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International audience; Gurmarin is a polypeptide isolated from the Indian plant Gymnema sylvestre, which specifically suppresses sweet taste in rodents without affecting responses to other basic taste stimuli, such as HCl, NaCl, and quinine. Although the exact mechanism of gurmarin inhibition is not known, it has been shown that gurmarin acts via the T1R2/T1R3 sweet taste receptor. The gurmarin molecule is made of 35 amino-acid residues and three intramolecular disulfide bridges. We report herein the 1.45 Å X-ray structure of gurmarin heterologously produced using the yeast Pichia pastoris. The structure revealed a typical knottin fold, which is compared with previously reported NMR solution structures. The atomic structure at this resolution allowed us to highlight a flexible region involving hydrophobic amino acid residues previously identified as a putative binding motif for the rat sweet taste receptor. By combining cellular based receptor assay and site-directed mutagenesis of gurmarin, we revealed that several amino acid residues located in this hydrophobic cluster of gurmarin severely affect rat sweet taste receptor inhibition. This study demonstrates that gurmarin can be used as a worthwhile tool to decipher the mechanism of sweet taste inhibition.

Gurmarin is a highly specific sweet-taste suppressing protein in rodents that is isolated from the Indian plant Gymnemasylvestre. Gurmarin consists of 35 amino acid residues containing three intramolecular disulfide bridges that form a cystine knot. Here, we report the crystal structure of gurmarin at a 1.45 Å resolution and compare it with previously reported NMR solution structures. The atomic structure at this resolution allowed us to identify a very flexible region consisting of hydrophobic residues. Some of these amino acid residues had been identified as a putative binding site for the rat sweet taste receptor in a previous study. By combining alanine-scanning mutagenesis of the gurmarin molecule and a functional cell-based receptor assay, we confirmed that some single point mutations in these positions drastically affect sweet taste receptor inhibition by gurmarin.

Nous percevons le goût sucré à l’aide d’un récepteur unique, appelé T1R2/T1R3. Ce récepteur nous permet de détecter tous les divers composés chimiques que nous percevons comme sucrés. De façon surprenante, des études récentes ont montré qu’il était présent dans de nombreux tissus et organes en dehors de la cavité orale. Parce qu’il joue un rôle dans le contrôle métabolique, ce récepteur représente une cible potentielle et prometteuse pour le traitement de l’obésité et des maladies métaboliques, telles que le diabète et l’obésité via notamment l’utilisation d’inhibiteurs spécifiques.

International audience

Functional architecture of sweet taste receptors. 10.wartburg symposium, Flavor chemistry and biology

