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Daniela Rotin - One of the best experts on this subject based on the ideXlab platform.
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the ubiquitin ligase NEDD4l regulates the na k 2cl co transporter nkcc1 slc12a2 in the colon
Journal of Biological Chemistry, 2017Co-Authors: Chong Jiang, Hiroshi Kawabe, Daniela RotinAbstract:Abstract The ubiquitin ligase NEDD4-like (NEDD4L, or NEDD4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out NEDD4L in mice intestinal epithelium (NEDD4Lf/f;Vil-CreERT2) we show here that NEDD4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl−, suggesting that Na+/K+/2Cl− (NKCC1/SLC12A2) co-transporter and ENaC are targets of NEDD4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the NEDD4L knock-out animals was increased, indicating that NEDD4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between NEDD4L and NKCC1, suggesting that NEDD4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in β and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for NEDD4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.
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The Ubiquitin Ligase NEDD4L Regulates the Na/K/2Cl Co-transporter NKCC1/SLC12A2 in the Colon
Journal of Biological Chemistry, 2017Co-Authors: Chong Jiang, Hiroshi Kawabe, Daniela RotinAbstract:Abstract The ubiquitin ligase NEDD4-like (NEDD4L, or NEDD4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out NEDD4L in mice intestinal epithelium (NEDD4Lf/f;Vil-CreERT2) we show here that NEDD4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl−, suggesting that Na+/K+/2Cl− (NKCC1/SLC12A2) co-transporter and ENaC are targets of NEDD4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the NEDD4L knock-out animals was increased, indicating that NEDD4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between NEDD4L and NKCC1, suggesting that NEDD4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in β and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for NEDD4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.
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Intestinal knockout of NEDD4 enhances growth of Apc^min tumors
Oncogene, 2016Co-Authors: C. Lu, C Thoeni, Steven Gallinger, A. O'connor, Hiroshi Kawabe, Daniela RotinAbstract:NEDD4 (NEDD4-1) is an E3 ubiquitin ligase that belongs to the HECT family and comprises a C2-WW(n)-HECT domain architecture. Although it has been reported to regulate growth factor receptors and cellular signaling, its role in cancer development has been controversial, with some studies proposing that it promotes cancer while others suggest it inhibits tumor growth. Here, we tested the effect of NEDD4 on intestinal tumor formation and growth using NEDD4-knockout mice ( NEDD4 floxed (fl) mice crossed to villin -Cre mice). Although we find that knockout of NEDD4 on its own does not cause tumor growth, its knockout in the context of Apc ^+/min-derived colorectal tumors leads to augmentation of tumor growth, suggesting that NEDD4 normally suppresses intestinal WNT signaling and growth of colonic tumors. WNT signaling microarray, immunoblotting and immunohistochemistry analyses of tumors derived from the Villin -Cre; NEDD4 ^fl/fl; Apc ^+/min colons demonstrated elevated expression of the WNT upstream effectors LEF1 (full length) and YY1 in these tumors relative to control ( Apc ^+/min alone) tumors. Together, these results suggest that NEDD4 suppresses colonic WNT signaling and tumor growth, at least in part, by suppressing the transcription factors LEF1 and YY1.
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Tyrosine phosphorylation of NEDD4 activates its ubiquitin ligase activity.
Science signaling, 2014Co-Authors: Avinash Persaud, Philipp Alberts, Sara Mari, Jiefei Tong, Ryan Murchie, Elena Maspero, Frozan Safi, Michael F. Moran, Simona Polo, Daniela RotinAbstract:Ligand binding to the receptor tyrosine kinase fibroblast growth factor (FGF) receptor 1 (FGFR1) causes dimerization and activation by transphosphorylation of tyrosine residues in the kinase domain. FGFR1 is ubiquitylated by the E3 ligase NEDD4 (also known as NEDD4-1), which promotes FGFR1 internalization and degradation. Although phosphorylation of FGFR1 is required for NEDD4-dependent endocytosis, NEDD4 directly binds to a nonphosphorylated region of FGFR1. We found that activation of FGFR1 led to activation of c-Src kinase–dependent tyrosine phosphorylation of NEDD4, enhancing the ubiquitin ligase activity of NEDD4. Using mass spectrometry, we identified several FGF-dependent phosphorylated tyrosines in NEDD4, including Tyr 43 in the C2 domain and Tyr 585 in the HECT domain. Mutating these tyrosines to phenylalanine to prevent phosphorylation inhibited FGF-dependent NEDD4 activity and FGFR1 endocytosis and enhanced cell proliferation. Mutating the tyrosines to glutamic acid to mimic phosphorylation enhanced NEDD4 activity. Moreover, the NEDD4 C2 domain bound the HECT domain, and the presence of phosphomimetic mutations inhibited this interaction, suggesting that phosphorylation of NEDD4 relieves an inhibitory intra- or intermolecular interaction. Accordingly, activation of FGFR1 was not required for activation of NEDD4 that lacked its C2 domain. Activation of c-Src by epidermal growth factor (EGF) also promoted tyrosine phosphorylation and enhanced the activity of NEDD4. Thus, we identified a feedback mechanism by which receptor tyrosine kinases promote catalytic activation of NEDD4 and that may represent a mechanism of receptor crosstalk.
