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1.
Cell Death Differ ; 24(12): 2150-2160, 2017 12.
Article in English | MEDLINE | ID: mdl-28862701

ABSTRACT

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.


Subject(s)
Kidney Diseases/enzymology , Nedd4 Ubiquitin Protein Ligases/deficiency , Amiloride/pharmacology , Animals , Epithelial Sodium Channel Blockers/pharmacology , Epithelial Sodium Channels/metabolism , Kidney Diseases/genetics , Kidney Diseases/metabolism , Kidney Diseases/pathology , Male , Mice , Mice, Transgenic , Nedd4 Ubiquitin Protein Ligases/genetics , Nedd4 Ubiquitin Protein Ligases/metabolism
2.
Behav Brain Res ; 303: 176-81, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26821291

ABSTRACT

The consolidation of short-term memory into long-term memory involves changing protein level and activity for the synaptic plasticity required for long-term potentiation (LTP). AMPA receptor trafficking is a key determinant of LTP and recently ubiquitination by Nedd4 has been shown to play an important role via direct action on the GluA1 subunit, although the physiological relevance of these findings are yet to be determined. We therefore investigated learning and memory in Nedd4(+/-) mice that have a 50% reduction in levels of Nedd4. These mice showed decreased long-term spatial memory as evidenced by significant increases in the time taken to learn the location of and subsequently find a platform in the Morris water maze. In contrast, there were no significant differences between Nedd4(+/+) and Nedd4(+/-) mice in terms of short-term spatial memory in a Y-maze test. Nedd4(+/-) mice also displayed a significant reduction in post-synaptic LTP measured in hippocampal brain slices. Immunofluorescence of Nedd4 in the hippocampus confirmed its expression in hippocampal neurons of the CA1 region. These findings indicate that reducing Nedd4 protein by 50% significantly impairs LTP and long-term memory thereby demonstrating an important role for Nedd4 in these processes.


Subject(s)
Endosomal Sorting Complexes Required for Transport/physiology , Hippocampus/physiology , Learning/physiology , Long-Term Potentiation , Spatial Memory/physiology , Ubiquitin-Protein Ligases/physiology , Animals , Endosomal Sorting Complexes Required for Transport/genetics , Heterozygote , Hippocampus/metabolism , Memory, Long-Term/physiology , Memory, Short-Term/physiology , Mice , Mice, Transgenic , Nedd4 Ubiquitin Protein Ligases , Neurons/metabolism , Receptors, AMPA/metabolism , Ubiquitin-Protein Ligases/genetics
3.
Gene ; 557(2): 113-22, 2015 Feb 25.
Article in English | MEDLINE | ID: mdl-25527121

ABSTRACT

Ubiquitination plays a crucial role in regulating proteins post-translationally. The focus of this review is on NEDD4, the founding member of the NEDD4 family of ubiquitin ligases that is evolutionarily conserved in eukaryotes. Many potential substrates of NEDD4 have been identified and NEDD4 has been shown to play a critical role in the regulation of a number of membrane receptors, endocytic machinery components and the tumour suppressor PTEN. In this review we will discuss the diverse pathways in which NEDD4 is involved, and the patho-physiological significance of this important ubiquitin ligase.


Subject(s)
Endosomal Sorting Complexes Required for Transport/physiology , Ubiquitin-Protein Ligases/physiology , Ubiquitination , Animals , Humans , Nedd4 Ubiquitin Protein Ligases , Neoplasms/enzymology , Oxidative Stress , PTEN Phosphohydrolase/metabolism , Signal Transduction , Ubiquitinated Proteins/metabolism
4.
Behav Brain Res ; 260: 15-24, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-24280120

ABSTRACT

Nedd4 is a widely expressed ubiquitin ligase that is necessary for normal neuronal development and function. However, largely due to the lethality of Nedd4 homozygous knockout mice, little is known about the physiological roles of Nedd4 in the adult brain. In this study we used Nedd4 heterozygous mice, which are viable and live to maturity, to assess for motor function and gait. Global motor function was not altered in these mice, a result consistent with the low level of Nedd4 expression observed in motor neurons of the spinal cord. However, Nedd4 heterozygous mice showed significant age-dependent changes in gait. The gait abnormalities included an overall extension of gait that was only evident in the 6 month old mice. We also observed distinct expression patterns of Nedd4, with pronounced staining in the Purkinje neurons of the cerebellum that are crucial for normal gait, and lower levels in other motor areas of the CNS. It has been recently shown that Nedd4 directly interacts with GluR1 containing AMPA receptors in an activity dependent manner to modulate receptor levels at the post-synaptic membrane. Using confocal immunohistochemistry, we found that there were subtle changes in GluR1 expression in 6 month old Nedd4 heterozygous mice. There appeared to be a redistribution of GluR1 into larger puncta in the molecular layer and in the membrane of the soma of the Purkinje neurons. This study is the first to show that a 50% reduction in Nedd4 levels is sufficient to produce significant gait defects in 6 month old mice. These defects may arise in part, from altered distribution of GluR1 in cerebellar neurons.


Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Gait Disorders, Neurologic/metabolism , Ubiquitin-Protein Ligases/metabolism , Age Factors , Animals , Blotting, Western , Body Weight , Brain/anatomy & histology , Brain/metabolism , Cerebellum/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Gait/physiology , Gait Disorders, Neurologic/genetics , Gene Expression , Heterozygote , Immunohistochemistry , Mice , Mice, Knockout , Microscopy, Confocal , Nedd4 Ubiquitin Protein Ligases , Organ Size , Purkinje Cells/metabolism , Receptors, AMPA/metabolism , Spinal Cord/metabolism , Subcellular Fractions/metabolism , Ubiquitin-Protein Ligases/genetics
5.
Biochem J ; 457(1): 27-31, 2014 Jan 01.
Article in English | MEDLINE | ID: mdl-24152020

ABSTRACT

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.


Subject(s)
Cerebral Cortex/metabolism , Endosomal Sorting Complexes Required for Transport/physiology , Neurons/metabolism , Sodium/pharmacology , Ubiquitin-Protein Ligases/physiology , Voltage-Gated Sodium Channels/metabolism , Animals , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/embryology , Embryo, Mammalian , Intracellular Space/metabolism , Mice , Mice, Knockout , Nedd4 Ubiquitin Protein Ligases , Neurons/drug effects , Patch-Clamp Techniques , Primary Cell Culture , Xenopus Proteins
6.
J Clin Invest ; 123(2): 657-65, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23348737

ABSTRACT

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human ß/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of ßENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of ß/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.


Subject(s)
Endosomal Sorting Complexes Required for Transport/deficiency , Hypertension/etiology , Kidney Tubules/physiopathology , Receptors, Drug/metabolism , Symporters/metabolism , Ubiquitin-Protein Ligases/deficiency , Animals , Blood Pressure , Disease Models, Animal , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Epithelial Sodium Channels/metabolism , Humans , Hypertension/genetics , Hypertension/physiopathology , Liddle Syndrome/etiology , Liddle Syndrome/genetics , Liddle Syndrome/physiopathology , Mice , Mice, Knockout , Nedd4 Ubiquitin Protein Ligases , Potassium/blood , Potassium/urine , Potassium Channels, Inwardly Rectifying/metabolism , Sodium/blood , Sodium/urine , Sodium, Dietary/administration & dosage , Sodium, Dietary/adverse effects , Solute Carrier Family 12, Member 3 , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
7.
Nat Commun ; 2: 287, 2011.
Article in English | MEDLINE | ID: mdl-21505443

ABSTRACT

The epithelial sodium channel (ENaC) is essential for sodium homoeostasis in many epithelia. ENaC activity is required for lung fluid clearance in newborn animals and for maintenance of blood volume and blood pressure in adults. In vitro studies show that the ubiquitin ligase Nedd4-2 ubiquitinates ENaC to regulate its cell surface expression. Here we show that knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung. This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2(-/-) animals, resulting in a failure to inflate lungs and perinatal lethality. A small percentage of Nedd4-2(-/-) animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung. Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.


Subject(s)
Endosomal Sorting Complexes Required for Transport/deficiency , Epithelial Sodium Channels/metabolism , Lung Diseases/genetics , Lung/pathology , Ubiquitin-Protein Ligases/deficiency , Animals , Blood Pressure , Blotting, Northern , Bronchoalveolar Lavage , Chemokines/metabolism , Cytokines/metabolism , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Endosomal Sorting Complexes Required for Transport/metabolism , Immunoblotting , Lung/embryology , Lung/metabolism , Lung Diseases/mortality , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Microscopy, Fluorescence , Nedd4 Ubiquitin Protein Ligases , Ubiquitin-Protein Ligases/metabolism
8.
Sci Signal ; 1(38): ra5, 2008 Sep 23.
Article in English | MEDLINE | ID: mdl-18812566

