{"gene":"NEDD4","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2007,"finding":"NEDD4-1 (NEDD4) was purified as the E3 ubiquitin ligase for PTEN; it catalyzes PTEN polyubiquitination in cells, leading to proteasomal degradation of PTEN and consequent activation of downstream PI3K/Akt signaling, potentiating cellular transformation.","method":"Biochemical purification of E3 ligase activity from cell lysates, in vitro ubiquitination assay, cell-based polyubiquitination assay, overexpression/knockdown with PTEN stability readout, xenograft tumor model","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus cell-based assays plus in vivo tumor model; widely cited foundational study with multiple orthogonal methods","pmids":["17218260"],"is_preprint":false},{"year":2008,"finding":"Subsequent study using two independently generated Nedd4-1 knockout mouse strains found no detectable interaction between Nedd4-1 and PTEN, and PTEN stability, ubiquitination, subcellular distribution, and downstream Akt activity were all unchanged in Nedd4-1-deficient cells, failing to support Nedd4-1 as the physiological E3 ligase for PTEN.","method":"Nedd4-1 knockout MEFs (two independent mouse strains), co-immunoprecipitation attempts, PTEN stability assay, PKB/Akt activity assay, nuclear PTEN function (Rad51 transactivation assay)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent KO mouse lines, multiple orthogonal functional readouts; directly contradicts PMID 17218260 regarding PTEN as physiological substrate","pmids":["18562292"],"is_preprint":false},{"year":2000,"finding":"NEDD4 binds the epithelial Na+ channel (ENaC) via its WW domains interacting with PY motifs (PPxY) in the C-termini of ENaC subunits, catalyzes ENaC ubiquitination via its HECT domain, and reduces ENaC surface expression; the C2 domain localizes NEDD4 to the apical plasma membrane in a Ca2+-dependent manner in polarized MDCK cells.","method":"Electrophysiology, co-immunoprecipitation, subcellular fractionation/localization, site-directed mutagenesis of PY motifs (Liddle's syndrome mutations), MDCK cell polarization experiments","journal":"Kidney international","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods, replicated across multiple studies and labs (PMID 10882424 corroborates), direct structure-function analysis","pmids":["10720933","10882424"],"is_preprint":false},{"year":2001,"finding":"SGK (serum- and glucocorticoid-regulated kinase) binds to NEDD4 (and NEDD4-2) via a PY motif in SGK; SGK phosphorylates NEDD4-2 (but not NEDD4), reducing NEDD4-2 binding to αENaC and thereby relieving NEDD4-2-mediated inhibition of epithelial Na+ absorption.","method":"Co-immunoprecipitation, in vitro kinase assay (phosphorylation of recombinant NEDD4-2), electrophysiology (Na+ current measurement), deletion/mutation analysis of PY motif","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus co-IP plus functional electrophysiology; single lab but multiple orthogonal methods","pmids":["11696533"],"is_preprint":false},{"year":2004,"finding":"GRK2 (G-protein-coupled receptor kinase 2) phosphorylates the C-terminus of the ENaC β-subunit and renders ENaC insensitive to inhibition by NEDD4-2, maintaining channels in an active state.","method":"Electrophysiology, in vitro phosphorylation assay, co-expression in Xenopus oocytes","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro phosphorylation assay plus electrophysiological functional readout; single lab","pmids":["15284439"],"is_preprint":false},{"year":2007,"finding":"GRK2 directly interacts with and phosphorylates NEDD4 (as well as NEDD4-2) at multiple sites, including threonine 466 in NEDD4 located in the WW3 domain region that binds ENaC.","method":"Co-immunoprecipitation, in vitro kinase assay with recombinant proteins, mass spectrometry of phosphorylation sites","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vitro kinase assay plus co-IP; single lab, single study","pmids":["17544362"],"is_preprint":false},{"year":2008,"finding":"NEDD4 acts as the E3 ubiquitin ligase for the β2-adrenergic receptor (β2AR); agonist stimulation triggers β-arrestin2-mediated recruitment of NEDD4 to activated β2AR, leading to NEDD4-catalyzed receptor ubiquitination and lysosomal degradation. β-arrestin2 is an essential adaptor, as its knockdown abolishes β2AR-NEDD4 interaction.","method":"Dominant-negative NEDD4 overexpression, siRNA knockdown (NEDD4, NEDD4-2, AIP4), co-immunoprecipitation of NEDD4–β-arrestin2–β2AR complex, ubiquitination assay, lysosomal trafficking assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, dominant-negative, siRNA knockdown with multiple functional readouts in same study; single lab","pmids":["18544533"],"is_preprint":false},{"year":2011,"finding":"NEDD4-1 directly binds and ubiquitylates activated FGFR1 via its WW3 domain interacting with a non-canonical non-PY motif on FGFR1, promoting FGFR1 endocytosis and attenuating downstream signaling (FRS2α, Akt, Erk1/2, PLCγ). FGFR1 lacking the Nedd4-1 recognition motif (FGFR1-Δ6) shows impaired endocytosis and sustained signaling; in neural stem cells promotes neuronal differentiation; in zebrafish disrupts anterior neuronal patterning.","method":"Direct binding assay, co-immunoprecipitation, in vitro/in vivo ubiquitination assay, receptor endocytosis assay, Nedd4-1 knockdown, FGFR1-Δ6 mutant in neural stem cells, zebrafish embryo expression","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro direct binding, in vitro ubiquitination, mutagenesis, cell-based endocytosis, and in vivo zebrafish validation in a single study","pmids":["21765395"],"is_preprint":false},{"year":2011,"finding":"NEDD4 is the E3 ubiquitin ligase responsible for AMPA receptor (AMPAR/GluA1) ubiquitination in neurons; NEDD4 co-localizes and associates with AMPARs in synaptoneurosomes, ubiquitinates GluA1 at K868, reduces AMPAR surface expression, and suppresses excitatory synaptic transmission. Knockdown of NEDD4 abolishes AMPAR ubiquitination.","method":"Co-immunoprecipitation, synaptosome fractionation, ubiquitination assay with lysine-to-arginine mutants (K868R), siRNA knockdown, electrophysiology (mEPSC recording)","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, site-directed mutagenesis, siRNA KD, electrophysiological functional readout; single lab with multiple orthogonal methods","pmids":["21338354"],"is_preprint":false},{"year":2014,"finding":"NEDD4-1 is an E3 ubiquitin ligase for Mdm2; it interacts with Mdm2 via the Mdm2 RING domain and catalyzes K63-linked polyubiquitination of Mdm2 (distinct from K48-linked chains that Mdm2/MdmX forms), thereby stabilizing Mdm2. Nedd4-1 KO MEFs show increased p53 levels and activity, more robust DNA damage response, and increased G1 arrest.","method":"In vitro E3 ligase assay with recombinant proteins, Nedd4-1 KO MEF lysates, co-immunoprecipitation, ubiquitin linkage-specific analysis, Mdm2 half-life