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Accueil > Agenda > Les séminaires Jean Roche > Contrôles génétiques et épigénétiques de la plasticité du sens chimique.

Contrôles génétiques et épigénétiques de la plasticité du (...)

Lundi 30 mai 2005, 11h, salle Lissitzky.

Bibliographie

1 : Reprod Biomed Online. 2004 Apr ;8(4):385-91.

Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor. Rao AJ, Ramachandra SG, Ramesh V, Couture L, Abdennebi L, Salesse R, Remy JJ.

Indian Institute of Science, Bangalore 560 012, India. ajrao biochem.iisc.ernet.in

Active immunization of proven fertile adult male bonnet monkeys (Macaca radiata) with phage-expressed follicle-stimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones.

2 : Eur J Biochem. 2003 Jul ;270(13):2905-12.

Ligand-specific dose-response of heterologously expressed olfactory receptors. Levasseur G, Persuy MA, Grebert D, Remy JJ, Salesse R, Pajot-Augy E.

INRA-Biotechnologies, Neurobiologie de l’Olfaction et de la Prise Alimentaire, Recepteurs et Communication Chimique, Jouy-en-Josas, France.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.

http://content.febsjournal.org/cgi/reprint/270/13/2905

3 : Biol Reprod. 2003 Jan ;68(1):323-7.

Maintenance of sexual immaturity in male mice and bucks by immunization against N-terminal peptides of the follicle-stimulating hormone receptor. Abdennebi L, Chun EY, Jammes H, Wei D, Remy JJ.

Laboratory of Molecular and Cellular Biology, I.N.R.A. Biotechnology, 78352 Jouy-en-Josas, France.

The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life ; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18-38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.

http://www.biolreprod.org/cgi/reprint/68/1/323

4 : Protein Expr Purif. 2002 Jun ;25(1):114-23.

Rescue of intracellularly trapped lutropin receptor exodomain by endodomain and reconstitution of a functional membrane receptor : interaction between exo- and endodomains. Bozon V, Couture L, Pajot-Augy E, Richard F, Remy JJ, Salesse R.

Laboratoire de Physiologie des Cellules Cardiaques et Vasculaires, CNRS-UMR 6542, Tours, France.

The lutropin receptor consists of an extracellular N-terminal half and a membrane-associated C-terminal half. hCG initially binds the exodomain with a high affinity and the resulting complex is thought to interact with the endodomain through a secondary contact generating a hormonal signal. Therefore, the exodomain and endodomain are likely to associate directly or indirectly with each other, but lack of fruitful materials and technology has hampered knowledge about their physical relationship and contact sites. In this work, we engineered a double-recombinant (separate exodomain and endodomain) baculovirus system successfully expressing on the surface of insect cells high levels of split LH receptor, binding the hormone with high affinity and inducing cAMP synthesis. In contrast, the exodomain and endodomain expressed separately were mostly trapped in cells. Our data indicate that the exodomain and endodomain are disulfide linked in the split receptor. When the disulfide links were reduced, the split receptor still induced cAMP up to 60%, which raises the intriguing possibility of a residual induction activity of the endodomain in the absence of high-affinity ligand binding. Our results also underscore that the targeting and transport of the LH receptor to plasma membrane require both domains, whereas each domain is independently sufficient for folding. The expression level of functional lutropin receptors is the highest ever reported. Our system may also be useful for future studies requiring a high amount of soluble secreted exodomain. Copyright 2002 Elsevier Science (USA).

http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WPJ-463H9JR-H-1&_cdi=6992&_user=113324&_orig=search&_coverDate=06%2F30%2F2002&_qd=1&_sk=999749998&view=c&wchp=dGLbVtb-zSkzS&md5=b8a112f134d2a91dff812de97ffe884d&ie=/sdarticle.pdf <http://www.sciencedirect.com/scienc...>

5 : Reproduction. 2002 Jun ;123(6):819-26. Differences in splicing of mRNA encoding LH receptor in theca cells according to breeding season in ewes. Abdennebi L, Lesport AS, Remy JJ, Grebert D, Pisselet C, Monniaux D, Salesse R.

Unite Recepteurs et Communication Cellulaire, INRA Biotechnologies, 78352 Jouy-en-Josas, France. latifa.abdennebi inserm.ipsc.u-psud.fr

Splice variants of mRNA encoding the LH receptor (LHR) during follicular development were characterized in cyclic and non-cyclic ewes. Granulosa and theca cells were collected from individual follicles. After amplification by RT-PCR of a region situated between exon 9 and exon 11 of the LHR gene, three distinct bands, LHR1 (full length), LHR2 (deletion of exon 10), LHR3 (deletion of 262 bp in exon 11), were observed in the granulosa and theca cells of ovine antral follicles of various sizes (2.5-6.0 mm). Expression of LHR mRNA in theca cells varied with the annual cycle of reproduction (P < 0.001), and was highly expressed in all classes of follicle collected from anoestrous ewes (1.3 +/- 0.1, n = 8 in small follicles ; 1.8 +/- 0.2, n = 8 in medium follicles ; 1.7 +/- 0.3, n = 4 in large follicles ; arbitrary units) compared with follicles collected from oestrous ewes (0.19 +/- 0.06, n = 8 in small follicles ; 0.2 +/- 0.04, n = 9 in medium follicles ; 0.18 +/- 0.04, n = 5 in large follicles). During the breeding season, no differences in the relative expression of the different splice variants were observed according to follicle size. In contrast, during anoestrus, LHR3 mRNA was significantly more abundant in large (6.0-6.5 mm) and medium (4.0-5.5 mm) than it was in small (2.5-3.5 mm) follicles. These results indicate that RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression in theca cells.

http://www.reproduction-online.org/cgi/reprint/123/6/819

6 : Eur J Biochem. 2001 Aug ;268(16):4570-9.

Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants. Dirnberger D, Steinkellner H, Abdennebi L, Remy JJ, van de Wiel D.

Zentrum fur Angewandte Genetik, Universitat fur Bodenkultur-Wien, Wien, Austria.

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex-type N-glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.

http://content.febsjournal.org/cgi/reprint/268/16/4570

7 : J Biol Chem. 2001 Jan 19 ;276(3):1681-7. Epub 2000 Oct 3.

Purification and structural analysis of a soluble human chorionogonadotropin hormone-receptor complex. Remy JJ, Nespoulous C, Grosclaude J, Grebert D, Couture L, Pajot E, Salesse R.

*Biologie Cellulaire et Moleculaire, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France. remy biotec.jouy.inra.fr

Receptors for the luteotropin/human chorionogonadotropin hormone belong to the G-protein-coupled receptor family by their membrane-anchoring domains. They also possess a large extracellular domain (ECD) responsible for most of the hormone-receptor interactions. Structure-function studies identified several contacts between hormone and receptor ECD, but the precise topology of the complex is still unknown because of the lack of suitable heterologous expression means. Receptor ECDs exhibit leucine repeats and have been modelized on the basis of the three-dimensional structure of the porcine ribonuclease inhibitor, the first structurally known leucine-rich repeats protein. Here we report overexpression (up to 20 mg per liter) and purification to homogeneity of a soluble human chorionogonadotropin-ECD receptor complex secreted by stably cotransfected Chinese hamster ovary cells. Biochemical analysis and surface plasmon resonance data were in favor of a unique dimer with a 1:1 ligand-receptor stoichiometry. Immunopurified complex was submitted to circular dichroism characterization ; CD spectra deconvolution indicated more than 25% alpha helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.

http://www.jbc.org/cgi/reprint/276/3/1681

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