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A splice isoform of DNEDD4, DNEDD4-long, negatively regulates neuromuscular synaptogenesis and viability in Drosophila.
PLoS ONE, 2011Co-Authors: Yunan Zhong, Alina Shtineman-kotler, Leo Nguyen, Konstantin G. Iliadi, Gabrielle L. Boulianne, Daniela RotinAbstract:Background Neuromuscular (NM) synaptogenesis is a tightly regulated process. We previously showed that in flies, Drosophila NEDD4 (dNEDD4/dNEDD4S) is required for proper NM synaptogenesis by promoting endocytosis of commissureless from the muscle surface, a pre-requisite step for muscle innervation. DNEDD4 is an E3 ubiquitin ligase comprised of a C2-WW(x3)-Hect domain architecture, which includes several splice isoforms, the most prominent ones are dNEDD4-short (dNEDD4S) and dNEDD4-long (dNEDD4Lo). Methodology/Principal Findings We show here that while dNEDD4S is essential for NM synaptogenesis, the dNEDD4Lo isoform inhibits this process and causes lethality. Our results reveal that unlike dNEDD4S, dNEDD4Lo cannot rescue the lethality of dNEDD4 null (DNEDD4T121FS) flies. Moreover, overexpression of UAS-dNEDD4Lo specifically in wildtype muscles leads to NM synaptogenesis defects, impaired locomotion and larval lethality. These negative effects of dNEDD4Lo are ameliorated by deletion of two regions (N-terminus and Middle region) unique to this isoform, and by inactivating the catalytic activity of dNEDD4Lo, suggesting that these unique regions, as well as catalytic activity, are responsible for the inhibitory effects of dNEDD4Lo on synaptogenesis. In accord with these findings, we demonstrate by sqRT-PCR an increase in dNEDD4S expression relative to the expression of dNEDD4Lo during embryonic stages when synaptogenesis takes place. Conclusion/Significance Our studies demonstrate that splice isoforms of the same dNEDD4 gene can lead to opposite effects on NM synaptogenesis.
Sharad Kumar - One of the best experts on this subject based on the ideXlab platform.
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Identification of novel interacting partners of the NEDD4 ubiquitin ligase in mouse testis.
Journal of proteomics, 2020Co-Authors: Jantina A Manning, Simon P. Windley, Jarrod J. Sandow, Sonia S. Shah, Patrick S Western, Dagmar Wilhelm, Sharad KumarAbstract:Posttranslational modification by ubiquitination targets proteins for degradation, recycling, stabilization or altered trafficking, and as such can alter cellular signaling pathways. The substrate specificity of this multistep process is controlled by ubiquitin ligases, including those of the HECT domain-containing NEDD4 family. In the testis, ubiquitination of many proteins contributes to organ development and maturation of spermatozoa and NEDD4 is known to be important in the control of spermatogonial stem cell homeostasis. However, a comprehensive understanding of NEDD4 substrates in testis development is lacking. Here we demonstrate high expression of NEDD4 in somatic cells of the mouse testis and in the murine Leydig cell-like cell line TM3. Immunoprecipitation of NEDD4 tagged with GFP at either the amino or carboxyl terminus was subjected to proteomic analysis for interacting proteins. We identified a substantial list of potential interaction partners, including known NEDD4 substrates, proteins involved in ubiquitination and proteins important for testis development and spermatogenesis. We confirmed the interaction of NEDD4 with a subset of these putative interacting proteins, validating the integrity of the dataset. These potential interactors may be further explored to reveal important roles of NEDD4-mediated ubiquitination in the testis. SIGNIFICANCE: Ubiquitination is important for testis development and function, and NEDD4 is known to ubiquitinate various proteins to affect cellular signaling and development, including those implicated in spermatogenesis. However, substrates of NEDD4 that are important during testis development remain to be identified. Here we report NEDD4 expression in the developing testis and TM3 testicular cell line. This study identifies a substantial list of NEDD4 interacting proteins in the TM3 testicular cell line, with validation of some of these interactions. Hence, this provides novel NEDD4 targets that may contribute to testis development and function that may be further explored.
