Régulation par la stimulation b3-adrénergique et les interactions protéines-protéines (implications putatives dans la mucoviscidose)

Au cours de ce travail, nous avons montré, par des approches pharmacologiques et biochimiques, que le récepteur 3-adrénergique régule la protéine CFTR indépendamment de la voie AMPc/PKA, voie classique de régulation de CFTR, mais que cette régulation implique la voie Gi/o-PI3K- ERK 1/2 MAPK. Cette voie de signalisation régule également CFTR indépendamment du récepteur 3-adrénergique. Nous avons également caractérisé l'expression du récepteur 3-adrénergique dans le poumon humain ainsi que dans les cellules épithéliales bronchiques humaines. Nous avons également montré que la surexpression de la protéine NHE-RF permet à la protéine mutée F508-CFTR de sortir du réticulum endoplasmique, finir son processus de maturation et s'acheminer enfin vers la membrane plasmique des cellules où elle a une activité chlore stimulée par l'AMPc.In the present work, Using pharmacological and biochemical approaches, we demonstrated that the 3-adrenoceptor regulates CFTR protein independently of AMPc/PKA pathway, but this regulation involved the Gi/o-PI3K- ERK 1/2 MAPK pathway. This signalling pathway is able to regulate CFTR independently of 3-adrenoceptor. We have characterized a 3-adrenoceptor expression in human lung and in bronchial human epithelial cells. We have also demonstrates that an overexpression of NHE-RF protein was able to restore the chloride activity of F508-CFTR. Furthermore, we have shown a significant increase in the maturation of F508-CFTR protein. NHE-RF enhanced also the trafficking of F508-CFTR protein towards the apical plasma membrane.NANTES-BU Sciences (441092104) / SudocSudocFranceF

NHE-RF1 protein rescues DeltaF508-CFTR function

(IF : 4,214)International audienceIn cystic fibrosis (CF), the DeltaF508-CFTR anterograde trafficking from the endoplasmic reticulum to the plasma membrane is inefficient. New strategies for increasing the delivery of DeltaF508-CFTR to the apical membranes are thus pathophysiologically relevant targets to study for CF treatment. Recent studies have demonstrated that PDZ-containing proteins play an essential role in determining polarized plasma membrane expression of ionic transporters. In the present study we have hypothesized that the PDZ-containing protein NHE-RF1, which binds to the carboxy terminus of CFTR, rescues DeltaF508-CFTR expression in the apical membrane of epithelial cells. The plasmids encoding DeltaF508-CFTR and NHE-RF1 were intranuclearly injected in A549 or Madin-Darby canine kidney (MDCK) cells, and DeltaF508-CFTR channel activity was functionally assayed using SPQ fluorescent probe. Cells injected with DeltaF508-CFTR alone presented a low chloride channel activity, whereas its coexpression with NHE-RF1 significantly increased both the basal and forskolin-activated chloride conductances. This last effect was lost with DeltaF508-CFTR deleted of its 13 last amino acids or by injection of a specific NHE-RF1 antisense oligonucleotide, but not by NHE-RF1 sense oligonucleotide. Immunocytochemical analysis performed in MDCK cells transiently transfected with DeltaF508-CFTR further revealed that NHE-RF1 specifically determined the apical plasma membrane expression of DeltaF508-CFTR but not that of a trafficking defective mutant potassium channel (KCNQ1). These data demonstrate that the modulation of the expression level of CFTR protein partners, like NHE-RF1, can rescue DeltaF508-CFTR activity

A systematic review on the genetics of male infertility in the era of next-generation sequencing

Objectives: To identify the role of next-generation sequencing (NGS) in male infertility, as advances in NGS technologies have contributed to the identification of novel genes responsible for a wide variety of human conditions and recently has been applied to male infertility, allowing new genetic factors to be discovered. Materials and methods: PubMed was searched for combinations of the following terms: ‘exome’, ‘genome’, ‘panel’, ‘sequencing’, ‘whole-exome sequencing’, ‘whole-genome sequencing’, ‘next-generation sequencing’, ‘azoospermia’, ‘oligospermia’, ‘asthenospermia’, ‘teratospermia’, ‘spermatogenesis’, and ‘male infertility’, to identify studies in which NGS technologies were used to discover variants causing male infertility. Results: Altogether, 23 studies were found in which the primary mode of variant discovery was an NGS-based technology. These studies were mostly focused on patients with quantitative sperm abnormalities (non-obstructive azoospermia and oligospermia), followed by morphological and motility defects. Combined, these studies uncover variants in 28 genes causing male infertility discovered by NGS methods. Conclusions: Male infertility is a condition that is genetically heterogeneous, and therefore remarkably amenable to study by NGS. Although some headway has been made, given the high incidence of this condition despite its detrimental effect on reproductive fitness, there is significant potential for further discoveries. Keywords: Male infertility, Next-generation sequencing, Genome-wide association stud

Association of Differing Qatari Genotypes with Vitamin D Metabolites

Objective. Genetic studies have identified four Qatari genotypes: Q1 Arab, Bedouin; Q2 Asian/Persian; Q3 African; and a fourth admixed group not fitting into the previous 3 groups. This study was undertaken to determine if there was an increased risk of deficiency of vitamin D and its metabolites associated with differing genotypes, perhaps due to genetic differences in skin pigmentation. Methods. 398 Qatari subjects (220 type 2 diabetes and 178 controls) had their genotype determined by Affymetrix 500 k SNP arrays. Total values of 1,25-dihydroxyvitamin D (1,25(OH)2D), 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D), and 25-hydroxy-3epi-vitamin D (3epi-25(OH)D) concentrations were measured by the LC-MS/MS analysis. Results. The distribution was as follows: 164 (41.2%) genotyped Q1, 149 (37.4%) genotyped Q2, 31 (7.8%) genotyped Q3, and 54 (13.6%) genotyped “admixed.” Median levels of 25(OH)D and 3epi-25(OH)D did not differ across Q1, Q2, Q3, and “admixed” genotypes, respectively. 1,25(OH)2D levels were lower (p<0.04) between Q2 and the admixed groups, and 24,25(OH)2D levels were lower (p<0.05) between Q1 and the admixed groups. Vitamin D metabolite levels were lower in females for 25(OH)D, 1,25(OH)2D (p<0.001), and 24,25(OH)2D (p<0.006), but 3epi-25(OH)D did not differ (p<0.26). Diabetes prevalence was not different between genotypes. Total 1,25(OH)2D (p<0.001), total 24,25(OH)2D (p<0.001), and total 3epi-25(OH)D (p<0.005) were all significantly lower in diabetes patients compared to controls whilst the total 25(OH)D was higher in diabetes than controls (p<0.001). Conclusion. Whilst 25(OH)D levels did not differ between genotype groups, 1,25(OH)2D and 24,25(OH)2D were lower in the admixed group, suggesting that there are genetic differences in vitamin D metabolism that may be of importance in a population that may allow a more targeted approach to vitamin D replacement. This may be of specific importance in vitamin D replacement strategies with the Q2 genotype requiring less, and the other genotypes requiring more to increase 1,25(OH)2D. Whilst overall the group was vitamin D deficient, total 25(OH)D was higher in diabetes, but 1,25(OH)2D, 24,25(OH)2D, and 3epi-25(OH)D were lower in diabetes that did not affect the relationship to genotype