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Dive into the research topics where Rony Nehmé is active.

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Featured researches published by Rony Nehmé.


Nature | 2011

The structural basis for agonist and partial agonist action on a β1-adrenergic receptor

Tony Warne; Rouslan Moukhametzianov; Jillian G. Baker; Rony Nehmé; Patricia C. Edwards; Andrew G. W. Leslie; Gebhard F. X. Schertler; Christopher G. Tate

β-adrenergic receptors (βARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline. Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) β1-adrenergic receptor (β1AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser5.42 and Ser5.46), but partial agonists only interact with Ser5.42 (superscripts refer to Ballesteros–Weinstein numbering). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.


Nature | 2016

Structure of the adenosine A2A receptor bound to an engineered G protein

Byron Carpenter; Rony Nehmé; Tony Warne; Andrew G. W. Leslie; Christopher G. Tate

G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary. Here we present the structure of the adenosine A2A receptor (A2AR) bound to an engineered G protein, mini-Gs, at 3.4 Å resolution. Mini-Gs binds to A2AR through an extensive interface (1,048 Å2) that is similar, but not identical, to the interface between Gs and the β2-adrenergic receptor. The transition of the receptor from an agonist-bound active-intermediate state to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A2AR–mini-Gs structure highlights both the diversity and similarity in G-protein coupling to GPCRs and hints at the potential complexity of the molecular basis for G-protein specificity.


PLOS ONE | 2014

The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor.

Jennifer Miller-Gallacher; Rony Nehmé; Tony Warne; Patricia C. Edwards; Gebhard F. X. Schertler; Andrew G. W. Leslie; Christopher G. Tate

The β1-adrenoceptor (β1AR) is a G protein-coupled receptor (GPCR) that is activated by the endogenous agonists adrenaline and noradrenaline. We have determined the structure of an ultra-thermostable β1AR mutant bound to the weak partial agonist cyanopindolol to 2.1 Å resolution. High-quality crystals (100 μm plates) were grown in lipidic cubic phase without the assistance of a T4 lysozyme or BRIL fusion in cytoplasmic loop 3, which is commonly employed for GPCR crystallisation. An intramembrane Na+ ion was identified co-ordinated to Asp872.50, Ser1283.39 and 3 water molecules, which is part of a more extensive network of water molecules in a cavity formed between transmembrane helices 1, 2, 3, 6 and 7. Remarkably, this water network and Na+ ion is highly conserved between β1AR and the adenosine A2A receptor (rmsd of 0.3 Å), despite an overall rmsd of 2.4 Å for all Cα atoms and only 23% amino acid identity in the transmembrane regions. The affinity of agonist binding and nanobody Nb80 binding to β1AR is unaffected by Na+ ions, but the stability of the receptor is decreased by 7.5°C in the absence of Na+. Mutation of amino acid side chains that are involved in the co-ordination of either Na+ or water molecules in the network decreases the stability of β1AR by 5–10°C. The data suggest that the intramembrane Na+ and associated water network stabilise the ligand-free state of β1AR, but still permits the receptor to form the activated state which involves the collapse of the Na+ binding pocket on agonist binding.


PLOS ONE | 2011

The hedgehog receptor patched is involved in cholesterol transport.

Michel Bidet; Olivier Joubert; Benoît Lacombe; Marine Ciantar; Rony Nehmé; Patrick Mollat; Lionel Bretillon; Hélène Faure; Robert Bittman; Martial Ruat; Isabelle Mus-Veteau

Background Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened. Methodology/Principal Findings Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol. Conclusion/Significance Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.


Molecular Membrane Biology | 2011

A class of mild surfactants that keep integral membrane proteins water-soluble for functional studies and crystallization

Jens Hovers; Meike Potschies; Ange Polidori; Bernard Pucci; Simon Raynal; Françoise Bonneté; Maria Josefa Serrano-Vega; Christopher G. Tate; Daniel Picot; Yves Pierre; Jean-Luc Popot; Rony Nehmé; Michel Bidet; Isabelle Mus-Veteau; Holger Bußkamp; Karl-Heinz Jung; Andreas Marx; Peter Timmins; Wolfram Welte

Abstract Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside (‘C12-b-M’) as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b6 f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.


Biochimica et Biophysica Acta | 2010

Stability study of the human G-protein coupled receptor, Smoothened.