measurement (CHX chase), p53 reporter assays, cell cycle analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstituted ubiquitination assay with recombinant proteins, KO cells, multiple functional readouts; single lab with multiple orthogonal approaches","pmids":["24413081"],"is_preprint":false},{"year":2014,"finding":"NEDD4-1 acts as an E3 ubiquitin ligase for Ras; Ras signaling stimulates NEDD4-1 expression, but oncogenic Ras mutations or EGF signaling prevent NEDD4-1-mediated Ras ubiquitination, leading to NEDD4-1 accumulation and subsequent PTEN degradation. This defines a Ras–NEDD4-1–PTEN regulatory loop.","method":"In vivo ubiquitination assay, co-immunoprecipitation, expression analysis in human cancer samples and cancer cell lines, genetic rescue experiments","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based ubiquitination assays, co-IP, human cancer samples; single lab","pmids":["24746824"],"is_preprint":false},{"year":2015,"finding":"NEDD4 ubiquitylates and destabilizes WW45 (SAV1) and LATS kinase (core Hippo pathway components), thereby suppressing Hippo signaling. MST1 protects WW45 but not LATS2 from NEDD4-mediated degradation. NEDD4 inactivation at high cell density enables contact inhibition via elevated Hippo activity. In Drosophila, NEDD4 promotes intestinal stem cell renewal by suppressing Hippo signaling.","method":"In vitro ubiquitination assay, co-immunoprecipitation, knockdown/overexpression with protein stability readouts, Drosophila genetic model (ISC proliferation)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro ubiquitination, reciprocal co-IP, cell-based and in vivo (Drosophila) validation; multiple orthogonal methods across two organisms","pmids":["25692647"],"is_preprint":false},{"year":2015,"finding":"NEDD4 monoubiquitinates IRS-2, promoting its association with ubiquitin-binding protein Epsin1 and recruitment of IRS-2 to the membrane, thereby enhancing IGF-I receptor-induced IRS-2 tyrosine phosphorylation, downstream signaling, and cell proliferation. In zebrafish, NEDD4 overexpression accelerates embryonic growth through IRS-2.","method":"Co-immunoprecipitation, in vitro/in vivo ubiquitination assay, siRNA knockdown, membrane fractionation, IGF-I signaling assays, zebrafish embryo experiments","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple biochemical methods plus in vivo zebrafish validation; single lab with multiple orthogonal methods","pmids":["25879670"],"is_preprint":false},{"year":2015,"finding":"Elevated NEDD4 is the E3 ubiquitin ligase responsible for HSF1 degradation induced by aggregated α-synuclein/proteotoxic stress; SIRT1-mediated deacetylation of HSF1 at K80 attenuates NEDD4-mediated HSF1 ubiquitination and stabilizes HSF1.","method":"Ubiquitination assay in neuroblastoma cells, siRNA knockdown of NEDD4, co-immunoprecipitation, pharmacological SIRT1 activation, site-directed mutagenesis (K80 acetylation), mouse model and human tissue validation","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based ubiquitination assay, KD, mutagenesis, in vivo confirmation; single lab","pmids":["26503960"],"is_preprint":false},{"year":2008,"finding":"NEDD4 acts as the E3 ubiquitin ligase for the ErbB4 JM-a/CYT-1 intracellular domain (ICD); NEDD4 binds via its WW domains to three PY motifs in the CYT-1 C-terminus, catalyzes CYT-1 ICD ubiquitination, and promotes its proteasomal and lysosomal degradation. Mutation of PY motifs impairs NEDD4 binding, increases CYT-1 ICD stability, and (for one PY mutant) enhances nuclear translocation.","method":"Co-immunoprecipitation, in vitro direct binding assay, in vitro ubiquitination assay, PY motif mutagenesis, proteasome/lysosome inhibitor experiments","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro direct binding, in vitro ubiquitination reconstitution, mutagenesis, cell-based stability assays; single lab with multiple orthogonal methods","pmids":["19193720"],"is_preprint":false},{"year":2010,"finding":"NEDD4-1 and β-arrestin-1 cooperate to ubiquitylate and internalize NHE1 (Na+/H+ exchanger 1) at the plasma membrane. β-arrestin-1 bridges NEDD4-1 to the NHE1 C-terminus; truncation of NHE1 C-terminus to aa550 abolishes β-arrestin-1 binding and NHE1 ubiquitylation. Knockout of either Nedd4-1 or β-arrestin-1 in mouse fibroblasts increases NHE1 surface levels and transport activity.","method":"Co-immunoprecipitation, siRNA knockdown, Nedd4-1 and β-arrestin-1 knockout mouse fibroblasts, ubiquitylation assay, surface biotinylation, ion transport assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse fibroblasts plus co-IP plus functional transport assay plus siRNA; multiple orthogonal methods","pmids":["20855896"],"is_preprint":false},{"year":2008,"finding":"NDFIP1 (Nedd4 family-interacting protein 1) recruits NEDD4 (and Nedd4-2 and Itch) into exosomes secreted from cells and primary neurons; without NDFIP1, these Nedd4 proteins are absent from exosomes. NDFIP1 increases overall exosome secretion.","method":"Exosome isolation, Western blot of exosomal fractions, co-transfection experiments, primary neuron exosome analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — biochemical fractionation with functional implication; single lab, replicated in both transfected cells and primary neurons","pmids":["18819914"],"is_preprint":false},{"year":2017,"finding":"NEDD4-1 undergoes K29-linked auto-ubiquitination at K1279, which serves as a scaffold to recruit the deubiquitinase USP13. The resulting NEDD4-1–USP13 complex removes K48-linked polyubiquitin chains from VPS34 at K419, stabilizing VPS34 and promoting autophagy flux. Knockout of either NEDD4-1 or USP13 increases K48-linked ubiquitination and degradation of VPS34.","method":"Co-immunoprecipitation, ubiquitination assay (linkage-specific), USP13/NEDD4-1 knockout cells, autophagosome formation assay, site-directed mutagenesis (K1279, K419)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, linkage-specific ubiquitination, KO cells, site-directed mutagenesis; single lab with multiple orthogonal methods","pmids":["32101753"],"is_preprint":false},{"year":2017,"finding":"NEDD4 ubiquitinates histone H3 on lysine 23/36/37 residues in a glucose-dependent manner; this H3 ubiquitination specifically recruits histone acetyltransferase GCN5 for subsequent H3 K9 acetylation at transcription start sites and enhancer regions, activating transcription of IL1α, IL1β, and GCLM, and promoting tumour sphere formation.","method":"ChIP-seq, microarray, in vitro ubiquitination assay with recombinant H3, site-directed mutagenesis of H3 lysines, GCN5 pulldown, tumour sphere formation assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of H3 ubiquitination, ChIP-seq genome-wide validation, mutagenesis, functional assays; single lab with multiple orthogonal methods","pmids":["28300060"],"is_preprint":false},{"year":2017,"finding":"NEDD4 ubiquitylation activity is