The sweet taste receptor is a heterodimer composed of two subunits called T1R2 and T1R3 whereas the T1R1 and the T1R3 subunits form a heterodimeric receptor for umami taste (the savory taste of monosodium glutamate). Each subunit belongs to the class C of G protein-coupled receptors (GPCRs) and is constituted by a large extracellular Nterminal domain (NTD) linked to the transmembrane domain by a cysteine-rich region. The NTD is composed of two lobes separated by a cleft in which ligands bind. T1R1- and T1R2-NTDs are able to bind sweeteners and umami compounds respectively and undergo ligand-dependent conformational changes (Zhang et al., 2008 ; Nie et al., 2005). However, the relative contribution of the two subunits to the heterodimeric receptor function remains largely unknown. To study the binding specificity of each subunit, a large amount of purified NTDs is suitable for biochemical and structural studies. To accomplish this goal, we expressed high level of T1R1- and T1R2-NTD as insoluble aggregated proteins (inclusion bodies), using Escherichia coli. The proteins were solubilized and in vitro refolded using suitable buffer and additives. The soluble proteins were then purified and characterized using electrophoresis, gel filtration, fluorescence spectroscopy and circular dichroism. The functionality of T1R1- and T1R2-NTD was measured the using isothermal microcalorimetry. Our data showed that the proteins are properly refolded and able to bind sweet or umami compounds with physiological relevant affinities. In summary, our expression system will allow large-scale production of active T1R1- and T1R2-NTD suitable for structural and functional studies. In addition to this work, we have studied a 35 residue polypeptide named gurmarin, well known to selectively inhibit responses to sweet substances without affecting responses to other basic taste stimuli, such as NaCl, HCl, and quinine in rodents. To further understand the structural basis of gurmarin recognition by T1R2-T1R3 receptor, we developed for the first time the heterologous expression of gurmarin using the methylotrophic yeast Pichia pastoris. This system allowed the expression of large quantities of recombinant gurmarin. The structural properties of gurmarin were checked by circular dichroism and nuclear magnetic resonance. We generated six mutants with single amino acids substitutions in the putative site of interaction between gurmarin and the rodent sweet taste receptor, using site-directed mutagenesis. In collaboration, the biological activity of was confirmed using a cell-based assay based on expression of rat T1R2-T1R3. Thanks to various combinations of human and rat T1R2-T1R3 chimeras, we showed that NTD of rat T1R3 is the major determinant of gurmarin’s inhibition., Le récepteur au goût sucré est un hétérodimère composé des sous-unités T1R2 et T1R3, alors que les sous-unités T1R1 et T1R3 s’assemblent pour composer le récepteur au goût umami. Chacune de ces sous-unités appartient à la classe C des récepteurs couplés aux protéines G (RCPG). Les membres de cette famille partagent une architecture commune, constituée d’un domaine N- terminal (DNT) qui est relié à un domaine transmembranaire par une région riche en cystéines. Le DNT est constitué de deux lobes qui forment le site orthostérique, qui est le site de liaison des principaux agonistes. Il a été montré que les DNT de T1R1 (DNT-T1R1) est capable de lier le L- glutamate alors que le DNT de T1R2 (DNT-T1R2) est capable de lier les sucres naturels et certains édulcorants (Zhang et al., 2008 ; Nie et al., 2005). Cependant les mécanismes moléculaires de détections des molécules sapides au niveau de ces domaines restent encore largement méconnus. Lors de cette étude, nous avons exprimé les DNT-T1R1, DNT-T1R2 humains et le DNT-T1R1 de souris à l’aide de la bactérie Escherichia coli, sous forme de protéines insolubles, appelés corps d’inclusion (CI). Les CI ont été purifiés puis solubilisés en utilisant un agent chaotrope. Ensuite, les protéines ont été repliées in vitro puis caractérisées par différentes approches parmi lesquelles l’électrophorèse, la filtration sur gel, la fluorescence et le dichroïsme circulaire. Leur fonctionnalité a ensuite été vérifiée par microcalorimétrie de titration isotherme. Nous avons montré que les protéines sont fonctionnelles et présentent des affinités en accord avec les données sensorielles. Les grandes quantités de protéines produites permettront de futures études structurales par cristallographie. Parallèlement à ce travail, nous avons étudié un polypeptide végétal, inhibiteur des goûts sucré et umami chez les rongeurs, appelé gurmarine. Afin de pouvoir étudier son interaction avec les DNT des récepteurs gustatifs de souris, nous avons mis au point l’expression et la purification de gurmarine recombinante produite en levure Pichia pastoris. Ce système d’expression a permis la production de quantités importantes de gurmarine dont les caractéristiques structurales ont été vérifiées par dichroïsme circulaire et résonnance magnétique nucléaire. Nous avons aussi, par mutagénèse dirigée, modifié différents acides aminés décrits comme étant potentiellement impliqués dans l’interaction avec les récepteurs aux goûts sucré et umami. En collaboration, l’activité de la gurmarine a été confirmée à l’aide d’un test cellulaire basé sur l’expression fonctionnelle du récepteur de rat T1R2/T1R3. A l’aide de différentes combinaisons des sous-unités humaines et de rongeurs, nous avons montré que l’inhibition par la gurmarine nécessitait la présence de la sous-unité T1R3 de rat.

Expression and biophysical characterization of the human T1R1 taste receptor subunit. 21. Congress of the european chemoreception research organisation (ECRO)

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De nombreux sens régissent notre comportement alimentaire. Parmi eux, la gustation joue un rôle déterminant pour orienter nos choix culinaires. Elle nous est familière et pourtant, elle constitue un des sens les plus complexes. Depuis une dizaine d’années, de notables progrès ont été réalisés dans sa connaissance, depuis la découverte des récepteurs gustatifs jusqu’à l’identification d’un polymorphisme interindividuel.