Rony Nehmé; Olivier Joubert; Michel Bidet; Benoît Lacombe; Ange Polidori; Bernard Pucci; Isabelle Mus-Veteau

Smoothened is a member of the G-protein coupled receptor (GPCR) family responsible for the transduction of the Hedgehog signal to the intracellular effectors of the Hedgehog signaling pathway. Aberrant regulation of this receptor is implicated in many cancers but also in neurodegenerative disorders. Despite the pharmacological relevance of this receptor, very little is known about its functional mechanism and its physiological ligand. In order to characterize this receptor for basic and pharmacological interests, we developed the expression of human Smoothened in the yeast Saccharomyces cerevisiae and Smoothened was then purified. Using Surface Plasmon Resonance technology, we showed that human Smoothened was in a native conformational state and able to interact with its antagonist, the cyclopamine, both at the yeast plasma membrane and after purification. Thermostability assays on purified human Smoothened showed that this GPCR is relatively stable in the classical detergent dodecyl-beta-d-maltoside (DDM). The fluorinated surfactant C(8)F(17)TAC, which has been proposed to be less aggressive towards membrane proteins than classical detergents, increased Smoothened thermostability in solution. Moreover, the replacement of a glycine by an arginine in the third intracellular loop of Smoothened coupled to the use of the fluorinated surfactant C(8)F(17)TAC during the mutant purification increased Smoothened thermostability even more. These data will be very useful for future crystallization assays and structural characterization of the human receptor Smoothened.


Biochimica et Biophysica Acta | 2009

Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast

Olivier Joubert; Rony Nehmé; Damien Fleury; Matthieu De Rivoyre; Michel Bidet; Ange Polidori; Martial Ruat; Bernard Pucci; Patrick Mollat; Isabelle Mus-Veteau

The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.


PLOS ONE | 2017

Mini-G proteins: Novel tools for studying GPCRs in their active conformation

Rony Nehmé; Byron Carpenter; Ankita Singhal; Annette Strege; Patricia C. Edwards; Courtney F. White; Haijuan Du; Reinhard Grisshammer; Christopher G. Tate

Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.


Methods of Molecular Biology | 2010

Heterologous Expression of Human Membrane Receptors in the Yeast Saccharomyces cerevisiae

Olivier Joubert; Rony Nehmé; Michel Bidet; Isabelle Mus-Veteau

Due to their implication in numerous diseases like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension, and Alzheimer disease, membrane proteins (MPs) represent around 50% of drug targets. However, only 204 crystal structures of MPs have been solved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. The yeast Saccharomyces cerevisiae is a convenient host for the production of mammalian MPs for functional and structural studies. Like bacteria, they are straightforward to manipulate genetically, are well characterized, can be easily cultured, and can be grown inexpensively in large quantities. The advantage of yeast compared to bacteria is that they have protein-processing and posttranslational modification mechanisms related to those found in mammalian cells. The recombinant rabbit muscle Ca(2+)-ATPase (adenosine triphosphatase), the first heterologously expressed mammalian MP for which the crystal structure was resolved, has been produced in S. cerevisiae. In this chapter, the focus is on expression of recombinant human integral MPs in a functional state at the plasma membrane of the yeast S. cerevisiae. Optimization of yeast culture and of MP preparations is detailed for two human receptors of the Hedgehog pathway: Patched and Smoothened.


Journal of Biological Chemistry | 2018

Mini G protein probes for active G protein– coupled receptors (GPCRs) in live cells

Qingwen Wan; Najeah Okashah; Asuka Inoue; Rony Nehmé; Byron Carpenter; Christopher G. Tate; Nevin A. Lambert

G protein–coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase–mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype–biased ligands.

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Christopher G. Tate

Laboratory of Molecular Biology

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Isabelle Mus-Veteau

University of Nice Sophia Antipolis

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Michel Bidet

University of Nice Sophia Antipolis

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Patricia C. Edwards

Laboratory of Molecular Biology

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Olivier Joubert

University of Nice Sophia Antipolis

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Byron Carpenter

Laboratory of Molecular Biology

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Andrew G. W. Leslie

Laboratory of Molecular Biology

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Tony Warne

Laboratory of Molecular Biology

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Bernard Pucci

Centre national de la recherche scientifique

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