restrained by an evolutionarily conserved allosteric auto-inhibition mechanism: ubiquitylation of a conserved lysine on the HECT domain α1-helix promotes NEDD4 oligomerization (trimerization) that renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation and causes unrestrained FGFR1 ubiquitylation and constitutive IKS channel downregulation.","method":"Biochemical and biophysical analysis (oligomerization assays), site-directed mutagenesis, in vitro ubiquitination assays, electrophysiology (IKS channel), TIRF microscopy, genetic experiments in yeast","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, mutagenesis, structural biophysics, electrophysiology, and genetic data across multiple substrates and organisms","pmids":["28069708"],"is_preprint":false},{"year":2019,"finding":"NEDD4 catalytic activity is autoinhibited by WW domain linker regions that engage the HECT domain; allosteric activation by NDFIP1 (and engineered ubiquitin variants) relieves this autoinhibition. The WW domain linker also influences ubiquitin chain linkage (K48 vs K63) and degree of polyubiquitination on substrates.","method":"In vitro ubiquitination assays with recombinant NEDD4-1, linker deletion/phosphomimic mutants, NDFIP1 binding assays, substrate ubiquitination assays (PTEN, p62, WBP2)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with multiple mutants and substrates; single lab with rigorous biochemical dissection","pmids":["31578285"],"is_preprint":false},{"year":1999,"finding":"NEDD4 interacts constitutively with the adapter protein Grb10 (which couples to insulin and IGF-I receptors) via the C2 domain of NEDD4 and a region including the SH2/BPS domain of Grb10, both in yeast two-hybrid and by endogenous co-immunoprecipitation in mouse embryo fibroblasts; the interaction is Ca2+-independent.","method":"Yeast two-hybrid, co-immunoprecipitation of endogenous proteins in MEFs, deletion mutagenesis of Grb10","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid confirmed by endogenous co-IP; single lab, no catalytic functional assay performed","pmids":["10446181"],"is_preprint":false},{"year":2005,"finding":"NEDD4 and NEDD4-2 share the same preferred ubiquitin-conjugating enzyme (E2) specificity: UbcH5b is the most efficient E2 for both ligases in vitro, with Ube2e3 used less efficiently.","method":"In vitro ubiquitination assay with panel of E2 enzymes and recombinant NEDD4/NEDD4-2","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution; single lab, single study","pmids":["16337426"],"is_preprint":false},{"year":2019,"finding":"NEDD4 and NEDD4L (NEDD4-2) ubiquitinate LGR5 receptor (and DVL2), targeting them for proteasomal and lysosomal degradation; loss of Nedd4/Nedd4l enhances ISC proliferation and sensitivity to R-spondin and accelerates tumour development in Apcmin mice, demonstrating that NEDD4/NEDD4L negatively regulate Wnt/β-catenin signaling through LGR5 degradation.","method":"In vitro ubiquitination assay, co-immunoprecipitation, conditional KO mouse models, organoid culture, Apcmin tumour model, protein stability assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro ubiquitination, KO mouse models, organoid/in vivo tumor phenotype; multiple orthogonal methods","pmids":["31867777"],"is_preprint":false},{"year":2020,"finding":"NEDD4 ubiquitinates VDAC2 and VDAC3 following erastin treatment, promoting their degradation; this suppresses erastin-induced ferroptosis. FOXM1-induced NEDD4 expression creates a negative feedback loop (FOXM1–NEDD4–VDAC2/3) that reduces ferroptosis sensitivity.","method":"siRNA knockdown of NEDD4, ubiquitination assay, co-immunoprecipitation, VDAC2/3 protein stability assay, ferroptosis assay (cell viability, lipid peroxidation)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, KD with functional ferroptosis readout; single lab with multiple orthogonal methods","pmids":["31974380"],"is_preprint":false},{"year":2021,"finding":"NEDD4, activated downstream of FGFR3, interacts with PD-L1 and catalyzes Lys48-linked polyubiquitination of PD-L1, targeting it for proteasomal degradation. Phosphorylation of NEDD4 by FGFR3 activates its E3 activity toward PD-L1.","method":"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), NEDD4 KO bladder cancer cell lines, FGFR3 inhibition experiments, tissue microarray, in vivo tumor mouse models","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, K48-specific ubiquitination assay, KO cells, in vivo tumor models; multiple orthogonal methods","pmids":["34753771"],"is_preprint":false},{"year":2020,"finding":"NEDD4 controls spermatogonial progenitor cell (SPC) homeostasis by targeting the RNA-binding protein NANOS2 for ubiquitination/destabilization, promoting spermatogonial differentiation. NEDD4 is also required for stress granule (SG) clearance via the endosomal-lysosomal pathway during recovery from stress.","method":"Nedd4 conditional KO mice (spermatogonia), co-immunoprecipitation, ubiquitination assay, immunofluorescence (SG dynamics), lysosomal pathway inhibitor experiments","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO mice with cellular phenotype, co-IP, ubiquitination assay, multiple functional readouts","pmids":["28585553"],"is_preprint":false},{"year":2013,"finding":"NEDD4-1 promotes glioma cell migration and invasion by physically interacting with CNrasGEF and promoting its polyubiquitination and proteasomal degradation, thereby removing a migration suppressor.","method":"Co-immunoprecipitation, in vivo ubiquitination assay, siRNA knockdown/overexpression with migration and invasion assays (Transwell, wound healing), glioma tissue expression analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus ubiquitination assay plus functional migration assays; single lab","pmids":["24340059"],"is_preprint":false},{"year":2017,"finding":"NEDD4 ubiquitinates and targets LATS1 (a core Hippo pathway kinase) for degradation in hepatocellular carcinoma cells, promoting tumor progression.","method":"siRNA knockdown/overexpression, co-immunoprecipitation, Western blot for LATS1 protein levels, cell proliferation/invasion assays","journal":"Cell cycle","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-IP and indirect protein level measurements; single lab, no in vitro ubiquitination assay performed","pmids":["29480061"],"is_preprint":false},{"year":2017,"finding":"NDFIP1 and NDFIP2 are essential adaptor proteins that bridge NEDD4 (and NEDD4L) to aquaporin-2 (AQP2), enabling NEDD4/NEDD4L-mediated AQP2 ubiquitination and lysosomal degradation; PY motifs in NDFIP1/2 are required for this function.","method":"siRNA knockdown in mpkCCD cells, Membrane Yeast Two-Hybrid (NDFIP2-AQP2 interaction), HEK293T co-transfection ubiquitination assay, NDFIP1/2 PY-motif mutants, AQP2 abundance assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Y2H, co-expression ubiquitination, KD in relevant epithelial cells, mutagenesis; single