Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.2.19 (ubiquitin-protein ligase)
799 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.
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PMID:Identification of novel human WW domain-containing proteins by cloning of ligand targets. 916 21

Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we report the assignment of backbone 1H alpha, 1HN, 15N, 13C', 13C alpha, and aliphatic 13C resonances of a fragment of rat Nedd4 (rNedd4) containing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-containing peptide derived from the beta subunit of rat ENaC, the betaP2 peptide. The secondary structures of these two WW domains, determined from chemical shifts of 13C alpha and 13C beta resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the peptidyl-prolyl isomerase Pin1. Triple resonance experiments that detect the 1H alpha chemical shift were necessary to complete the chemical shift assignment, owing to the large number of proline residues in this fragment of rNedd4. A new experiment, which correlates sequential residues via their 15N nuclei and also detects 1H alpha chemical shifts, is introduced and its utility for the chemical shift assignment of sequential proline residues is discussed. Data collected on the WW(II+III)-betaP2 complex indicate that these WW domains have different affinities for the betaP2 peptide.
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PMID:NMR studies of tandem WW domains of Nedd4 in complex with a PY motif-containing region of the epithelial sodium channel. 992 3

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PY motif of either the beta or gamma subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind alpha-, beta-, and gamma-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+ channel and of WW domain 1 to an unknown associated protein.
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PMID:All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+. 1021 29

The epithelial Na(+) channel (ENaC) regulates Na(+) absorption in epithelial tissues including the lung, colon and sweat gland, and in the distal nephrons of the kidney. When Na(+)-channel function is disrupted, salt and water homoeostasis is affected. The cytoplasmic regions of the Na(+)-channel subunits provide binding sites for other proteins to interact with and potentially regulate Na(+)-channel activity. Previously we showed that a proline-rich region of the alpha subunit of the Na(+) channel bound to a protein of 116 kDa from human lung cells. Here we report the identification of this protein as human Nedd4, a ubiquitin-protein ligase that binds to the Na(+)-channel subunits via its WW domains. Further, we show that WW domains 2, 3 and 4 of human Nedd4 bind to the alpha, beta and gamma Na(+)-channel subunits but not to a mutated beta subunit. In addition, when co-expressed in Xenopus oocytes, human Nedd4 down-regulates Na(+)-channel activity.
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PMID:Human Nedd4 interacts with the human epithelial Na+ channel: WW3 but not WW1 binds to Na+-channel subunits. 1064 8

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY, motif which is believed to be important for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome, where mutations delete or alter the PPxY motif of either the beta or gamma subunits, has been proposed to result in increased ENaC activity. Here we present evidence that KIAA0439 protein, a close relative of Nedd4, is also a potential regulator of ENaC. We demonstrate that KIAA0439 WW domains bind all three ENaC subunits. We show that a recombinant KIAA0439 WW domain protein acts as a dominant negative mutant that can interfere with the Na(+)-dependent feedback inhibition of ENaC in whole-cell patch clamp experiments. We propose that KIAA0439 and Nedd4 proteins either play a redundant role in ENaC regulation or function in a tissue- and/or signal-specific manner to down-regulate ENaC.
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PMID:The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel. 1124 92

The amiloride-sensitive epithelial Na(+) channels (ENaC) in the intralobular duct cells of mouse mandibular glands are inhibited by the ubiquitin-protein ligase, Nedd4, which is activated by increased intracellular Na(+). In this study we have used whole-cell patch clamp methods in mouse mandibular duct cells to investigate the role of the C termini of the alpha-, beta-, and gamma-subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the beta- and gamma-subunits, but not the alpha-subunit, inhibited the activity of the Na(+) channels. This mechanism did not involve Nedd4 and probably resulted from the exogenous C termini interfering competitively with the protein-protein interactions that keep the channels active. In the case of the C terminus of mouse beta-ENaC, the interacting motif included betaSer(631), betaAsp(632), and betaSer(633). In the C terminus of mouse gamma-ENaC, it included gammaSer(640). Once these motifs were deleted, we were able to use the C termini of beta- and gamma-ENaC to prevent Nedd4-mediated down-regulation of Na(+) channel activity. The C terminus of the alpha-subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na(+) channels. We conclude that mouse Nedd4 interacts with the beta- and gamma-subunits of ENaC.
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PMID:Roles of the C termini of alpha -, beta -, and gamma -subunits of epithelial Na+ channels (ENaC) in regulating ENaC and mediating its inhibition by cytosolic Na+. 1127 74

The epithelial Na(+) channel (ENaC), which plays an essential role in renal Na(+) handling, is composed of three subunits (alpha beta gamma), each containing a conserved PY motif at the C terminus. In Liddle's syndrome, an inherited form of salt-sensitive hypertension, the PY motifs of either beta or gamma ENaC are deleted or modified. We have recently shown that a ubiquitin-protein ligase Nedd4 binds via its WW domains to these PY motifs on ENaC, that ENaC is regulated by ubiquitination, and that Xenopus laevis Nedd4 (xNedd4) controls the cell surface pool of ENaC when coexpressed in Xenopus oocytes. Interestingly, Na(+) transporting cells, derived from mouse cortical collecting duct, express two different Nedd4 isoforms, which we have termed mNedd4-1 and mNedd4-2. Only mNedd4-2, which is orthologous to xNedd4, but not mNedd4-1, is able to regulate ENaC activity, and this property correlates with the capability to bind to the ENaC complex. Hence, Nedd4-2 may be encoded by a novel susceptibility gene for arterial hypertension.
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PMID:Liddle's syndrome: a novel mouse Nedd4 isoform regulates the activity of the epithelial Na(+) channel. 1147 28