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Deletion of NEDD4-2 results in progressive kidney disease in mice.
Cell death and differentiation, 2017Co-Authors: Tanya L Henshall, Natasha A. Boase, Pranay Goel, Jantina A Manning, Hiroshi Kawabe, Omri S. Alfassy, Sharad KumarAbstract:NEDD4-2 (NEDD4L), a ubiquitin protein ligase of the NEDD4 family, is a key regulator of cell surface expression and activity of the amiloride-sensitive epithelial Na+ channel (ENaC). While hypomorphic alleles of NEDD4-2 in mice show salt-sensitive hypertension, complete knockout results in pulmonary distress and perinatal lethality due to increased cell surface levels of ENaC. We now show that NEDD4-2 deficiency in mice also results in an unexpected progressive kidney injury phenotype associated with elevated ENaC and Na+Cl- cotransporter expression, increased Na+ reabsorption, hypertension and markedly reduced levels of aldosterone. The observed nephropathy is characterized by fibrosis, tubule epithelial cell apoptosis, dilated/cystic tubules, elevated expression of kidney injury markers and immune cell infiltration, characteristics reminiscent of human chronic kidney disease. Importantly, we demonstrate that the extent of kidney injury can be partially therapeutically ameliorated in mice with nephron-specific deletions of NEDD4-2 by blocking ENaC with amiloride. These results suggest that increased Na+ reabsorption via ENaC causes kidney injury and establish a novel role of NEDD4-2 in preventing Na+-induced nephropathy. Contrary to some recent reports, our data also indicate that ENaC is the primary in vivo target of NEDD4-2 and that NEDD4-2 deletion is associated with hypertension on a normal Na+ diet. These findings provide further insight into the critical function of NEDD4-2 in renal pathophysiology.
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NEDD4 2 NEDD4l the ubiquitin ligase for multiple membrane proteins
Gene, 2015Co-Authors: Pranay Goel, Jantina A Manning, Sharad KumarAbstract:Abstract NEDD4-2 (also known as NEDD4L, neural precursor cell expressed developmentally down-regulated 4-like) is a ubiquitin protein ligase of the NEDD4 family which is known to bind and regulate a number of membrane proteins to aid in their internalization and turnover. Several of the NEDD4-2 substrates include ion channels, such as the epithelial and voltage-gated sodium channels. Given the critical function of NEDD4-2 in regulating membrane proteins, this ligase is essential for the maintenance of cellular homeostasis. In this article we review the biology and function of this important ubiquitin-protein ligase and discuss its pathophysiological significance.
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NEDD4 2 NEDD4l controls intracellular na mediated activity of voltage gated sodium channels in primary cortical neurons
Biochemical Journal, 2014Co-Authors: Jenny Ekberg, Natasha A. Boase, Jantina A Manning, Philip Poronnik, Grigori Y Rychkov, Sharad KumarAbstract:NEDD4-2, a HECT (homologous with E6-associated protein C-terminus)-type ubiquitin protein ligase, has been implicated in regulating several ion channels, including Navs (voltage-gated sodium channels). In Xenopus oocytes NEDD4-2 strongly inhibits the activity of multiple Navs. However, the conditions under which NEDD4-2 mediates native Nav regulation remain uncharacterized. Using NEDD4-2-deficient mice, we demonstrate in the present study that in foetal cortical neurons NEDD4-2 regulates Navs specifically in response to elevated intracellular Na(+), but does not affect steady-state Nav activity. In dorsal root ganglia neurons from the same mice, however, NEDD4-2 does not control Nav activities. The results of the present study provide the first physiological evidence for an essential function of NEDD4-2 in regulating Navs in the central nervous system.