lab with multiple methods","pmids":["28931009"],"is_preprint":false},{"year":2019,"finding":"NEDD4 E3 ligase and β-arrestins regulate mGlu7 receptor ubiquitination and trafficking: upon agonist stimulation, β-arrestins recruit NEDD4 to mGlu7, facilitating NEDD4-mediated ubiquitination that promotes mGlu7 endocytosis and degradation by both proteasomal and lysosomal pathways; NEDD4 is also required for mGlu7-dependent MAPK signaling.","method":"Co-immunoprecipitation in HEK293 cells and rat neurons, ubiquitination assay, surface expression assay, endocytosis assay, MAPK signaling readout, siRNA knockdown","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP in neurons, ubiquitination assay, functional endocytosis and signaling readouts; single lab with multiple orthogonal methods","pmids":["31373553"],"is_preprint":false},{"year":2020,"finding":"NEDD4 promotes oligodendrocyte differentiation and developmental myelination by stabilizing VHL via K63-linked ubiquitination; the Daam2-VHL-NEDD4 axis governs OPC differentiation during development and remyelination after white matter injury.","method":"Conditional KO mouse models (Nedd4 KO), proteomic analysis of Daam2-VHL complex, K63-linked ubiquitination assay, myelination/remyelination assays, human MS lesion analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO mice, proteomics, K63-specific ubiquitination assay, in vivo demyelination models, and human tissue validation","pmids":["32792353"],"is_preprint":false},{"year":2016,"finding":"LRAD3, a member of the LDL receptor family, activates NEDD4 E3 ligase activity via its intracellular PPxY motifs binding to the WW domains of NEDD4; binding to the terminal PPxY motif of LRAD3 is required to promote NEDD4 auto-ubiquitination and subsequent proteasomal degradation.","method":"Co-immunoprecipitation, in vitro ubiquitination (auto-ubiquitination) assay, PPxY mutagenesis, proteasome inhibitor experiments","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro auto-ubiquitination assay, co-IP, site-directed mutagenesis; single lab","pmids":["26854353"],"is_preprint":false},{"year":2024,"finding":"Lactate inhibits NEDD4-mediated ubiquitination of Caspase-11 by inducing K33 lactylation of NEDD4, which impairs the interaction between NEDD4 and Caspase-11, thereby allowing Caspase-11 accumulation and promoting non-canonical pyroptosis in macrophages during acetaminophen-induced liver injury.","method":"Co-immunoprecipitation, ubiquitination assay, lactylation detection, site-directed mutagenesis (NEDD4 K33), macrophage pyroptosis assay, mouse APAP liver injury model","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, site-directed mutagenesis of lactylation site, in vivo mouse model; single lab","pmids":["38385085"],"is_preprint":false},{"year":2022,"finding":"NEDD4 ubiquitinates and promotes proteasomal degradation of NLRP3 by interacting with adaptor protein SF3A2, which bridges NEDD4 to NLRP3; this inhibits NLRP3 inflammasome activation and macrophage pyroptosis.","method":"Co-immunoprecipitation (NEDD4-SF3A2-NLRP3), Western blot for NLRP3 levels after NEDD4 overexpression/knockdown, pyroptosis assay, mouse MI/R model","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP, protein level assays, functional pyroptosis readout in cells and mice; single lab","pmids":["36732831"],"is_preprint":false},{"year":2024,"finding":"NEDD4-1 interacts with the PPTY motif of VDAC1 via its WW domain and catalyzes K48-linked ubiquitination of VDAC1, promoting its proteasomal degradation; hepatocyte-specific Nedd4-1 KO increases VDAC1 accumulation and oligomerization, worsening APAP-induced mitochondrial damage and liver injury.","method":"Co-immunoprecipitation, in vivo/in vitro ubiquitination assay (K48-linkage specific), hepatocyte-specific KO and overexpression mouse models, VDAC1 knockdown rescue experiments","journal":"Acta pharmaceutica Sinica. B","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, K48-specific ubiquitination assay, hepatocyte KO mouse, overexpression, and genetic rescue; multiple orthogonal methods","pmids":["37139424"],"is_preprint":false},{"year":2021,"finding":"NEDD4 is the E3 ubiquitin ligase for E-cadherin; N4BP3 acts as an adaptor that promotes NEDD4-mediated K48/K63-linked polyubiquitination and degradation of E-cadherin, driving breast cancer metastasis via EMT.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, in vivo metastasis assay, N4BP3 depletion experiments","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, in vivo metastasis model; single lab","pmids":["36162713"],"is_preprint":false},{"year":2022,"finding":"NEDD4 promotes TGFBR1 signaling by binding directly to TGF-β type I receptor (TGFBR1) and forming K27-linked ubiquitin at lysine 391, thereby enhancing TGF-β signal transduction and tumor cell migration in hepatocellular carcinoma.","method":"Co-immunoprecipitation, in vivo ubiquitination assay (K27-specific), NEDD4 knockdown with HCC migration/invasion assays, PDX model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, linkage-specific ubiquitination, in vivo PDX model; single lab","pmids":["35501461"],"is_preprint":false},{"year":2024,"finding":"NEDD4 interacts with and ubiquitinates DMT1 (divalent metal transporter 1) at lysine residues 6, 69, and 277, facilitating DMT1 degradation and reducing iron accumulation/ferroptosis after intracerebral hemorrhage; NEDD4 overexpression alleviated ferroptosis and improved neurological outcomes.","method":"Co-immunoprecipitation, ubiquitination assay (site-directed mutagenesis of K6/K69/K277), lentiviral Nedd4 overexpression, ferroptosis markers (GPX4, MDA, iron content), mouse ICH model","journal":"CNS neuroscience & therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, site-directed mutagenesis of ubiquitination sites, in vivo mouse model; single lab","pmids":["38634270"],"is_preprint":false},{"year":2024,"finding":"NEDD4 catalyzes K48-linked ubiquitination of TAK1 at sites in TAK1, promoting its proteasomal degradation; THBS1 protects TAK1 from NEDD4-mediated ubiquitination by interacting with TAK1, and THBS1 silencing upregulates NEDD4 expression, leading to increased TAK1 degradation and necroptosis in trophoblasts.","method":"Co-immunoprecipitation, K48-specific ubiquitination assay, NEDD4 knockdown/overexpression, THBS1 silencing, mouse PE model","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, K48-specific ubiquitination assay, in vivo mouse model; single lab","pmids":["38569496"],"is_preprint":false},{"year":2023,"finding":"NEDD4 binds to and mediates ubiquitination and proteasomal degradation of CSF1R in macrophages; IFN-I signaling increases NEDD4-mediated CSF1R ubiquitination, and USP18 impairs this by interrupting NEDD4 binding to CSF1R.