Biosynthese et metabolisme des steroiedes ovariens chez l'anguille (Anguilla anguilla L.) argentee

SIGLECNRS T 54603 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Unconventional Actions of Glycoprotein Hormone Subunits: A Comprehensive Review

International audienceThe glycoprotein hormones (GPH) are heterodimers composed of a common alpha subunit and a specific beta subunit. They act by activating specific leucine-rich repeat G protein-coupled receptors. However, individual subunits have been shown to elicit responses in cells devoid of the receptor for the dimeric hormones. The alpha subunit is involved in prolactin production from different tissues. The human chorionic gonadotropin beta subunit (beta hCG) plays determinant roles in placentation and in cancer development and metastasis. A truncated form of the thyrotropin (TSH) beta subunit is also reported to have biological effects. The GPH alpha- and beta subunits are derived from precursor genes (gpa and gpb, respectively), which are expressed in most invertebrate species and are still represented in vertebrates as GPH subunit paralogs (gpa2 and gpb5, respectively). No specific receptor has been found for the vertebrate GPA2 and GPB5 even if their heterodimeric form is able to activate the TSH receptor in mammals. Interestingly, GPA and GPB are phylogenetically and structurally related to cysteine-knot growth factors (CKGF) and particularly to a group of antagonists that act independently on any receptor. This review article summarizes the observed actions of individual GPH subunits and presents the current hypotheses of how these actions might be induced. New approaches are also proposed in light of the evolutionary relatedness with antagonists of the CKGF family of proteins

Les précurseurs évolutifs des gènes des sous-unités a et b des hormones glycoprotéiques (implication dans le développement)

Deux protéines structurellement proches des sous-unités a et b des hormones glycoprotéiques hypophysaires (GpHs) des vertébrés ont été identifiées dans des génomes de vertébrés et de Protostomes et ont été dénommées glycoprotéines a2 (GPA2) et b5 (GPB5). Chez les Mammifères, l hétérodimère de ces deux protéines recombinantes active le récepteur à la thyrotropine et a donc été nommé thyrostimuline. Cependant le rôle et le mode d action de ces protéines restaient très incertains. Nous avons entrepris une étude visant à déterminer quand ces protéines sont apparues et à explorer leur expression embryonnaire. Nous avons pu montrer que GPA2 et GPB5 sont apparues avec l émergence des Bilatériens suggérant qu elles pouvaient être associées à un caractère apparu avec ce groupe. Nous apportons aussi des arguments suggérant que les sous-unités des GpHs résultent de la duplication, peu avant la radiation des vertébrés, d un locus contenant gpa2 et gpb5. Pour initier leur étude fonctionnelle, nous nous sommes d abord intéressés à un représentant d un groupe taxinomique ayant émergé avant cette duplication, l amphioxus. Nous avons analysé l expression des gènes gpa2 et gpb5 en parallèle avec celle du récepteur homologue aux récepteurs des GpHs des vertébrés (gphrr) aux stades embryonnaire et larvaire. Nous montrons que l expression de gpb5 est faible mais quasi ubiquitaire, tandis que gpa2 est exprimé dans deux structures apparues avec les Bilatériens, le système nerveux central (SNC) et l intestin où son expression est partiellement recouverte par celle de gphrr. Notre deuxième étude chez deux vertébrés (le médaka et la souris) a permis de confirmer l expression embryonnaire des gènes gpa2 et gpb5 dans le SNC et l intestin. Elle a également permis de mettre en évidence leur expression dans les organes du système visuel, les arcs branchiaux ou encore dans les bourgeons de membres. L ensemble de ces données montre que ces protéines pourraient jouer un rôle, individuel ou en partenariat, dans la mise en place de certaines structures. Par ailleurs, l expression de ces gènes dans certaines structures comme l intestin adulte ou le thymus néonatal suggèrent que GPA2 et GPB5 pourraient participer aux mécanismes de défenses immunitaires innée et spécifique.Two proteins structurally related to glycoprotein hormone (GpH) a and b subunits have been identified in vertebrate and Protostome genomes and have been named glycoproteins a2 (GPA2) and bche mti5 (GPB5). The mammalian recombinant heterodimer of these two proteins was shown to activate the thyrotropin receptor and was therefore named thyrostimulin. However their function and mechanism of action still remained unknown. We developed a study to determine when these proteins appeared and to explore their embryonic expression. We showed that GPA2 and GPB5 appeared with the emergence of bilateria suggesting they could be involved in a novelty of this taxa. We gave some evidences supporting that GpH subunits arose from a duplication of a locus containing both gpa2 and gpb5 just prior the vertebrate s radiation. In view of initiating their functional study, we focused on amphioxus, a member of a taxinomic group that emerged before this duplication. We analyzed gpa2 and gpb5 genes expression in parallel with the vertebrate GpHs receptor homolog (gphrr) during development. We demonstrated that the gpb5 gene expression was weak but essentially ubiquitous while gpa2 was expressed in two structures that appeared with bilateria, the central nervous system (CNS) and the intestine where its expression was partially overlapped by that of gphrr. A second study in two distinct vertebrate species (medaka and mouse) allowed us to confirm the embryonic expression of gpa2 and gpb5 genes in CNS and intestine. Their expression was also detected in occular system organs, in branchial arches or in limb buds. All these data show that these proteins could act, individually or in partnership, in the development of some structures. Moreover, the expression of these genes in adult intestine or the postnatal thymus for example, together with data from the literature, suggest that GPA2 and GPB5 could be involved in innate and specific immunity defense mechanisms.PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor