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NEDD4-2 (NEDD4L) controls intracellular Na+-mediated activity of voltage-gated sodium channels in primary cortical neurons
Biochemical Journal, 2013Co-Authors: Jenny Ekberg, Natasha A. Boase, Jantina A Manning, Philip Poronnik, Grigori Y Rychkov, Sharad KumarAbstract:NEDD4-2, a HECT (homologous with E6-associated protein C-terminus)-type ubiquitin protein ligase, has been implicated in regulating several ion channels, including Navs (voltage-gated sodium channels). In Xenopus oocytes NEDD4-2 strongly inhibits the activity of multiple Navs. However, the conditions under which NEDD4-2 mediates native Nav regulation remain uncharacterized. Using NEDD4-2-deficient mice, we demonstrate in the present study that in foetal cortical neurons NEDD4-2 regulates Navs specifically in response to elevated intracellular Na(+), but does not affect steady-state Nav activity. In dorsal root ganglia neurons from the same mice, however, NEDD4-2 does not control Nav activities. The results of the present study provide the first physiological evidence for an essential function of NEDD4-2 in regulating Navs in the central nervous system.
Olivier Staub - One of the best experts on this subject based on the ideXlab platform.
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the role of intercalated cell NEDD4 2 in bp regulation ion transport and transporter expression
Journal of The American Society of Nephrology, 2018Co-Authors: Masayoshi Nanami, Truyen D Pham, Roy L Sutliff, Karen I Lopezcayuqueo, Janet D Klein, Olivier Staub, Baoli Yang, Régine Chambrey, Annie Y ParkAbstract:BackgroundNEDD4-2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between NEDD4-2 and BP. In this study, we explored the effect of intercalated cell (IC) NEDD4-2 gene ablation on IC transporter abundance and function and on BP.Methods We generated IC NEDD4-2 knockout mice using Cre-lox technology and produced global pendrin/NEDD4-2 null mice by breeding global NEDD4-2 null (NEDD4-2-/- ) mice with global pendrin null (Slc26a4-/- ) mice. Mice ate a diet with 1%-4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl- and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.Results IC NEDD4-2 gene ablation markedly increased electroneutral Cl-/HCO3- exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC NEDD4-2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, or the Na+-dependent Cl-/HCO3- exchanger (Slc4a8). However, IC NEDD4-2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC NEDD4-2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global NEDD4-2 knockout mice.Conclusions IC NEDD4-2 regulates Cl-/HCO3- exchange in ICs., NEDD4-2 gene ablation increases BP in part through its action in these cells.
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The Role of Intercalated Cell NEDD4–2 in BP Regulation, Ion Transport, and Transporter Expression
Journal of the American Society of Nephrology : JASN, 2018Co-Authors: Masayoshi Nanami, Truyen D Pham, Roy L Sutliff, Janet D Klein, Olivier Staub, Baoli Yang, Régine Chambrey, Young-hee Kim, Karen I. López-cayuqueo, Annie Y ParkAbstract:BackgroundNEDD4-2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between NEDD4-2 and BP. In this study, we explored the effect of intercalated cell (IC) NEDD4-2 gene ablation on IC transporter abundance and function and on BP.Methods We generated IC NEDD4-2 knockout mice using Cre-lox technology and produced global pendrin/NEDD4-2 null mice by breeding global NEDD4-2 null (NEDD4-2-/- ) mice with global pendrin null (Slc26a4-/- ) mice. Mice ate a diet with 1%-4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl- and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.Results IC NEDD4-2 gene ablation markedly increased electroneutral Cl-/HCO3- exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC NEDD4-2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, or the Na+-dependent Cl-/HCO3- exchanger (Slc4a8). However, IC NEDD4-2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC NEDD4-2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global NEDD4-2 knockout mice.Conclusions IC NEDD4-2 regulates Cl-/HCO3- exchange in ICs., NEDD4-2 gene ablation increases BP in part through its action in these cells.