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, USP18 KO macrophage experiments, CSF1R protein stability assay","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, genetic KO; single lab with multiple methods","pmids":["38100351"],"is_preprint":false},{"year":2021,"finding":"NEDD4 triggers ubiquitination and proteasomal degradation of the transcription factor FOXA1; FOXA1 transcriptionally activates miR-340-5p which suppresses ATF1, creating a regulatory axis downstream of NEDD4 in colon cancer progression.","method":"Co-immunoprecipitation, ubiquitination assay, chromatin immunoprecipitation, luciferase reporter assay, in vivo xenograft model","journal":"RNA biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, ChIP, luciferase, in vivo model; single lab","pmids":["33530829"],"is_preprint":false},{"year":2020,"finding":"YOD1 (deubiquitinase) binds to and deubiquitinates NEDD4; YOD1 removes K63-linked polyubiquitin chains from NEDD4 and suppresses NEDD4-induced cell proliferation.","method":"Co-immunoprecipitation, deubiquitination assay, K63-specific ubiquitin chain analysis, cell proliferation assay with YOD1/NEDD4 overexpression","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP, deubiquitination assay, functional proliferation readout; single lab","pmids":["31916733"],"is_preprint":false},{"year":2023,"finding":"NEDD4-1 ubiquitinates SR-BI at K500 and K508, promoting its degradation; hepatocyte lipocalin-2 (Lcn2) blocks this Nedd4-1-mediated SR-BI ubiquitination, improving HDL metabolism and reducing atherosclerosis in mice.","method":"Co-immunoprecipitation, ubiquitination assay (site-directed mutagenesis K500/K508), hepatocyte-specific Lcn2 KO and overexpression, Nedd4-1 KO mice, SR-BI K500A/K508A knock-in mice","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, site-directed mutagenesis of ubiquitination sites, multiple conditional KO mouse models including knock-in; strong in vivo validation","pmids":["37863040"],"is_preprint":false},{"year":2024,"finding":"GSK-3β phosphorylates PGC-1α at Thr295, creating a recognition motif for NEDD4-1, which then catalyzes ubiquitin-dependent proteasomal degradation of PGC-1α; mutation of Thr295 negates shikonin's suppressive effects on mitochondrial biogenesis and TNBC metastasis.","method":"Co-immunoprecipitation, ubiquitination assay, phosphorylation site mutagenesis (Thr295), LC-MS/MS identification of phosphorylation, in vivo TNBC metastasis model","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation-dependent co-IP, ubiquitination assay, mutagenesis, in vivo model; single lab","pmids":["38937832"],"is_preprint":false},{"year":2017,"finding":"Adaptor protein Numb binds NEDD4-1 and regulates its E3 ligase activity and its association with PTEN, thereby controlling PTEN ubiquitination; Numb regulates cell proliferation and glucose metabolism in a PTEN-dependent manner.","method":"Co-immunoprecipitation (Numb-NEDD4-1 interaction), ubiquitination assay for PTEN, Numb knockdown with PTEN stability readout, cell proliferation and glucose metabolism assays","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP, ubiquitination assay, knockdown with functional readout; single lab","pmids":["28437168"],"is_preprint":false},{"year":2018,"finding":"NEDD4 TV3 (a specific transcript variant) activates the NF-κB pathway through its association with the adaptor protein RIP, contributing to inflammation in keloid formation.","method":"Forced expression and selective knockdown of NEDD4 TV3, NF-κB reporter assay, co-immunoprecipitation with RIP, in vivo keloid mouse model","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP of NEDD4 TV3 with RIP, functional NF-κB reporter, in vivo mouse model; single lab","pmids":["30273597"],"is_preprint":false}],"current_model":"NEDD4 (NEDD4-1) is a HECT-domain E3 ubiquitin ligase that recognizes substrates primarily via WW domain–PY motif (PPxY) interactions; it ubiquitinates a broad array of membrane proteins (ENaC, FGFR1, AMPAR/GluA1, β2AR, mGlu7, NHE1, VDAC1/2/3, LGR5, PD-L1, SR-BI, DMT1), nuclear substrates (histone H3, HSF1, FOXA1), and signaling proteins (Mdm2, PTEN, Ras, WW45/LATS, VPS34, Caspase-11, NLRP3, TAK1, CSF1R, TGFBR1), using K48-, K63-, K27-, or K29-linked ubiquitin chains to direct substrates to proteasomal or lysosomal degradation or to scaffold non-degradative signaling; its own HECT domain is subject to intramolecular autoinhibition by WW domain linkers, and allosteric activation is achieved through NDFIP1 binding, PPxY-containing adaptors (LRAD3), or phosphorylation (by GRK2 or FGFR3), while K29-linked auto-ubiquitination recruits the deubiquitinase USP13 to form a complex that stabilizes VPS34 and promotes autophagy, revealing both degradative and non-degradative scaffolding functions of the ligase."},"narrative":{"mechanistic_narrative":"NEDD4 (NEDD4-1) is a HECT-domain E3 ubiquitin ligase that controls the abundance and trafficking of membrane receptors, ion channels, and signaling proteins, governing diverse processes from epithelial Na+ transport and synaptic transmission to growth-factor signaling, tumor suppression, and cell death [PMID:10720933, PMID:10882424, PMID:21338354, PMID:25692647]. Substrate selection is driven principally by recognition of PY (PPxY) motifs through its WW domains, as established for ENaC subunits, ErbB4 CYT-1, and VDAC1 [PMID:10720933, PMID:10882424, PMID:19193720, PMID:37139424], while its C2 domain mediates Ca2+-dependent membrane localization and constitutive adaptor binding (Grb10) [PMID:10720933, PMID:10882424, PMID:10446181]. The ligase assembles ubiquitin chains of multiple linkages to dictate substrate fate: K48-linked chains target PD-L1, VDAC1, TAK1, and Mdm2 for proteasomal degradation [PMID:34753771, PMID:37139424, PMID:38569496], whereas K63- and K27-linked chains can be non-degradative, stabilizing Mdm2 and VHL or potentiating TGFBR1 signaling [PMID:24413081, PMID:32792353, PMID:35501461]. Beyond catalyzing degradation, NEDD4 acts as a non-degradative scaffold: K29-linked auto-ubiquitination at K1279 recruits the deubiquitinase USP13 to protect VPS34 from K48 chains and promote autophagy [PMID:32101753]. NEDD4 ubiquitinates numerous receptors for endocytic downregulation, frequently using arrestin or NDFIP adaptors to bridge substrate recognition—β-arrestin recruits NEDD4 to the agonist-activated β2-adrenergic and mGlu7 receptors, and to NHE1, while NDFIP1/2 bridge it to aquaporin-2 [PMID:18544533, PMID:31373553, PMID:20855896, PMID:28931009]. Its activity is tightly autoregulated: WW-domain linker regions and an oligomerization-promoting HECT α1 lysine impose intramolecular autoinhibition that is relieved by NDFIP1, PPxY adaptors (LRAD3), or substrate-directed phosphorylation by GRK2 and FGFR3 [PMID:28069708, PMID:31578285, PMID:17544362, PMID:26854353, PMID:34753771]. NEDD4 also ubiquitinates nuclear substrates including histone H3, where glucose-dependent H3 ubiquitination recruits GCN5 to activate transcription [PMID:28300060]. NEDD4 is reported as an E3 ligase for PTEN, but two independent