Heterogeneity in the processing of ClC-5 mutants related to Dent disease

International audienceMutations in the electrogenic Cl-/H+ exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in X. laevis oocytes and in HEK293 cells of 9 previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC 5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were non-functional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss-of-function in Dent disease

Physiopathologie des ClC-Ks (syndrome de Bartter/Gitelman et hypertension)

PARIS-BIUP (751062107) / SudocSudocFranceF

Rôle de la protéine CFTR et du canal K+ TASK2 dans les phénomènes de régulation du volume des cellules du tubule proximal de rein de souris

Le but de cette étude était de comprendre d une part le mécanisme d activation du canal K+ TASK2 au cours de la RVD et d autre part le rôle de TASK2 et de CFTR dans un autre phénomène de régulation cellulaire intervenant durant l apoptose : l AVD (Apoptotic Volume Decrease). Pour cela, nous avons utilisé des lignées de cellules proximales provenant de rein de souris sauvages ou invalidées respectivement pour les gènes codant pour ces 2 protéines d intérêt (TASK2 et CFTR). Lors de l étude du mécanisme de régulation de TASK2 pendant la RVD (Regulatory Volume Decrease), nous avons montré que le choc hypotonique induit un flux de Cl- à travers du canal Cl- de type VRCC qui serait une driving force pour la sortie de HCO3- par l échangeur Cl-/HCO3- entraînant l alcalinisation du pH externe indispensable à l activation du canal TASK2. Lors de l étude du rôle de TASK2 et de CFTR dans l apoptose des cellules proximales, nous avons montré que l application d un agent apoptotique provoquerait un efflux de glutathion relié à l activité de CFTR ce qui permettrait une augmentation des ROS. Au final, cette élévation activerait, par un mécanisme encore inconnu, l augmentation des conductances Cl- de type VRCC et K+ de type TASK2 induisant ainsi une diminution du volume cellulaire indispensable à l entrée des cellules en apoptose. Par contre, dans les cellules task2 -/-, incapables de réguler leur volume lors d un choc hypotonique, nous avons pu mesurer une AVD et une activité de la caspase-3 réduites de 60% comparées aux cellules sauvages. Nous avons mis en évidence une compensation du canal TASK2 par un canal K+ de large conductance sensible au calcium, Maxi-K channel.NICE-BU Sciences (060882101) / SudocSudocFranceF

KCNQ1 K⁺ Channels are Involved in Lipopolysaccharide-induced Apoptosis of Distal Kidney Cells

International audienceMost bacteria initiate host inflammatory responses through interactions with epithelial cells. Lipopolysaccharide (LPS), a component of the bacterial cell wall is a major cause of septic shock in emergency care units and in the pathogenesis of acute renal failure. Kidney cells exposed to LPS undergo apoptotic changes, including cell volume decrease, phosphatidylserine exposure, caspase-3- and membrane K+ conductance -activation. Whole-cell configuration was used to identify K+ channels in primary and immortalized culture of mice distal convoluted tubules. LPS exposure induced a 3 fold increase in intracellular cAMP concentration and the activation of an outwardly rectifying K+ conductance in both immortalized and primary culture of distal cells. This LPS-induced current exhibited KCNQ1 K+ channel characteristics, i.e. inhibition by quinidine, chromanol293B and low dose of HMR1556 (IC50<1 microM) and insensitive to TEA and charybdotoxin. The background-like biophysical properties of the current suggest that the KCNQ1 pore-forming subunit is associated with a KCNE2 or KCNE3 ancillary subunit. RT-PCR experiments confirmed the presence of KCNQ1 and KCNE3 mRNA transcripts in primary culture of distal segments. Activation of the KCNQ1/KCNE3 K+ current appeared to be an essential step in the LPS-induced apoptosis process since HMR1556 blocked the LPS-induced- cell volume decrease, -caspase-3 activation and -phosphatidylserine exposure

A Ral Guanine Exchange Factor-Ral Pathway Is Conserved in Drosophila melanogaster and Sheds New Light on the Connectivity of the Ral, Ras, and Rap Pathways