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Renal Tubular Ubiquitin-Protein Ligase NEDD4-2 Is Required for Renal Adaptation during Long-Term Potassium Depletion
Journal of The American Society of Nephrology, 2017Co-Authors: Al-qusairi, Arohan R. Subramanya, Renuga Devi Rajaram, Denis Basquin, Marc Maillard, Olivier StaubAbstract:Adaptation of the organism to potassium (K + ) deficiency requires precise coordination among organs involved in K + homeostasis, including muscle, liver, and kidney. How the latter performs functional and molecular changes to ensure K + retention is not well understood. Here, we investigated the role of ubiquitin-protein ligase NEDD4-2, which negatively regulates the epithelial sodium channel (ENaC), Na + /Cl − cotransporter (NCC), and with no-lysine-kinase 1 (WNK1). After dietary K + restriction for 2 weeks, compared with control littermates, inducible renal tubular NEDD4-2 knockout ( NEDD4L Pax8/LC1 ) mice exhibited severe hypokalemia and urinary K + wasting. Notably, expression of the ROMK K + channel did not change in the distal convoluted tubule and decreased slightly in the cortical/medullary collecting duct, whereas BK channel abundance increased in principal cells of the connecting tubule/collecting ducts. However, K + restriction also enhanced ENaC expression in NEDD4L Pax8/LC1 mice, and treatment with the ENaC inhibitor, benzamil, reversed excessive K + wasting. Moreover, K + restriction increased WNK1 and WNK4 expression and enhanced SPAK-mediated NCC phosphorylation in NEDD4L Pax8/LC1 mice, with no change in total NCC. We propose a mechanism in which NEDD4-2 deficiency exacerbates hypokalemia during dietary K + restriction primarily through direct upregulation of ENaC, whereas increased BK channel expression has a less significant role. These changes outweigh the compensatory antikaliuretic effects of diminished ROMK expression, increased NCC phosphorylation, and enhanced WNK pathway activity in the distal convoluted tubule. Thus, NEDD4-2 has a crucial role in K + conservation through direct and indirect effects on ENaC, distal nephron K + channels, and WNK signaling.
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the kcnq1 potassium channel is down regulated by ubiquitylating enzymes of the NEDD4 NEDD4 like family
Cardiovascular Research, 2007Co-Authors: Thomas Jespersen, Olivier Staub, Mathieu Membrez, Celine S Nicolas, Bruno Pitard, Sorenpeter Olesen, Isabelle Baro, Hugues AbrielAbstract:OBJECTIVE: The voltage-gated KCNQ1 potassium channel regulates key physiological functions in a number of tissues. In the heart, KCNQ1 alpha-subunits assemble with KCNE1 beta-subunits forming a channel complex constituting the delayed rectifier current I(Ks). In epithelia, KCNQ1 channels participate in controlling body electrolyte homeostasis. Several regulatory mechanisms of the KCNQ1 channel complexes have been reported, including protein kinase A (PKA)-phosphorylation and beta-subunit interactions. However, the mechanisms controlling the membrane density of KCNQ1 channels have attracted less attention. METHODS AND RESULTS: Here we demonstrate that KCNQ1 proteins expressed in HEK293 cells are down-regulated by NEDD4/NEDD4-like ubiquitin-protein ligases. KCNQ1 and KCNQ1/KCNE1 currents were reduced upon co-expression of NEDD4-2, the isoform among the nine members of the NEDD4/NEDD4-like family displaying the highest expression level in human heart. In vivo expression of a catalytically inactive form of NEDD4-2, able to antagonize endogenous NEDD4-2 in guinea-pig cardiomyocytes, increased I(Ks) significantly, but did not modify I(K1). Concomitant with the reduction in current induced by NEDD4-2, an increased ubiquitylation as well as a decreased total level of KCNQ1 proteins were observed in HEK293 cells. Pull-down and co-immunoprecipitation experiments showed that NEDD4-2 interacts with the C-terminal part of KCNQ1. The NEDD4/NEDD4-like-mediated regulation of the KCNQ1 channel complexes is strictly dependent on a PY motif located in the distal part of the C-terminal domain. When this motif was mutated, the current and ubiquitylation levels were unaffected by NEDD4-2, and NEDD4-2 proteins were neither pulled-down nor co-immunoprecipitated. CONCLUSIONS: These results suggest that KCNQ1 internalization and stability is physiologically regulated by its NEDD4/NEDD4-like-dependent ubiquitylation. This mechanism may thereby be important in regulating the surface density of the KCNQ1 channels in cardiomyocytes and other cell types.
