all the therian mammal species that have been tested. In order to determine if binding is lost in the oldest surviving mammalian lineage of monotremes we investigated whether the melatonin receptor has the ability to bind melatonin in the platypus (Ornithorhynchus anatinus), and evaluated its pharmacological profile. Sequence and phylogenetic analysis showed that platypus has in fact retained the ancestral Mel1c and has the capacity to bind melatonin similar to other mammalian melatonin receptors (MT1 and MT2), with an affinity in the 1 nM range. We also investigated the binding of a set of melatoninergic ligands used previously to characterize the molecular pharmacology of the melatonin receptors from sheep, rats, mice, and humans and found that the general profiles of these compounds make Mel1c resemble human MT1 more than MT2. This work shows that the loss of GPR50 binding evolved after the divergence of monotremes less than 190MYA in therian mammals.

Published
2018
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7. Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles

Author

Jacky Marie, Gilles Ferry, Christel Logez, Olivier Nosjean, Jean Martinez, Clémence Dupré, Mélody Guéry, Benjamin Fould, Jean-Alain Fehrentz, Jean A. Boutin, Renaud Wagner, Jean-Louis Banères, Sophie Mary, Céline Legros, Marjorie Damian, Céline M'Kadmi, Biotechnologie et signalisation cellulaire (BSC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biotechnologies, Pharamcologie Moléculaire et Cellulaire, Institut de Recherches Servier, Pharmacologie Moléculaire et Cellulaire, Institut de Recherche Servier, and Centre de Recherche de Croissy

Subjects
0301 basic medicine, Models, Molecular, G protein, Receptors, Melatonin, CHO Cells, Biochemistry, Pichia, Pichia pastoris, 03 medical and health sciences, Cricetulus, GTP-Binding Proteins, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Receptor, Receptors, Ghrelin, Integral membrane protein, ComputingMilieux_MISCELLANEOUS, G protein-coupled receptor, biology, Maleates, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, Ligand (biochemistry), Lipids, Cell biology, Nanostructures, 030104 developmental biology, Membrane, Solubility, Liposomes, Polystyrenes, Signal transduction
Abstract

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.

Published
2015
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8. Description of the constitutive activity of cloned human melatonin receptors hMT1 and hMT2 and discovery of inverse agonists

Author

Devavry, Séverine, Legros, Celine, Brasseur, Chantal, Delagrange, Philippe, Spadoni, Gilberto, Cohen, William, Malpaux, Benoit, Boutin, J.A., Nosjean, Olivier, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Biotechnologies, Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, Département des Sciences Expérimentales, Istituto di Chimica Farmaceutica e Tossicologica, Università degli Studi di Urbino 'Carlo Bo', Biotechnologies, Pharamcologie Moléculaire et Cellulaire, Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], binding, biochemical description, recombinant melatonin receptor, inverse agonist, constitutive activity, hormones, hormone substitutes, and hormone antagonists
Abstract

Melatonin receptors have been described to activate different G proteindependent signaling pathways, both in laboratory, heterologous, cellular models and in physiological conditions. Furthermore, the constitutive activity of G proteincoupled receptors has been shown to be key in physiological and pathological conditions. In the case of melatonin receptors, information is rather scare and concerns only MT1 receptors. In the present report, we show that the G proteincoupled melatonin receptors do have a constitutive, nonmelatonin-induced signaling activity using two cellular models of different origins, the Chinese hamster ovary cell line and Neuro2A, a neuroblastoma cell line. Furthermore, we show that this constitutive activity involves mainly Gi proteins, which is consistent with the common knowledge on the melatonin receptors. Importantly, we also describe, for the first time, inverse agonist properties for melatonin ligands. Although it is clear than more in-depth, biochemistry-based studies will be required to better understand by which pathway(s) the constitutively active melatonin receptors transfer melatonin information into intracellular biochemical events; our data open interesting perspectives for understanding the importance of the constitutive activity of melatonin receptors in physiological conditions.

Published
2012
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