Nedd4-1 knockout mouse lines found no effect on PTEN interaction, stability, or downstream Akt activity, leaving the physiological relevance of this substrate unresolved [PMID:17218260, PMID:18562292].","teleology":[{"year":2000,"claim":"Established the core substrate-recognition logic of NEDD4 by showing WW-domain engagement of PY motifs drives ubiquitination and surface downregulation of a physiological ion channel.","evidence":"Co-IP, electrophysiology, PY-motif mutagenesis (Liddle's mutations), and C2-domain localization in polarized MDCK cells for ENaC","pmids":["10720933","10882424"],"confidence":"High","gaps":["Did not resolve ubiquitin chain linkage type on ENaC","Regulation of NEDD4 activity itself not addressed"]},{"year":2001,"claim":"Showed that kinase phosphorylation can regulate NEDD4-family channel control, defining a signaling input to the ENaC pathway.","evidence":"Co-IP, in vitro kinase assay, and Na+ current measurement of SGK acting on NEDD4/NEDD4-2","pmids":["11696533"],"confidence":"High","gaps":["SGK phosphorylates NEDD4-2 but not NEDD4, leaving NEDD4 regulation here open","Structural basis of phospho-dependent affinity change not defined"]},{"year":2007,"claim":"Proposed PTEN as a NEDD4 substrate linking the ligase to PI3K/Akt and transformation, the first oncogenic role assigned.","evidence":"Biochemical E3 purification, in vitro and cell ubiquitination, and xenograft model","pmids":["17218260"],"confidence":"High","gaps":["Physiological relevance contested by subsequent KO data","In vivo requirement not tested with genetic loss-of-function in this study"]},{"year":2008,"claim":"Directly challenged the PTEN-substrate model using genetic loss-of-function, establishing that NEDD4 substrate assignments require in vivo validation.","evidence":"Two independent Nedd4-1 KO mouse strains, co-IP attempts, and PTEN stability/Akt readouts in MEFs","pmids":["18562292"],"confidence":"High","gaps":["Does not exclude context- or tissue-specific PTEN regulation","Cannot rule out redundancy with NEDD4L"]},{"year":2008,"claim":"Defined arrestin-bridged recruitment as a mechanism for NEDD4 targeting of GPCRs, generalizing beyond direct PY recognition.","evidence":"β-arrestin2 knockdown, dominant-negative NEDD4, complex co-IP, and lysosomal trafficking assay for β2AR","pmids":["18544533"],"confidence":"High","gaps":["Ubiquitin chain linkage on β2AR not determined","Structural basis of arrestin-NEDD4 bridging not resolved"]},{"year":2011,"claim":"Showed NEDD4 attenuates receptor tyrosine kinase signaling via endocytic downregulation, and that recognition can occur through non-canonical non-PY motifs.","evidence":"Direct binding, in vitro/in vivo ubiquitination, endocytosis assay, FGFR1-Δ6 mutant, and zebrafish neuronal patterning for FGFR1","pmids":["21765395"],"confidence":"High","gaps":["Generality of non-PY recognition across substrates unclear","Chain linkage directing FGFR1 endocytosis not specified"]},{"year":2011,"claim":"Extended NEDD4 control to neuronal AMPA receptors, linking the ligase to synaptic strength.","evidence":"Synaptosome co-IP, GluA1 K868R mutagenesis, siRNA knockdown, and mEPSC recording","pmids":["21338354"],"confidence":"High","gaps":["Upstream signal triggering NEDD4-AMPAR engagement not defined","In vivo behavioral consequence not tested"]},{"year":2014,"claim":"Revealed linkage-specific non-degradative ubiquitination by showing NEDD4 stabilizes Mdm2 via K63 chains, distinct from degradative K48 control.","evidence":"Reconstituted in vitro ligase assay, Nedd4-1 KO MEFs, linkage analysis, CHX chase, and p53/cell-cycle readouts","pmids":["24413081"],"confidence":"High","gaps":["How K63 stabilization opposes Mdm2 autoubiquitination mechanistically unclear","Physiological p53 contexts not fully mapped"]},{"year":2015,"claim":"Placed NEDD4 as a negative regulator of the Hippo tumor-suppressor pathway and a positive regulator of growth signaling, defining opposing pro-proliferative roles.","evidence":"In vitro ubiquitination, co-IP, protein stability, Drosophila ISC models for WW45/LATS, and IRS-2 monoubiquitination/Epsin1 recruitment with zebrafish growth assays","pmids":["25692647","25879670"],"confidence":"High","gaps":["Switch between degradative and signaling-promoting outputs not unified mechanistically","Cross-talk with autoinhibition state not addressed"]},{"year":2017,"claim":"Uncovered NEDD4 as a non-degradative scaffold in autophagy, where self-K29-ubiquitination recruits a deubiquitinase to protect a third protein.","evidence":"Linkage-specific ubiquitination, NEDD4-1/USP13 KO cells, K1279/K419 mutagenesis, and autophagosome assays for the VPS34 complex","pmids":["32101753"],"confidence":"High","gaps":["Trigger inducing K29 auto-ubiquitination unknown","Structural basis of USP13 recruitment by K29 chains not defined"]},{"year":2017,"claim":"Identified a nuclear/chromatin function for NEDD4, coupling glucose-dependent histone H3 ubiquitination to GCN5 recruitment and transcriptional activation.","evidence":"In vitro H3 ubiquitination, ChIP-seq, H3 lysine mutagenesis, GCN5 pulldown, and tumorsphere assays","pmids":["28300060"],"confidence":"High","gaps":["How a cytoplasmic membrane ligase accesses chromatin not resolved","Metabolic signal linking glucose to NEDD4 activity unclear"]},{"year":2019,"claim":"Defined the molecular basis of NEDD4 autoinhibition and its allosteric relief, explaining how the ligase is held inactive and switched on.","evidence":"Oligomerization biophysics and HECT α1 lysine mutagenesis (28069708); WW-linker deletion/phosphomimic mutants and NDFIP1 activation across PTEN/p62/WBP2 (31578285)","pmids":["28069708","31578285"],"confidence":"High","gaps":["In vivo prevalence of the oligomeric inactive state unknown","How phosphorylation by GRK2/FGFR3 intersects with linker autoinhibition not fully mapped"]},{"year":2020,"claim":"Established physiological roles of NEDD4 in development and cell-fate via genetic models, including spermatogonial homeostasis and myelination.","evidence":"Conditional Nedd4 KO mice with NANOS2 ubiquitination and stress-granule clearance (28585553); Daam2-VHL-NEDD4 K63 axis in oligodendrocyte differentiation with human MS lesion analysis (32792353)","pmids":["28585553","32792353"],"confidence":"High","gaps":["Direct WW-PY recognition of these substrates not always demonstrated","Linkage-to-outcome rules across these substrates not unified"]},{"year":2021,"claim":"Linked NEDD4 to immune checkpoint and cell-death control, showing FGFR3-activated NEDD4 degrades PD-L1 and connecting it to ferroptosis regulation.","evidence":"K48-specific ubiquitination, NEDD4 KO cancer cells, in vivo tumor models for PD-L1 (34753771); VDAC2/3 ubiquitination in the FOXM1-NEDD4 ferroptosis loop (31974380)","pmids":["34753771","31974380"],"confidence":"High","gaps":["FGFR3-dependent phospho-activation mechanism on NEDD4 not structurally defined","Selectivity