The epithelial Na(+) channel (ENaC) is regulated via PY motif-WW domain interaction by the mouse (m) ubiquitin-protein ligase mNedd4-2 but not by its close relative mNedd4-1. Whereas mNedd4-1 is composed of one C2, three WW, and one HECT domain, mNedd4-2 comprises four WW domains and one HECT domain. Both proteins have human (h) homologs, hNedd4-1 and hNedd4-2; however, both of them include four WW domains. Therefore, we characterized hNedd4-1 and hNedd4-2 in Xenopus laevis oocytes with respect to ENaC binding and interaction. We found that hNedd4-2 binds to and abrogates ENaC activity, whereas hNedd4-1 does not coimmunoprecipitate with ENaC and has only modest effects on ENaC activity. Structure-function studies revealed that the C2 domain of hNedd4-1 prevents this protein from downregulating ENaC and that WW domains 3 and 4, involved in interaction with ENaC, do not by themselves provide specificity for ENaC recognition. Taken together, our data demonstrate that hNedd4-2 inhibits ENaC, implying that this protein is a modulator of salt homeostasis, whereas hNedd4-1 is not primarily involved in ENaC regulation.
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PMID:Distinct characteristics of two human Nedd4 proteins with respect to epithelial Na(+) channel regulation. 1150 96

The activity of the epithelial Na(+) channel (ENaC) is required for the maintenance of salt and water balance in the body. Channel activity is regulated by the ubiquitin-protein ligase Nedd4 ['neuronal precursor cell-expressed developmentally down-regulated (gene 4)'] that interacts with the channel via its WW domains. Mutations in channel subunits that disrupt this interaction cause Liddle's syndrome, a severe inherited form of hypertension. In previous studies we showed that WW domains 2, 3 and 4 of human Nedd4 bound to the human ENaC (hENaC) subunits, whereas WW domain 1 did not. Here we extend this observation to determine the binding affinities of the human Nedd4 WW domains for hENaC C-terminal peptides. We show that WW domains 2, 3 and 4 bind with differing affinities to Na(+) channel subunit peptides. WW domain 3 has the highest affinity and we predict that WW domain 3 contributes most of the binding because a construct containing the three WW domains bound no better than WW domain 3 alone. Further, a single amino acid change (Arg(165)-->Thr) in WW domain 1 enables binding to the alpha subunit of the channel to occur, with an affinity comparable with that of WW domain 4. Differential binding propensities between the various WW domains and Na(+) channel subunit peptides are explained on the basis of quantitative structural modelling of the complexes and their isolated components.
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PMID:A single WW domain is the predominant mediator of the interaction between the human ubiquitin-protein ligase Nedd4 and the human epithelial sodium channel. 1180 77

The validation of full-length cDNA represents a crucial step in gene identification and subsequent functional analysis. In searching for candidate genes for bipolar disorder on chromosome 18q21, a novel gene homologous to NEDD4 (Neural precursor cells expressed developmentally down-regulated) was identified using exon trapping and cDNA cloning. This novel gene is termed NEDD4L (Human Gene Nomenclature Committee symbol). Typical NEDD4 orthologues that contain a C2 (Ca(2+)/lipid-binding) and a HECT (Homologous to the E6-AP Carboxyl Terminus) ubiquitin-protein ligase domain, and multiple WW domains have been shown to regulate the epithelial sodium channel (ENaC). In mice, Nedd4 has two distinct isoforms termed Nedd4-1 that belongs to the typical NEDD4 class, and Nedd4-2 that is homologous to Nedd4-1 but lacks the C2 domain. NEDD4L contains the WW and HECT domains seen in the NEDD4 gene family, but lacks the C2 domain in the N-terminus. BLAST database search showed that the deduced polypeptide of NEDD4L has 97 and 62% sequence identity to mouse Nedd4-2 and human NEDD4, respectively. Multiple forms of transcripts of NEDD4L have been isolated, which differ in transcription start and termination sites together with the presence or absence of an alternative spliced exon. Northern blot analysis showed a 3.4 kb mRNA species was specifically expressed in heart and skeletal muscle, while a 3.2 kb band and/or an additional 3.6 kb band is seen in other tissues tested. Striking homology of NEDD4L to mouse Nedd4-2 suggests it is the human homologue of mouse Nedd4-2. Its position in a region of linkage for autosomal dominant orthostatic hypotensive disorder and its potential role in regulating ENaC make NEDD4L a candidate gene for this disorder.
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PMID:NEDD4L on human chromosome 18q21 has multiple forms of transcripts and is a homologue of the mouse Nedd4-2 gene. 1184 Jan 94


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