National audience

National audience

International audience; Umami is the typical taste induced by monosodium glutamate,which is thought to be detected by a heterodimeric G-proteincoupled receptors, T1R1 and T1R3. The most unique feature ofumami taste is its potentiation by purine nucleotidemonophosphates (IMP, GMP), which also elicit umami taste bytheir own. Zhang et al. (Proc. Natl Acad. Sci. USA, 2008) haverecently proposed a cooperative ligand-binding model involvingT1R1 N-terminal domain (NTD), where L-glutamate (L-glu)binds close to the hinge region, and purine nucleotides bind to anadjacent site close to the opening of the Venus flytrap domain. Tofurther understand the structural basis of umami stimulirecognition by T1R1, a large amount of purified T1R1 NTD issuitable for biochemical and structural studies. Here, we reportthe successful expression and purification of the soluble NTD ofhuman T1R1. The protein was expressed as insoluble aggregatedprotein (inclusion bodies) expressed in high level in Escherichiacoli. The protein was solubilized and in vitro refolded usingsuitable buffer and additives. We then measured the binding ofumami stimuli to T1R1 NTD using fluorescence spectroscopy.Fluorescent assay demonstrated that T1R1 NTD is properlyrefolded and able to bind L-glu with physiological relevantaffinity. The mode of interaction was specific since T1R1 NTDdid not bind sweet stimuli like fructose, sucralose or glycine. Inaddition, we observed that L-glu binding affinity is enhanced inpresence of IMP. To further validate our results, we havegenerated single amino-acid changes in L-Glu and IMP bindingsites. In summary, our expression system will allow large scaleproduction of active protein suitable for structural and functionalstudies.

International audience

International audience; Several drug-metabolizing enzymes (DME), such as cytochrome P450- dependent monooxygenases (CYP) and transferases have been characterized in the olfactory epithelium. Some of them are preferentially expressed in this tissue, while others are similar to those present in the liver. The role of these enzymes remains unclear. Since the olfactory mucosa is in direct contact with the external environment, these enzymes can contribute to the detoxification of chemical compounds. In addition, these enzymes could be involved in the olfaction process, especially in the biotransformation of odorants. Indeed, the rapid inactivation and clearance of odorants is a prerequisite for the capability of the olfactory system to receive iterative incoming signals. If such a hypothesis is plausible,it can be assumed that induction or inhibition of DME would have an impact on clearance of odorants and therefore on the olfactory signal. The aim of the present study was to identify treatments which modulate olfactory DME in the rat. The effects of known inducers of hepatic DME (arochlor, dexamethasone,phenobarbital, methylcholanthrene and ethoxyquin) on activity and mRNA expression of a panel of DME have been evaluated.All these treatments caused changes in activity and expression of several DME in the olfactory mucosa. The effects were found to be lower than those observed in the liver. Administration of dexamethasone resulted in significant increases in CYP-dependent activities and in CYP2G1 and CYP3A9 expression in the olfactory mucosa. Significant induction of UDP-glucuronosyltransferase (UGT) activities and UGT2A1 expression was also observed after dexamethasone treatment. Ethoxyquin administration induced a significant enhancement of quinone reductase and a decrease of CYP-dependent activities. The other treatments(arochlor, phenobarbital, methylcholanthrene) slightly increased the activities of UGT and quinone reductase. This study demonstrated that it is possible to modulate drug-metabolizing enzymes in the rat olfactory mucosa by chemical treatments.The impact of dexamethasone and ethoxyquin on olfactory signal will be further investigated.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the beta-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions. Fonds Forton, Welbio [CR-2012S-04R]; Vaincre la Mucoviscidose, Mukoviszidose e.V; Association Luxembourgeoise de Lutte contre la Mucoviscidose; ABCF2; Chiesi FondationChiesi Pharmaceuticals Inc; Cystic Fibrosis Foundation; Italian Cystic Fibrosis Research Foundation; Fibrosis FoundationItalian Cystic Fibrosis Research Foundation; [BB/M012573/1]; CF Foundation Therapeutics; Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI); Instruct-ULTRA 731005; FWO