CARACTERISATION FONCTIONNELLE DU SITE ACTIF DE L'AMINOPEPTIDASE A PAR MUTAGENESE DIRIGEE. MISE EN EVIDENCE DE NOUVEAUX MOTIFS CONSENSUS DES AMINOPEPTIDASES MONOZINCS

PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Elabela/Toddler and apelin bind differently to the apelin receptor

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Etudes structure-fonction du récepteur de l'apéline par modélisation moléculaire et mutagenèse dirigée (recherche d'agonistes et/ou d'antagonistes de ce récepteur)

L'apéline est le ligand naturel du récepteur orphelin humain APJ, un récepteur à sept domaines transmembranaires, couplé aux protéines G (RCPG). L apéline et son récepteur jouent un rôle important dans le maintien de l équilibre hydrique et dans la régulation des fonctions cardiovasculaires et métaboliques. Afin de développer des agonistes ou des antagonistes de ce récepteur, inexistants lorsque j ai commencé mes travaux de thèse, il est important d avoir des informations sur l organisation structurale et fonctionnelle du récepteur ainsi que de son ligand, afin de comprendre comment l apéline interagit avec celui-ci et comment elle l active. Pour cela, nous avons construit en collaboration avec le Dr B. Maigret, le premier modèle tridimensionnel (3D) du récepteur de l'apéline complexé avec pE13F et K17F, par homologie avec le modèle validé du récepteur de la cholécystokinine de type 1. Le modèle obtenu a permis de visualiser plusieurs interactions entre l apéline et son récepteur. Parmi celles-ci, deux arginines de l apéline interagissent en surface du récepteur avec des résidus acides. La phénylalanine C-terminale de l'apéline, nécessaire à l internalisation du récepteur de l'apéline et à l effet hypotenseur de l apéline, s insère dans une poche aromatique composée des résidus aromatiques Trp 152 et Trp 259 et Phe 255, située au fond du site de liaison du récepteur. Nous avons vérifié ces interactions par des études structure-fonction par mutagenèse dirigée sur le récepteur de l'apéline. Nous avons ainsi montré que la Phe 255 et le Trp 259 interagissent avec la phénylalanine C-terminale de l apéline et qu ils jouent un rôle clé dans l'internalisation du récepteur de l'apéline, sans jouer de rôle dans la liaison de l apéline à son récepteur, ni dans le couplage du récepteur à la protéine Gi. Cette étude représente une première validation du modèle 3D du récepteur de l apéline. Nous nous sommes ensuite intéressés aux résidus du récepteur de l'apéline impliqués dans la liaison de l apéline à son récepteur. Suite à la publication récente des structures cristallographiques de RCPG, nous avons construit avec le Dr B. Maigret, deux nouveaux modèles 3D du récepteur de l apéline sur la base des structures cristallographiques des récepteurs b2-adrénergique et CXCR4. Dans ces trois modèles, nous avons observé une superposition parfaite de la poche aromatique décrite ci-dessus alors que de grandes différences sont apparues dans la nature des résidus interagissant avec l'apéline à la surface du récepteur. Par mutagenèse dirigée, nous avons montré que l Asp92, le Glu172 et l Asp282 sont des résidus essentiels à la liaison de l apéline à son récepteur. Ceci nous a permis d'apporter une seconde validation du modèle 3D du récepteur de l'apéline et de sélectionner celui fondé sur la structure du CXCR4. Ce modèle pourra être utilisé pour réaliser des campagnes de criblage in silico de chimiothèques virtuelles ainsi que pour développer un pharmacophore d agoniste ou d antagoniste du récepteur de l apéline.Apelin is the endogenous ligand of the receptor APJ, a seven-transmembrane domain receptor coupled to G-protein. Apelin and its receptor play an important role in the regulation of body fluid homeostasis and cardiovascular functions. In order to design agonists or antagonists for the apelin receptor, nonexistent at the beginning of my thesis, it was important to define the structural elements in apelin and its receptor, required for apelin binding and subsequent receptor activation. To this end, we built, in collaboration with Dr. B. Maigret, the first three-dimensional (3D) model of the human apelin receptor complexed with pE13F or K17F, using a validated model of the cholecystokinin receptor-1 as a template. This model has shown different interactions between the apelin and its receptor. Among them, two arginines of the peptide interact with acidic residues at the top of the receptor. In addition, the C-terminal Phe of apelin, crucial for the internalization of the receptor and for the hypotensive effect of the apelin, is embedded in a hydrophobic pocket constituted by Trp 152, Phe 255 and Trp 259 and located at the bottom of the receptor. The reality of these interactions was evaluated by structure-function studies by site-directed mutagenesis on the apelin receptor. We then demonstrated that Phe 255 and Trp 259, by interacting with the C-terminal Phe of the peptide, are crucial for apelin receptor internalization without playing a role in apelin binding nor in the Gai coupling. This study represents the first validation of the apelin receptor 3D model. In order to pursue this validation, we focused our interest on the receptor surface to identify the residues involved in the apelin binding. For this purpose, two additional 3D models based on the b2-adrenergic and CXCR4 crystallographic structures recently published were built. The comparison of the three models showed a perfect superimposition of the hydrophobic pocket whereas the organization and the nature of the acidic residues implicated in the apelin binding are very different. In order to validate definitively one of these 3D models, we evaluated the role of these residues in the apelin binding by site-directed-mutagenesis. This study resulted in the identification of three key residues: Asp 92, Glu172 and Asp 282. These results validated the 3D model based on the CXCR4 crystallographic structure. This validated model will be used to perform in silico screening of virtual chemical libraries and may lead to the identification of agonists or antagonists of the apelin receptor.PARIS-BIUP (751062107) / SudocSudocFranceF