among VDAC paralogs not fully explained"]},{"year":2024,"claim":"Demonstrated post-translational modification of NEDD4 itself (lactylation) as a substrate-selective regulatory switch, expanding the modes controlling its activity.","evidence":"Co-IP, ubiquitination assay, NEDD4 K33 lactylation detection/mutagenesis, and APAP liver injury model for Caspase-11","pmids":["38385085"],"confidence":"Medium","gaps":["Single lab, single substrate context for lactylation","Whether lactylation affects other substrates not tested"]},{"year":null,"claim":"How NEDD4 integrates its autoinhibition state, adaptor repertoire, and diverse post-translational modifications to select among its many substrates and chain linkages in a given cellular context remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model predicting substrate choice or chain-linkage output","Tissue-specific adaptor and modification landscapes uncharacterized","Physiological vs. overexpression-based substrate assignments not consistently distinguished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,9,17,25,35]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[2,14,18,25,35]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,9,25,35]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[19,20]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,6,15]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[18]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[16,26]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,9,25,35]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,7,11,30,37]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[17]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[2,15,29,38]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[25,33,34,40]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[18]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[24,33,34,38,39]}],"complexes":["NEDD4-USP13 complex"],"partners":["NDFIP1","ARRB2","USP13","GRK2","SGK","GRB10","LRAD3","YOD1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P46934","full_name":"E3 ubiquitin-protein ligase NEDD4","aliases":["Cell proliferation-inducing gene 53 protein","HECT-type E3 ubiquitin transferase NEDD4","Neural precursor cell expressed developmentally down-regulated protein 4","NEDD-4"],"length_aa":1319,"mass_kda":149.1,"function":"E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. Specifically ubiquitinates 'Lys-63' in target proteins (PubMed:19920177, PubMed:21399620, PubMed:23644597). Involved in the pathway leading to the degradation of VEGFR-2/KDFR, independently of its ubiquitin-ligase activity. Monoubiquitinates IGF1R at multiple sites, thus leading to receptor internalization and degradation in lysosomes (By similarity). Ubiquitinates FGFR1, leading to receptor internalization and degradation in lysosomes (PubMed:21765395). Promotes ubiquitination of RAPGEF2 (PubMed:11598133). According to PubMed:18562292 the direct link between NEDD4 and PTEN regulation through polyubiquitination described in PubMed:17218260 is questionable. Involved in ubiquitination of ERBB4 intracellular domain E4ICD (By similarity). Part of a signaling complex composed of NEDD4, RAP2A and TNIK which regulates neuronal dendrite extension and arborization during development (By similarity). Ubiquitinates TNK2 and regulates EGF-induced degradation of EGFR and TNF2 (PubMed:20086093). Ubiquitinates BRAT1 and this ubiquitination is enhanced in the presence of NDFIP1 (PubMed:25631046). Ubiquitinates DAZAP2, leading to its proteasomal degradation (PubMed:11342538). Ubiquitinates POLR2A (PubMed:19920177). Functions as a platform to recruit USP13 to form an NEDD4-USP13 deubiquitination complex that plays a critical role in cleaving the 'Lys-48'-linked ubiquitin chains of VPS34 and then stabilizing VPS34, thus promoting the formation of autophagosomes (PubMed:32101753) (Microbial infection) Involved in the ubiquitination of Ebola virus protein VP40 which plays a role in viral budding","subcellular_location":"Cytoplasm; Nucleus; Cell membrane","url":"https://www.uniprot.org/uniprotkb/P46934/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NEDD4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NEDD4","total_profiled":1310},"omim":[{"mim_id":"621542","title":"ARRESTIN DOMAIN-CONTAINING PROTEIN 2; ARRDC2","url":"https://www.omim.org/entry/621542"},{"mim_id":"621529","title":"TRANSMEMBRANE PROTEIN 168; TMEM168","url":"https://www.omim.org/entry/621529"},{"mim_id":"621053","title":"NEDD4-BINDING PROTEIN 2-LIKE 1; N4BP2L1","url":"https://www.omim.org/entry/621053"},{"mim_id":"620048","title":"RETENTION IN ENDOPLASMIC RETICULUM SORTING RECEPTOR 1; RER1","url":"https://www.omim.org/entry/620048"},{"mim_id":"619788","title":"ARRESTIN DOMAIN-CONTAINING PROTEIN 4; ARRDC4","url":"https://www.omim.org/entry/619788"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skeletal muscle","ntpm":63.3},{"tissue":"tongue","ntpm":54.3}],"url":"https://www.proteinatlas.org/search/NEDD4"},"hgnc":{"alias_symbol":["KIAA0093","MGC176705","NEDD4-1","RPF1"],"prev_symbol":[]},"alphafold":{"accession":"P46934","domains":[{"cath_id":"3.90.1750.10","chopping":"955-1108_1155-1198","consensus_level":"high","plddt":84.0912,"start":955,"end":1198},{"cath_id":"3.30.2410.10","chopping":"1205-1312","consensus_level":"high","plddt":78.3946,"start":1205,"end":1312}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P46934","model_url":"https://alphafold.ebi.ac.uk/files/AF-P46934-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P46934-F1-predicted_aligned_error_v6.png","plddt_mean":53.34},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NEDD4","jax_strain_url":"https://www.jax.org/strain/search?query=NEDD4"},"sequence":{"accession":"P46934","fasta_url":"https://rest.uniprot.org/uniprotkb/P46934.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P46934/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P46934"}},"corpus_meta":[{"pmid":"17218260","id":"PMC_17218260","title":"NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN.","date":"2007","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/17218260","citation_count":600,"is_preprint":false},{"pmid":"31974380","id":"PMC_31974380","title":"Nedd4 ubiquitylates VDAC2/3 to suppress erastin-induced ferroptosis in melanoma.