ABSTRACT

The ubiquitin ligase Nedd4 has been proposed to regulate a number of signaling pathways, but its physiological role in mammals has not been characterized. Here we present an analysis of Nedd4-null mice to show that loss of Nedd4 results in reduced insulin-like growth factor 1 (IGF-1) and insulin signaling, delayed embryonic development, reduced growth and body weight, and neonatal lethality. In mouse embryonic fibroblasts, mitogenic activity was reduced, the abundance of the adaptor protein Grb10 was increased, and the IGF-1 receptor, which is normally present on the plasma membrane, was mislocalized. However, surface expression of IGF-1 receptor was restored in homozygous mutant mouse embryonic fibroblasts after knockdown of Grb10, and Nedd4(-/-) lethality was rescued by maternal inheritance of a disrupted Grb10 allele. Thus, in vivo, Nedd4 appears to positively control IGF-1 and insulin signaling partly through the regulation of Grb10 function.


Subject(s)
Insulin-Like Growth Factor I/physiology , Receptor, IGF Type 1/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/physiology , Animals , Cell Membrane/metabolism , Cells, Cultured , Endosomal Sorting Complexes Required for Transport , GRB10 Adaptor Protein/metabolism , Insulin/physiology , Mice , Mice, Mutant Strains , Nedd4 Ubiquitin Protein Ligases , Phosphorylation
9.
J Biol Chem ; 282(16): 12135-42, 2007 Apr 20.
Article in English | MEDLINE | ID: mdl-17322297

ABSTRACT

The muscarine-sensitive K(+) current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K(+) currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system.


Subject(s)
Gene Expression Regulation , KCNQ Potassium Channels/physiology , KCNQ2 Potassium Channel/physiology , KCNQ3 Potassium Channel/physiology , Ubiquitin-Protein Ligases/physiology , Amino Acid Motifs , Animals , Down-Regulation , Endosomal Sorting Complexes Required for Transport , Humans , Membrane Potentials , Nedd4 Ubiquitin Protein Ligases , Oocytes/metabolism , Protein Binding , Rats , Ubiquitin/chemistry , Xenopus Proteins , Xenopus laevis
10.
Mol Microbiol ; 53(3): 929-40, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15255903

ABSTRACT

In Aspergillus nidulans, it is known that creB encodes a deubiquitinating enzyme that forms a complex with the WD40 motif containing protein encoded by creC, that mutations in these genes lead to altered carbon source utilization and that the creD34 mutation suppresses the phenotypic effects of mutations in creC and creB. Therefore, creD was characterized in order to dissect the regulatory network that involves the CreB-CreC deubiquitination complex. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p proteins from Saccharomyces cerevisiae. An additional gene was identified in the A. nidulans genome that also encodes an arrestin and PY motif-containing protein, which we have designated apyA, and thus two similar proteins also exist in A. nidulans. In S. cerevisiae, Rod1p and Rog3p interact with the ubiquitin ligase Rsp5p, and so the A. nidulans homologue of Rsp5p was identified, and the gene encoding this HECT ubiquitin ligase was designated hulA. CreD and ApyA were tested for protein-protein interactions with HulA via the bacterial two-hybrid system, and ApyA showed strong interaction, and CreD showed weak interaction, with HulA in this system.


Subject(s)
Aspergillus nidulans/genetics , Carrier Proteins/genetics , Escherichia coli Proteins , Fungal Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Amino Acid Sequence , Aspergillus nidulans/enzymology , Base Sequence , Cloning, Molecular , DNA Primers , Genetic Complementation Test , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , Protein Kinases/genetics , Restriction Mapping , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics
11.
Genetics ; 164(1): 95-104, 2003 May.
Article in English | MEDLINE | ID: mdl-12750323

ABSTRACT

Mutations in the acrB gene, which were originally selected through their resistance to acriflavine, also result in reduced growth on a range of sole carbon sources, including fructose, cellobiose, raffinose, and starch, and reduced utilization of omega-amino acids, including GABA and beta-alanine, as sole carbon and nitrogen sources. The acrB2 mutation suppresses the phenotypic effects of mutations in the creB gene that encodes a regulatory deubiquitinating enzyme, and in the creC gene that encodes a WD40-repeat-containing protein. Thus AcrB interacts with a regulatory network controlling carbon source utilization that involves ubiquitination and deubiquitination. The acrB gene was cloned and physically analyzed, and it encodes a novel protein that contains three putative transmembrane domains and a coiled-coil region. AcrB may play a role in the ubiquitination aspect of this regulatory network.


Subject(s)
Acriflavine/metabolism , Aspergillus nidulans/genetics , Amino Acid Sequence , Aspergillus nidulans/metabolism , Base Sequence , Molecular Sequence Data , Mutation
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