Mapping the H+ (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation

V-ATPases (H+ ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology

Caractérisation fonctionnelle de la pendrine (paramètres cinétiques et voies de régulation)

Le syndrome de Pendred est une maladie à transmission autosomique récessive traditionnellement définie par l'association d'une surdité et d'un goitre ; La pendrine a été découverte en 1997 à la suite d études génétiques visant à identifier le gène responsable du syndrome décrit un siècle auparavant. La pendrine appartient à la famille SLC26 des échangeurs d anion sodium indépendants. La pendrine est exprimée dans la thyroïde, l oreille interne et le rein. Elle participe à la synthèse des hormones thyroïdiennes par son activité d échange Cl-/I-, à l homéostasie de l endolymphe dans l oreille interne par la sécrétion de HCO3- et à l équilibre acide-base et réabsorption de NaCl dans le rein en tant qu échangeur Cl-/HCO3-. Malgré l importance de la pendrine il existe peu de données concernant ses propriétés intrinsèques et ses voies de régulation. Les objectifs de ce travail de thèse ont été :1) d étudier les propriétés intrinsèques de la pendrine en caractérisant ses paramètres cinétiques, 2) de caractériser la régulation de la pendrine par le pH et 3) d étudier la régulation de la pendrine du canal collecteur par l AMPc. Nous avons montré pour la première fois que l activité de la pendrine dépend du pH intra et extracellulaire. Cette sensibilité de la pendrine au pH peut être un élément clé dans le contrôle de l échange ionique dans le CCD du rein et dans le sac endolymphatique de l oreille interne. Nous avons également démontré une régulation de la pendrine par l AMPc/PKA. Cette régulation semble particulièrement importante dans le contexte de l hypertension rénale sel-sensible et dans la régulation de la synthèse hormonale par la thyroïdePARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Acidosis‐induced activation of distal nephron principal cells triggers Gdf15 secretion and adaptive proliferation of intercalated cells

International audienceType A intercalated cells of the renal collecting duct participate in the maintenance of the acid/base balance through their capacity to adapt proton secretion to homeostatic requirements. We previously showed that increased proton secretion stems in part from the enlargement of the population of proton secreting cells in the outer medullary collecting duct through division of fully differentiated cells, and that this response is triggered by growth/differentiation factor 15. This study aimed at deciphering the mechanism of acid load-induced secretion of Gdf15 and its mechanism of action

Renal Intercalated Cells Sense and Mediate Inflammation via the P2Y14 Receptor

Uncontrolled inflammation is one of the leading causes of kidney failure. Pro-inflammatory responses can occur in the absence of infection, a process called sterile inflammation. Here we show that the purinergic receptor P2Y14 (GPR105) is specifically and highly expressed in collecting duct intercalated cells (ICs) and mediates sterile inflammation in the kidney. P2Y14 is activated by UDP-glucose, a damage-associated molecular pattern molecule (DAMP) released by injured cells. We found that UDP-glucose increases pro-inflammatory chemokine expression in ICs as well as MDCK-C11 cells, and UDP-glucose activates the MEK1/2-ERK1/2 pathway in MDCK-C11 cells. These effects were prevented following inhibition of P2Y14 with the small molecule PPTN. Tail vein injection of mice with UDP-glucose induced the recruitment of neutrophils to the renal medulla. This study identifies ICs as novel sensors, mediators and effectors of inflammation in the kidney via P2Y14

Altered V-ATPase expression in renal intercalated cells isolated from B1-subunit deficient mice by fluorescence activated cell sorting

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1(-/-)) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1(-/-) and wild type mice. However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild type and Atp6v1b1(-/-) mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1(+/+) mice) with Atp6v1b1(-/-) mice to generate novel EGFP-B1(-/-) mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1(+/+) and EGFP-B1(-/-) mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly down-regulated in EGFP-B1(-/-) mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions the B2 up-regulation compensates for the lack of B1, and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are down-regulated

P2Y₁₄ activation by UDP-glucose increases ERK1/2-phosphorylation in MDCK-C11 cells.

<p>Representative immunoblots showing triplicates of ERK1/2 phosphorylation (upper lane) versus total ERK1/2 (lower lane) in cells pretreated with vehicle or the P2Y₁₄ antagonist PPTN (10 μM), in the absence (CTRL) or presence of 100 μM UDP-glucose (UDP-glu). Quantification of the ratio of p-ERK/total ERK showed that UDP-glucose induced a significant increase in ERK1/2 phosphorylation (lower left panel, n = 7) and that PPTN prevented the increase in ERK1/2 phosphorylation induced by UDP-glucose (lower right panel, n = 5). Values are represented, relative to either control or PPTN alone, as means ± SEM, * p < 0.005.</p

Quantitative PCR detection of pro-inflammatory mediators in EGFP(+) cells.

<p>EGFP(+) cells were isolated by FACS from B1-EGFP mice 4h after an i.v. injection with saline (sham) or with saline containing 100 μM UDP-glucose (UDP-glu). All values are normalized to GAPDH. Data are represented as % changes relative to control. Values are mean ± SEM (n = 4), *P<0.05, ** P<0.001.</p

Sequence of the primers used for RT-PCR detection.

<p>Sequence of the primers used for RT-PCR detection.</p

Quantitative PCR detection of pro-inflammatory mediators in MDCK-C11 cells.

<p>(A) Detection of mRNA transcripts specific for IL-8, CCL2, CCL4, CCL5, IL1b and TNFa under control conditions and 4h after 100 μM UDP-glucose treatment. Data are represented as % changes relative to control. All values are normalized to GAPDH and are shown as Means ± SEM (n = 5), ** P<0.001. (B) Quantification of changes in IL-8 (left) and CCL2 (right) mRNA expression in MDCK-C11 cells pretreated with the vehicle only or with the MEK inhibitor, PD98059 (50 μM) for 30 minutes in the absence (CTRL) or presence of 100 μM UDP-glucose (UDP-glu). (C) Quantification of changes in IL-8 (<b>left</b>) and CCL2 (<b>right</b>) mRNA expression in MDCK-C11 cells pretreated with the vehicle only or with PPTN (10 μM) for 30 minutes, in the absence (CTRL) or presence of 100 μM UDP-glucose (UDP-glu). Data are represented as % changes relative to control. Values are means ± SEM (n = 3), * P<0.05, **P<0.001.</p