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the kcnq1 potassium channel is down regulated by ubiquitylating enzymes of the NEDD4 NEDD4 like family
Cardiovascular Research, 2007Co-Authors: Thomas Jespersen, Olivier Staub, Mathieu Membrez, Celine S Nicolas, Bruno Pitard, Sorenpeter Olesen, Isabelle Baro, Hugues AbrielAbstract:Objective : The voltage-gated KCNQ1 potassium channel regulates key physiological functions in a number of tissues. In the heart, KCNQ1 α-subunits assemble with KCNE1 β-subunits forming a channel complex constituting the delayed rectifier current I Ks. In epithelia, KCNQ1 channels participate in controlling body electrolyte homeostasis. Several regulatory mechanisms of the KCNQ1 channel complexes have been reported, including protein kinase A (PKA)-phosphorylation and β-subunit interactions. However, the mechanisms controlling the membrane density of KCNQ1 channels have attracted less attention. Methods and results : Here we demonstrate that KCNQ1 proteins expressed in HEK293 cells are down-regulated by NEDD4/NEDD4-like ubiquitin-protein ligases. KCNQ1 and KCNQ1/KCNE1 currents were reduced upon co-expression of NEDD4-2, the isoform among the nine members of the NEDD4/NEDD4-like family displaying the highest expression level in human heart. In vivo expression of a catalytically inactive form of NEDD4-2, able to antagonize endogenous NEDD4-2 in guinea-pig cardiomyocytes, increased I Ks significantly, but did not modify I K1. Concomitant with the reduction in current induced by NEDD4-2, an increased ubiquitylation as well as a decreased total level of KCNQ1 proteins were observed in HEK293 cells. Pull-down and co-immunoprecipitation experiments showed that NEDD4-2 interacts with the C-terminal part of KCNQ1. The NEDD4/NEDD4-like-mediated regulation of the KCNQ1 channel complexes is strictly dependent on a PY motif located in the distal part of the C-terminal domain. When this motif was mutated, the current and ubiquitylation levels were unaffected by NEDD4-2, and NEDD4-2 proteins were neither pulled-down nor co-immunoprecipitated. Conclusions : These results suggest that KCNQ1 internalization and stability is physiologically regulated by its NEDD4/NEDD4-like-dependent ubiquitylation. This mechanism may thereby be important in regulating the surface density of the KCNQ1 channels in cardiomyocytes and other cell types.
Hiroshi Kawabe - One of the best experts on this subject based on the ideXlab platform.
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the e3 ubiquitin ligase NEDD4 enhances killing of membrane perturbing intracellular bacteria by promoting autophagy
Autophagy, 2017Co-Authors: Gang Pei, Hiroshi Kawabe, Hellen Buijze, Haipeng Liu, Pedro Mouraalves, Christian Goosmann, Volker Brinkmann, Anca Dorhoi, Stefan H E KaufmannAbstract:The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1.
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Deletion of NEDD4-2 results in progressive kidney disease in mice.
Cell death and differentiation, 2017Co-Authors: Tanya L Henshall, Natasha A. Boase, Pranay Goel, Jantina A Manning, Hiroshi Kawabe, Omri S. Alfassy, Sharad KumarAbstract:NEDD4-2 (NEDD4L), a ubiquitin protein ligase of the NEDD4 family, is a key regulator of cell surface expression and activity of the amiloride-sensitive epithelial Na+ channel (ENaC). While hypomorphic alleles of NEDD4-2 in mice show salt-sensitive hypertension, complete knockout results in pulmonary distress and perinatal lethality due to increased cell surface levels of ENaC. We now show that NEDD4-2 deficiency in mice also results in an unexpected progressive kidney injury phenotype associated with elevated ENaC and Na+Cl- cotransporter expression, increased Na+ reabsorption, hypertension and markedly reduced levels of aldosterone. The observed nephropathy is characterized by fibrosis, tubule epithelial cell apoptosis, dilated/cystic tubules, elevated expression of kidney injury markers and immune cell infiltration, characteristics reminiscent of human chronic kidney disease. Importantly, we demonstrate that the extent of kidney injury can be partially therapeutically ameliorated in mice with nephron-specific deletions of NEDD4-2 by blocking ENaC with amiloride. These results suggest that increased Na+ reabsorption via ENaC causes kidney injury and establish a novel role of NEDD4-2 in preventing Na+-induced nephropathy. Contrary to some recent reports, our data also indicate that ENaC is the primary in vivo target of NEDD4-2 and that NEDD4-2 deletion is associated with hypertension on a normal Na+ diet. These findings provide further insight into the critical function of NEDD4-2 in renal pathophysiology.
