Foncteurs dérivés de l'algèbre symétrique (application au calcul de certains groupes d'homologie fonctorielle des espaces K (B, n))

PARIS13-BU Sciences (930792102) / SudocSudocFranceF

Confocal and TIRF microscopy based approaches to visualize arrestin trafficking in living cells

International audienceArrestins are key proteins that serve as versatile scaffolds to control and mediate G protein coupled receptors (GPCR) activity. Arrestin control of GPCR functions involves their recruitment from the cytosol to plasma membrane-localized GPCRs and to endosomal compartments, where they mediate internalization, sorting and signaling of GPCRs. Several methods can be used to monitor trafficking of arrestins; however, live fluorescence imaging remains the method of choice to both assess arrestin recruitment to ligand-activated receptors and to monitor its dynamic subcellular localization. Here, we present two approaches based on Total Internal Fluorescence (TIRF) microscopy and confocal microscopy to visualize arrestin trafficking in live cells in real time and to assess their co-localization with the GPCR of interest and their localization at specific subcellular locations

Récepteur V2 de la vasopressine (à la recherche de partenaires d'interaction et d'outils thérapeutiques)

L arginine vasopressine (AVP) est une hormone qui au niveau du rein présente une activité antidiurétique permettant le contrôle de l osmolalité, du volume sanguin et de la pression artérielle. Cette action est médiée par le récepteur membranaire V2 (RV2) localisé dans les cellules principales du tubule collecteur du rein. Une pathologie génétique rare, le diabète insipide néphrogénique congénital (DINc), est associée à une absence de réponse à l AVP et est due majoritairement à des mutations du RV2. A ce jour, plus de 200 mutations du gène du RV2 ont été identifiées dont la plupart conduisent à une séquestration intracellulaire du récepteur, ce qui l empêche de répondre à l AVP. Dans ce contexte, nous avons cherché à mieux comprendre et à caractériser les mécanismes impliqués dans la séquestration intracellulaire du RV2 dans le DINc. Nous avons aussi recherché les partenaires protéiques potentiellement impliqués dans cette rétention intracellulaire. Enfin, d un point de vue thérapeutique, nous avons caractérisé de nouveaux outils pour le traitement du DINc. Nous avons donc étudié le rôle probable de deux clusters arginines situés sur les boucles intracellulaires i1 et i3 du RV2 dans les mécanismes de séquestration. Nous avons constaté que l absence du cluster de la boucle i1 est létale pour le repliement du récepteur tandis que celui de la boucle i3 semble à l origine d un contrôle du trafic vers la membrane plasmique. Par ailleurs, à l aide d un peptide mimétique de la boucle i3 et/ou avec le récepteur entier, nous avons identifié de probables acteurs protéiques impliqués dans la rétention intracellulaire. Nous nous sommes notamment intéressés à la protéine P32 (gC1qR) pour laquelle nous avons validé l interaction directe de cette protéine avec le cluster arginines de la boucle i3 du RV2 et vérifié qu elle pouvait interagir avec le récepteur entier. Nous avons aussi pu montrer une interaction de P32 avec les mutants du DINc suggérant ainsi le rôle de cette protéine dans la régulation du trafic. Enfin, l absence de traitements spécifiques et efficaces pour le DINc nous a conduit à caractériser de nouvelles molécules permettant de rétablir la fonction biologique. Nous avons donc montré que de nouveaux ligands agonistes nonpeptidiques étaient capables de restaurer l adressage à la membrane plasmique et la fonction de plusieurs mutants du DINc. Nos travaux ont ainsi permis de caractériser de nouvelles pharmacochaperones potentiellement efficaces pour plusieurs mutants du DINc.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

Design and synthesis of cyclic and linear peptide-agarose tools for baiting interacting protein partners of GPCRs

International audienceA ligation strategy for the synthesis of cyclic and linear peptides covalently linked to agarose beads designed as baits to identify new interacting partners of intracellular loops of the V2 vasopressin receptor, a member of the G-protein-coupled receptor family, is reported. The peptide-resin conjugates were subsequently shown to interact specifically with a fraction of proteins present in cellular lysates

Intracellular VHHs to monitor and modulate GPCR signaling

International audienceSingle-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reflects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientific community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010's. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or b-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the field of GPCR signaling and trafficking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery

Tag retention and effects of PIT-tagging on survival and growth of Salmo trutta : preliminary results

Tag retention and effects of PIT-tagging on survival and growth of Salmo trutta : preliminary results. 1. International Workshop on PIT Telemetr

Tag retention and effects of PIT-tagging on survival and growth of Salmo trutta : preliminary results

Tag retention and effects of PIT-tagging on survival and growth of Salmo trutta : preliminary results. 1. International Workshop on PIT Telemetr

Biased GPCR signaling by the native parathyroid hormone–related protein 1 to 141 relative to its N-terminal fragment 1 to 36

International audienceThe parathyroid hormone (PTH)–related protein (PTHrP) is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling, and causes hypercalcemia in patients with malignancy. Although mature forms of PTHrP in the body consist of splice variants of 139, 141, and 173 amino acids, our current understanding on how endogenous PTHrP transduces signals through its cognate G-protein coupled receptor (GPCR), the PTH type 1 receptor (PTHR), is largely derived from studies done with its N-terminal fragment, PTHrP1-36. Here, we demonstrate using various fluorescence imaging approaches at the single cell level to measure kinetics of (i) receptor activation, (ii) receptor signaling via Gs and Gq, and (iii) receptor internalization and recycling that the native PTHrP1-141 displays biased agonist signaling properties that are not mimicked by PTHrP1-36. Although PTHrP1–36 induces transient cAMP production, acute intracellular Ca2+ (iCa2+) release and β-arrestin recruitment mediated by ligand–PTHR interactions at the plasma membrane, PTHrP1-141 triggers sustained cAMP signaling from the plasma membrane and fails to stimulate iCa2+ release and recruit β-arrestin. Furthermore, we show that the molecular basis for biased signaling differences between PTHrP1-36 and properties of native PTHrP1-141 are caused by the stabilization of a singular PTHR conformation and PTHrP1-141 sensitivity to heparin, a sulfated glycosaminoglycan. Taken together, our results contribute to a better understanding of the biased signaling process of a native protein hormone acting in conjunction with a GPCR

The Polycystin-1, Lipoxygenase, and α -Toxin Domain Regulates Polycystin-1 Trafficking

International audienc

Loi relative à l'enseignement

Bedeau Marie Alphonse, Arnaud Frédéric, Chapot Jean-Jacques François, Lacaze , Peupin , Bérard , Bonaparte Louis-Napoléon. Loi relative à l'enseignement. In: Bulletin administratif de l'instruction publique. Tome 1 n°3, mars 1850. pp. 57-80