International audienceBackground: Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor , a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypo-gonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood. Results: Using ATAC chromatin accessibility analyses on three cell lines recapitulating gradual stages of gonadotrope differentiation and in vivo on developing pituitaries, we demonstrate that a yet undescribed enhancer is transiently recruited during gonadotrope specification. Using CRISPR/Cas9, we show that this enhancer is mandatory for the emergence of Nr5a1 during gonadotrope specification. Furthermore, we identify a highly conserved estrogen-binding element and demonstrate that the enhancer activation is dependent upon estrogen acting through ERα. Lastly, we provide evidence that binding of ERα is crucial for chromatin remodeling of Nr5a1 enhancer and promoter, leading to RNA polymerase recruitment and transcription. Conclusion: This study identifies the earliest regulatory sequence involved in gonadotrope lineage specification and highlights the key epigenetic role played by ERα in this differentiation process

Discovery of novel dengue virus NS5 methyltransferase non-nucleoside inhibitors by fragment-based drug design

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Molecular characterization of sea bass gonadotropin subunits (α, FSHβ, and LHβ) and their expression during the reproductive cycle

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass (Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain-pituitary cDNA library. The nucleotide sequences of the common α, the FSHβ, and the LHβ subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHβ is more similar to higher vertebrate FSHβ's (35-37%) than to LHβ's (26-30%), whereas sea bass LHβ is more similar to LHβ's (40-53%) than to FSHβ's (26-41%). Phylogenetic analysis of fish GTH sequences grouped the β subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.We acknowledge the Spanish Ministry of Education and Science for funding a fellowship to J. Mateos and the European Community for funding a Marie-Curie research grant (MCFI-1999-01335) to E. Mañanós. This work was financially supported by research projects funded by the European Community (FAIR CT97-3785) and by the Spanish Ministry of Education and Science (CICYT, MARI1998-1542-CE)

microARN et inactivation de la fonction gonadotrope hypophysaire

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Novel 2-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,3,4-oxadiazole and 3-phenyl-5-[(E)-2-(thiophen-2-yl)ethenyl]-1,2,4-oxadiazole derivatives as dengue virus inhibitors targeting NS5 polymerase

International audienceno abstrac

Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay

International audienceAlphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m7GMP releasing pyrophosphate (GT reaction) and the m7GMP is next transferred onto the 5'-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m7GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses

Novel Class of Chikungunya Virus Small Molecule Inhibitors That Targets the Viral Capping Machinery

Despite the worldwide reemergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we aimed to identify the target of a novel class of CHIKV inhibitors, i.e., the CHVB series. CHVB compounds inhibit the in vitro replication of CHIKV isolates with 50% effective concentrations in the low-micromolar range. A CHVB-resistant variant (CHVBres) was selected that carried two mutations in the gene encoding nsP1 (responsible for viral RNA capping), one mutation in nsP2, and one mutation in nsP3. Reverse genetics studies demonstrated that both nsP1 mutations were necessary and sufficient to achieve ∼18-fold resistance, suggesting that CHVB targets viral mRNA capping. Interestingly, CHVBres was cross-resistant to the previously described CHIKV capping inhibitors from the MADTP series, suggesting they share a similar mechanism of action. In enzymatic assays, CHVB inhibited the methyltransferase and guanylyltransferase activities of alphavirus nsP1 proteins. To conclude, we identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity makes this chemical scaffold a potential candidate for CHIKV drug development.status: publishe