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the ubiquitin ligase NEDD4l regulates the na k 2cl co transporter nkcc1 slc12a2 in the colon
Journal of Biological Chemistry, 2017Co-Authors: Chong Jiang, Hiroshi Kawabe, Daniela RotinAbstract:Abstract The ubiquitin ligase NEDD4-like (NEDD4L, or NEDD4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out NEDD4L in mice intestinal epithelium (NEDD4Lf/f;Vil-CreERT2) we show here that NEDD4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl−, suggesting that Na+/K+/2Cl− (NKCC1/SLC12A2) co-transporter and ENaC are targets of NEDD4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the NEDD4L knock-out animals was increased, indicating that NEDD4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between NEDD4L and NKCC1, suggesting that NEDD4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in β and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for NEDD4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.
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The Ubiquitin Ligase NEDD4L Regulates the Na/K/2Cl Co-transporter NKCC1/SLC12A2 in the Colon
Journal of Biological Chemistry, 2017Co-Authors: Chong Jiang, Hiroshi Kawabe, Daniela RotinAbstract:Abstract The ubiquitin ligase NEDD4-like (NEDD4L, or NEDD4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out NEDD4L in mice intestinal epithelium (NEDD4Lf/f;Vil-CreERT2) we show here that NEDD4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl−, suggesting that Na+/K+/2Cl− (NKCC1/SLC12A2) co-transporter and ENaC are targets of NEDD4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the NEDD4L knock-out animals was increased, indicating that NEDD4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between NEDD4L and NKCC1, suggesting that NEDD4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in β and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for NEDD4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.
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Intestinal knockout of NEDD4 enhances growth of Apc^min tumors
Oncogene, 2016Co-Authors: C. Lu, C Thoeni, Steven Gallinger, A. O'connor, Hiroshi Kawabe, Daniela RotinAbstract:NEDD4 (NEDD4-1) is an E3 ubiquitin ligase that belongs to the HECT family and comprises a C2-WW(n)-HECT domain architecture. Although it has been reported to regulate growth factor receptors and cellular signaling, its role in cancer development has been controversial, with some studies proposing that it promotes cancer while others suggest it inhibits tumor growth. Here, we tested the effect of NEDD4 on intestinal tumor formation and growth using NEDD4-knockout mice ( NEDD4 floxed (fl) mice crossed to villin -Cre mice). Although we find that knockout of NEDD4 on its own does not cause tumor growth, its knockout in the context of Apc ^+/min-derived colorectal tumors leads to augmentation of tumor growth, suggesting that NEDD4 normally suppresses intestinal WNT signaling and growth of colonic tumors. WNT signaling microarray, immunoblotting and immunohistochemistry analyses of tumors derived from the Villin -Cre; NEDD4 ^fl/fl; Apc ^+/min colons demonstrated elevated expression of the WNT upstream effectors LEF1 (full length) and YY1 in these tumors relative to control ( Apc ^+/min alone) tumors. Together, these results suggest that NEDD4 suppresses colonic WNT signaling and tumor growth, at least in part, by suppressing the transcription factors LEF1 and YY1.
W. Weissenhorn - One of the best experts on this subject based on the ideXlab platform.
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ebola virus matrix protein vp40 interaction with human cellular factors tsg101 and NEDD4
Journal of Molecular Biology, 2003Co-Authors: Joanna Timmins, G. Schoehn, S. Scianimanico, T. Vernet, Rw Ruigrok, Sylvie Ricardblum, W. WeissenhornAbstract:Abstract The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human NEDD4 and Tsg101 in vitro. We show that WW domain 3 of human NEDD4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of NEDD4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin enzyme variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNEDD4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.
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Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and NEDD4.
Journal of Molecular Biology, 2003Co-Authors: Joanna Timmins, G. Schoehn, S. Ricard-blum, S. Scianimanico, T. Vernet, Rw Ruigrok, W. WeissenhornAbstract:The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human NEDD4 and Tsg101 in vitro. We show that WW domain 3 of human NEDD4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of NEDD4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin enzyme variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNEDD4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human NEDD4 and Tsg101 in vitro. We show that WW domain 3 of human NEDD4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of NEDD4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin enzyme variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNEDD4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.