Contribution of Molecular Modeling and Site-directed Mutagenesis to the Identification of Two Structural Residues, Arg-220 and Asp-227, in Aminopeptidase A

International audienceAminopeptidase A is a zinc metalloenzyme involved in the formation of brain angiotensin III, which exerts a tonic stimulatory action on the central control of blood pressure. Thus, central inhibitors of aminopeptidase A constitute putative central antihypertensive agents. Mutagenic studies have been performed to investigate organization of the aminopeptidase A active site, with a view to designing such inhibitors. The structure of one monozinc aminopeptidase (leukotriene A(4) hydrolase) was recently resolved and used to construct a three-dimensional model of the aminopeptidase A ectodomain. This new model, highly consistent with the results of mutagenic studies, showed a critical structural interaction between two conserved residues, Arg-220 and Asp-227. Mutagenic replacement of either of these two residues disrupted maturation and subcellular localization and abolished the enzymatic activity of aminopeptidase A, confirming the critical structural role of these residues. In this study, we generated the first three-dimensional model of a strict aminopeptidase, aminopeptidase A. This model constitutes a new tool to probe further the active site of aminopeptidase A and to design new inhibitors of this enzyme

Multiple Cross Talk between Angiotensin II, Bradykinin, and Insulin Signaling in the Cortical Thick Ascending Limb of Rat Kidney

International audienceCortical thick ascending limb (CTAL) naturally expresses the angiotensin II (AngII) receptor type 1A (AT1 -R), the bradykinin (BK) receptor type 2 (B2 -R), and the insulin receptor. This segment is made of a single morphologically distinct cell type. AngII and BK are involved in same transduction pathways but differ markedly in their physiological actions on Na transport. Besides, the insulin signaling intersects with those of AngII and BK at multiple levels and especially by stimulation on Na reabsorption. Thus, the CTAL is a biologically suitable model to study the cross talk between G protein-coupled receptors or G protein-coupled receptors and receptor tyrosine kinase. In this work, the cross talks between AngII, BK, and insulin signaling are studied in rat CTAL by measuring changes in [Ca2]i . We show that BK exerts negative modulatory effects on AngII-induced [Ca2]i responses dependent on tyrosine kinase and MAPK pathways. Moreover, in the presence of BK, AngII-induced Na transport is suppressed. These effects suggest an interaction between AT1 -R and B 2 -R. We show a positive interaction between the insulin receptor and the AT1 -R through a protein kinase A-dependent mechanism that involves MAPK cascade, leading to the stimulation of the Ca2 influx induced by AngII. The presence of such interactions brings additional arguments for a complex and fine regulation of CTAL functions and puts forward the potentially beneficial effect of BK across this segment, in case of hyperinsulinemia or insulin resistance, by its negative feedback on AngII actions

Study of Asparagine 353 in Aminopeptidase A: Characterization of a Novel Motif (GXMEN) Implicated in Exopeptidase Specificity of Monozinc Aminopeptidases

International audienceAminopeptidase A (EC 3.4.11.7, APA) is a 160 kDa membrane-bound zinc enzyme that contains the HEXXH consensus sequence found in members of the zinc metalloprotease family, the zincins. In addition, the monozinc aminopeptidases are characterized by another conserved motif, GXMEN, the glutamate residue of which has been shown to be implicated in the exopeptidase specificity of aminopeptidase A [Vazeux G. (1998) Biochem. J. 334, 407-413]. In carboxypeptidase A (EC 3.4.17.1, CPA), the exopeptidase specificity is conferred by an arginine residue (Arg-145) and an asparagine residue (Asn-144). Thus, we hypothesized that Asn-353 of the GXMEN motif in APA plays a similar role to Asn-144 in CPA and contributes to the exopeptidase specificity of APA. We investigated the functional role of Asn-353 in APA by substituting this residue with a glutamine (Gln-353), an alanine (Ala-353) or an aspartate (Asp-353) residue by site-directed mutagenesis. Expression of wild-type and mutated APAs revealed that Gln-353 and Ala-353 are similarly routed and glycosylated to the wild-type APA, whereas Asp-353 is trapped intracellularly and partially glycosylated. Kinetic studies, using alpha-L-glutamyl-beta-naphthylamide (GluNA) as a substrate showed that the K(m) values of the mutants Gln-353 and Ala-353 were increased 11- and 8-fold, respectively, whereas the k(cat) values were decreased (2-fold) resulting in a 24- and 14-fold reduction in cleavage efficiency. When alpha-L-aspartyl-beta-naphthylamide or angiotensin II were used as substrates, the mutations had a greater effect on k(cat), leading to a similar decrease in cleavage efficiencies as that observed with GluNA. We then measured the inhibitory potencies of several classes of inhibitors, glutamate thiol, glutamine thiol and two isomers (L- or D-) of glutamate phosphonate to explore the functional role of Asn-353. The data indicate that Asn-353 is critical for the integrity and catalytic activity of APA. This residue is involved in substrate binding via interactions with the free N-terminal part and with the P1 carboxylate side chain of the substrate. In conclusion, Asn-353 of the GXMEN motif, together with Glu-352, contributes to the exopeptidase specificity of APA and plays an equivalent role to Asn-144 in CPA