Marie-Annick Persuy

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein‐coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian Gα subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian Golf compared to the Gα15 subunit, resulting in dose‐dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast‐borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17‐40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.

Quantitative assessment of olfactory receptors activity in immobilized nanosomes: a novel concept for bioelectronic nose

We describe how mammalian olfactory receptors (ORs) could be used as sensing elements of highly specific and sensitive bioelectronic noses. An OR and an appropriate G(alpha) protein were co-expressed in Saccharomyces cerevisiae cells from which membrane nanosomes were prepared, and immobilized on a sensor chip. By Surface Plasmon Resonance, we were able to quantitatively evaluate OR stimulation by an odorant, and G protein activation. We demonstrate that ORs in nanosomes discriminate between odorant ligands and unrelated odorants, as in whole cells. This assay also provides the possibility for quantitative assessment of the coupling efficiency of the OR with different G(alpha) subunits, without the interference of the cellular transduction pathway. Our findings will be useful to develop a new generation of electronic noses for detection and discrimination of volatile compounds, particularly amenable to micro- and nano-sensor formats.

Localization of orexins and their receptors in the rat olfactory system: possible modulation of olfactory perception by a neuropeptide synthetized centrally or locally

Orexin-A and -B, also known as hypocretins, are two neuropeptides acting on feeding and sleep. They are specific ligands for two different receptors belonging to the G-protein coupled receptors family. Orexin fibers and orexin receptor neurons have been previously described in the forebrain olfactory system. Using immunocytochemistry, we showed that both orexin-A and -B as well as their receptors were present at different levels of the olfactory system, from the nasal mucosa to nuclei of the amygdala. A punctuated staining for orexins and their receptors was detected at the apical part of the olfactory epithelium; in the lamina propria of the mucosa, the staining was localized around olfactory nerves. At the ultrastructural level, olfactory neurons and supporting cells were found immunoreactive for orexins and their receptors. The labeling was localized in dendritic knobs and cilia of neurons, in the apical part and microvilli of supporting cells. The finding of immunolabeled cisternae of reticulum strongly suggests a local synthesis of both peptides and receptors, confirmed by RT-PCR experiments. In forebrain and amygdala regions, we detected numerous orexin fibers. Orexin receptors were present in mitral-tufted cells of the bulb and in many neuronal perikarya in the anterior olfactory nuclei, piriform cortex and amygdala nuclei. Altogether, these results show that orexins and their receptors are present at all levels of the olfactory system, from cilia where odors bind to their receptors to central regions where integration of olfactory signals occurs. They suggest a possible modulation of olfactory perception by these neuropeptides.

Complete nucleotide sequence of ovine β-casein cDNA: inter-species comparison

Abstract The complete nucleotide sequence of ovine β-casein mRNA has been determined by sequencing, according to Sanger-Messing, both a recombinant clone isolated from a mammary cDNA pUC 18 library and a single-stranded cDNA generated by reverse transcription from a synthetic 17-mer primer complementary to the 5′ part of the mRNA coding frame. The 1088 nucleotide long β-casein mRNA, excluding the poly(A) tail, contains a coding frame of 669 nucleotides including the stop codon, flanked by 60 adn 359 nucleotides in the 5′ and 3′ untranslated regions, respectively. It arises from the splicing of 9 exons as deduced from gene sequence data. The deduced amino acid sequence differs at 3 positions from that previously determined by direct sequencing of mature β-casein. Comparison of the ovine, bovine, rat, mouse, and rabbit β-casein mRNA sequences shows a higher homology in the 3′ and 5′ untranslated regions. The most conserved regions in the open reading frame are essentially those encoding the signal peptide and the major phosphorylation site.

Relationship between Homo-oligomerization of a Mammalian Olfactory Receptor and Its Activation State Demonstrated by Bioluminescence Resonance Energy Transfer

G-protein-coupled receptor homo-oligomerization has been increasingly reported. However, little is known regarding the relationship between activation of the receptor and its association/conformational states. The mammalian olfactory receptors (ORs) belong to the G protein-coupled receptor superfamily. In this study, the homo-oligomerization status of the human OR1740 receptor and its involvement in receptor activation upon odorant ligand binding were addressed by co-immunoprecipitation and bioluminescence resonance energy transfer approaches using crude membranes or membranes from different cellular compartments. For the first time, our data clearly show that mammalian ORs constitutively self-associate into homodimers at the plasma membrane level. This study also demonstrates that ligand binding mediates a conformational change and promotes an inactive state of the OR dimers at high ligand concentrations. These findings support and validate our previously proposed model of OR activation/inactivation based on the tripartite odorant-binding protein-odorant-OR partnership.