Ras GTPases are central to many physiological and pathological signaling pathways and act via a combination of effectors. In mammals, at least three Ral exchange factors (RalGEFs) contain a Ras association domain and constitute a discrete subgroup of Ras effectors. Despite their ability to bind activated Rap as well as activated Ras, they seem to act downstream of Ras but not downstream of Rap. We have revisited the Ras/Rap-Ral connections in Drosophila melanogaster by using iterative two-hybrid screens with these three GTPases as primary baits and a subsequent genetic approach. We show that (i) the Ral-centered protein network appears to be extremely conserved in human and flies, (ii) in this network, RGL is a functional Drosophila orthologue of RalGEFs, and (iii) the RGL-Ral pathway functionally interacts with both the Ras and Rap pathways. Our data do not support the paradigmatic model where Ral is in the effector pathway of Ras. They reveal a signaling circuitry where Ral is functionally downstream of the Rap GTPase, at odds with the pathways described for mammalian cell lines. Thus, in vivo data show variations in the connectivity of pathways described for cell lines which might display only a subset of the biological possibilities

Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-beta/Smad signaling.

Members of the TGF-beta family of growth factors signal from the cell surface through serine/threonine kinase receptors. Intracellular propagation of the signal occurs by phosphorylation of intracellular proteins of the Smad family. Smad7 belongs to the subclass of inhibitory Smads that function as antagonists of TGF-beta signaling. A yeast two-hybrid screen of a human placental cDNA expression library using full-length mouse Smad7 as bait identified Yes-Associated Protein (YAP65) as a novel Smad7-interacting protein. The association of Smad7 with YAP65 was confirmed using co-expressed tagged proteins in COS-7 cells. Deletion of the PY motif of Smad7 reduced but did not abolish YAP65-Smad7 association, suggesting the existence of several interacting domains. We demonstrate that YAP65 potentiates the inhibitory activity of Smad7 against TGF-beta-induced, Smad3/4-dependent, gene transactivation. Furthermore, YAP65 augments the association of Smad7 to activated TGF-beta receptor type I (TbetaRI), whereas YAP65(1-301), which exerts a dominant-negative effect against Smad7-driven inhibition of TGF-beta signaling, reduces these interactions. Together, these data provide the first evidence that YAP65 is a Smad7 partner that facilitates the recruitment of the latter to activated TbetaRI, and enhances the inhibitory activity of Smad7 against TGF-beta signaling

Stratégie de conception d'expérience patient

National audienceRéflexion collective, codesign, construction collaborative, conception participative ou encore design thinking, les sciences participatives révèlent toute leur pertinence dans une époque où les enjeux écosystémiques sont prégnants. L'intelligence collective, sur laquelle est basée cette culture de la création par et pour les usagers, nécessite la participation de toutes les parties prenantes de votre projet. Cette interdisciplinarité au cœur de la e-santé est une thématique peu abordée dans la littérature non-scientifique. Expliquée et intégrée à la démarche de conception d'expérience utilisateur en santé, elle recèle des atouts précieux pour concevoir une expérience patient éthique, souveraine et durable. Les dimensions d'épanouissement et de liberté individuelle y sont centrales. Un livre incontournable si vous êtes entrepreneur du numérique ou designer d'expérience en santé

The novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for degradation

Ubiquitin-dependent degradation plays an important role in the negative regulation of TGF-β signaling. Here, we identify Tiul1 (for TGIF interacting ubiquitin ligase 1), a novel E3 ubiquitin ligase that inhibits TGF-β signaling by targeting both the activated receptor and Smad2 for degradation. Tiul1 associates constitutively with Smad7 and induces degradation of the activated type I receptor without affecting the expression levels of Smad7. Tiul1 can also interact with Smad2 and the nuclear corepressor TGIF upon activation of TGF-β signaling. Like Smad7, the steady-state levels of TGIF are not affected by Tiul1, but the interaction of Tiul1 with TGIF allows this ubiquitin ligase to target Smad2 for degradation. Consistent with this, overexpression of Tiul1 suppressed TGF-β-induced growth arrest and transcriptional responses. In addition, silencing of Tiul1 or TGIF genes by siRNA resulted in suppression of the TGF-β-dependent degradation of Smad2 and an enhancement of TGF-β-mediated gene expression. These results reveal a new role for TGIF as a component of a ubiquitin ligase complex that mediates the degradation of Smad2 in response to TGF-β signaling

Novel CLCN5 mutations in patients with Dent’s disease result in altered ion currents or impaired exchanger processing

International audienc