Heterogeneity in the ribosomal family of the yeast Yarrowia lipolytica: genomic organization and segregation studies

The cloned r-DNA units of Yarrowia lipolytica [Van Heerikhuizen et al., 39 (1985) 213-222] and their restriction fragments have been used to probe blots of genomic DNA of this yeast. Wild-type and laboratory strains were shown to contain two-to-five types of repeated units, each strain displaying a specific pattern. By comparing their restriction patterns, we could localize the differences between units within their spacer region. Tetrad analysis strongly suggested a clustered organization of each type of repeat as well as the occurrence of meiotic exchanges within the r-DNA family. Chromosome loss was induced by benomyl and allowed to map several r-DNA clusters on the same chromosome. All those results indicate that the Y. lipolytica r-DNA gene family is quite different from other yeasts.

Force measurements on natural membrane nanovesicles reveal a composition-independent, high Young's modulus

Mechanical properties of nano-sized vesicles made up of natural membranes are crucial to the development of stable, biocompatible nanocontainers with enhanced functional, recognition and sensing capabilities. Here we measure and compare the mechanical properties of plasma and inner membrane nanovesicles ∼80 nm in diameter obtained from disrupted yeast Saccharomyces cerevisiae cells. We provide evidence of a highly deformable behaviour for these vesicles, able to support repeated wall-to-wall compressions without irreversible deformations, accompanied by a noticeably high Youngs modulus (∼300 MPa) compared to that obtained for reconstituted artificial liposomes of similar size and approaching that of some virus particles. Surprisingly enough, the results are approximately similar for plasma and inner membrane nanovesicles, in spite of their different lipid compositions, especially on what concerns the ergosterol content. These results point towards an important structural role of membrane proteins in the mechanical response of natural membrane vesicles and open the perspective to their potential use as robust nanocontainers for bioapplications.

Diffusion-controlled deposition of natural nanovesicles containing G-protein coupled receptors for biosensing platforms

Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for sensing biodevices. This is particularly true for the case of G-protein coupled receptors (GPCRs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. However, the controlled production of natural vesicles containing GPCRs and their reproducible deposition on biosensor surfaces are among the outstanding challenges in the road map to realize practical biomolecular devices based on GPCRs. In this work we present the production and characterization of membrane nanovesicles from Saccharomyces cerevisiae containing heterologously expressed olfactory receptors – a member of the family of GPCRs – and study their deposition onto substrates used as biosensor supports. We show by direct observation with Atomic Force Microscopy that nanovesicles deposit and flatten without rupturing on glass substrates following approximately a diffusive law. We show that surface coverages larger than 20–25% of the substrate can be reproducibly achieved under practical nanovesicle concentrations and reasonable time scales, while keeping to the minimum the presence of background residuals coming from the nanovesicles production process. Surface chemistry modification of gold substrates indicates a higher affinity of natural nanovesicles for acid modified surfaces as compared to amino or alcohol modified surfaces. Present results constitute an important step in the practical realization of biosensor devices based on natural nanovesicles integrating G-protein coupled membrane receptors.

Mammalian olfactory receptors: molecular mechanisms of odorant detection, 3D-modeling, and structure-activity relationships.

This chapter describes the main characteristics of olfactory receptor (OR) genes of vertebrates, including generation of this large multigenic family and pseudogenization. OR genes are compared in relation to evolution and among species. OR gene structure and selection of a given gene for expression in an olfactory sensory neuron (OSN) are tackled. The specificities of OR proteins, their expression, and their function are presented. The expression of OR proteins in locations other than the nasal cavity is regulated by different mechanisms, and ORs display various additional functions. A conventional olfactory signal transduction cascade is observed in OSNs, but individual ORs can also mediate different signaling pathways, through the involvement of other molecular partners and depending on the odorant ligand encountered. ORs are engaged in constitutive dimers. Ligand binding induces conformational changes in the ORs that regulate their level of activity depending on odorant dose. When present, odorant binding proteins induce an allosteric modulation of OR activity. Since no 3D structure of an OR has been yet resolved, modeling has to be performed using the closest G-protein-coupled receptor 3D structures available, to facilitate virtual ligand screening using the models. The study of odorant binding modes and affinities may infer best-bet OR ligands, to be subsequently checked experimentally. The relationship between spatial and steric features of odorants and their activity in terms of perceived odor quality are also fields of research that development of computing tools may enhance.

A novel platform based on immobilized histidine tagged olfactory receptors, for the amperometric detection of an odorant molecule characteristic of boar taint

We report a dose-dependent detection of androstenone in solution, as one of the boar taint compounds, based on related OR7D4 olfactory receptors immobilized on a gold electrode through their 6-His tag and NTA-copper complex, as visualized through fluorescence microscopy. Square wave voltammetry (SWV) is for the first time, the method used to monitor the olfactory receptor/odorant recognition process. The relative variation of the Cu(I)-Cu(II) current peak increases linearly versus log (concentration of androstenone) from 10(-14)M to 10(-4)M, in buffer solution. Negative tests were performed, using an unrelated odorant, helional, itself a ligand of OR 1740. Cross-selectivity was also tested after immobilization of OR 1740.