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31974380","citation_count":391,"is_preprint":false},{"pmid":"11696533","id":"PMC_11696533","title":"Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11696533","citation_count":357,"is_preprint":false},{"pmid":"10882424","id":"PMC_10882424","title":"Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases.","date":"2000","source":"The Journal of membrane biology","url":"https://pubmed.ncbi.nlm.nih.gov/10882424","citation_count":302,"is_preprint":false},{"pmid":"19557014","id":"PMC_19557014","title":"Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions.","date":"2010","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/19557014","citation_count":194,"is_preprint":false},{"pmid":"17726579","id":"PMC_17726579","title":"The Nedd4-like family of E3 ubiquitin ligases and cancer.","date":"2007","source":"Cancer metastasis reviews","url":"https://pubmed.ncbi.nlm.nih.gov/17726579","citation_count":186,"is_preprint":false},{"pmid":"10322449","id":"PMC_10322449","title":"Nedd4-like proteins: an emerging family of ubiquitin-protein ligases implicated in diverse cellular functions.","date":"1999","source":"Trends in cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10322449","citation_count":184,"is_preprint":false},{"pmid":"10720933","id":"PMC_10720933","title":"Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination.","date":"2000","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/10720933","citation_count":183,"is_preprint":false},{"pmid":"17502380","id":"PMC_17502380","title":"Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17502380","citation_count":179,"is_preprint":false},{"pmid":"18544533","id":"PMC_18544533","title":"Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the beta2-adrenergic receptor.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18544533","citation_count":165,"is_preprint":false},{"pmid":"12167587","id":"PMC_12167587","title":"Concerted action of ENaC, Nedd4-2, and Sgk1 in transepithelial Na(+) transport.","date":"2002","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/12167587","citation_count":156,"is_preprint":false},{"pmid":"18562292","id":"PMC_18562292","title":"The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization.","date":"2008","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/18562292","citation_count":156,"is_preprint":false},{"pmid":"25433090","id":"PMC_25433090","title":"NEDD4-2 (NEDD4L): the ubiquitin ligase for multiple membrane proteins.","date":"2014","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/25433090","citation_count":149,"is_preprint":false},{"pmid":"21338354","id":"PMC_21338354","title":"Nedd4-mediated AMPA receptor ubiquitination regulates receptor turnover and trafficking.","date":"2011","source":"Journal of 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catalyzes PTEN polyubiquitination in cells, leading to proteasomal degradation of PTEN and consequent activation of downstream PI3K/Akt signaling, potentiating cellular transformation.\",\n      \"method\": \"Biochemical purification of E3 ligase activity from cell lysates, in vitro ubiquitination assay, cell-based polyubiquitination assay, overexpression/knockdown with PTEN stability readout, xenograft tumor model\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus cell-based assays plus in vivo tumor model; widely cited foundational study with multiple orthogonal methods\",\n      \"pmids\": [\"17218260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Subsequent study using two independently generated Nedd4-1 knockout mouse strains found no detectable interaction between Nedd4-1 and PTEN, and PTEN stability, ubiquitination, subcellular distribution, and downstream Akt activity were all unchanged in Nedd4-1-deficient cells, failing to support Nedd4-1 as the physiological E3 ligase for PTEN.\",\n      \"method\": \"Nedd4-1 knockout MEFs (two independent mouse strains), co-immunoprecipitation attempts, PTEN stability assay, PKB/Akt activity assay, nuclear PTEN function (Rad51 transactivation assay)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent KO mouse lines, multiple orthogonal functional readouts; directly contradicts PMID 17218260 regarding PTEN as physiological substrate\",\n      \"pmids\": [\"18562292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"NEDD4 binds the epithelial Na+ channel (ENaC) via its WW domains interacting with PY motifs (PPxY) in the C-termini of ENaC subunits, catalyzes ENaC ubiquitination via its HECT domain, and reduces ENaC surface expression; the C2 domain localizes NEDD4 to the apical plasma membrane in a Ca2+-dependent manner in polarized MDCK cells.\",\n      \"method\": \"Electrophysiology, co-immunoprecipitation, subcellular fractionation/localization, site-directed mutagenesis of PY motifs (Liddle's syndrome mutations), MDCK cell polarization experiments\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods, replicated across multiple studies and labs (PMID 10882424 corroborates), direct structure-function analysis\",\n      \"pmids\": [\"10720933\", \"10882424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SGK (serum- and glucocorticoid-regulated kinase) binds to NEDD4 (and NEDD4-2) via a PY motif in SGK; SGK phosphorylates NEDD4-2 (but not NEDD4), reducing NEDD4-2 binding to αENaC and thereby relieving NEDD4-2-mediated inhibition of epithelial Na+ absorption.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay (phosphorylation of recombinant NEDD4-2), electrophysiology (Na+ current measurement), deletion/mutation analysis of PY motif\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus co-IP plus functional electrophysiology; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"11696533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"GRK2 (G-protein-coupled receptor kinase 2) phosphorylates the C-terminus of the ENaC β-subunit and renders ENaC insensitive to inhibition by NEDD4-2, maintaining channels in an active state.\",\n      \"method\": \"Electrophysiology, in vitro phosphorylation assay, co-expression in Xenopus oocytes\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro phosphorylation assay plus electrophysiological functional readout; single lab\",\n      \"pmids\": [\"15284439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"GRK2 directly interacts with and phosphorylates NEDD4 (as well as NEDD4-2) at multiple sites, including threonine 466 in NEDD4 located in the WW3 domain region that binds ENaC.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay with recombinant proteins, mass spectrometry of phosphorylation sites\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vitro kinase assay plus co-IP; single lab, single study\",\n      \"pmids\": [\"17544362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"NEDD4 acts as the E3 ubiquitin ligase for the β2-adrenergic receptor (β2AR); agonist stimulation triggers β-arrestin2-mediated recruitment of NEDD4 to activated β2AR, leading to NEDD4-catalyzed receptor ubiquitination and lysosomal degradation. β-arrestin2 is an essential adaptor, as its knockdown abolishes β2AR-NEDD4 interaction.\",\n      \"method\": \"Dominant-negative NEDD4 overexpression, siRNA knockdown (NEDD4, NEDD4-2, AIP4), co-immunoprecipitation of NEDD4–β-arrestin2–β2AR complex, ubiquitination assay, lysosomal trafficking assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, dominant-negative, siRNA knockdown with multiple functional readouts in same study; single lab\",\n      \"pmids\": [\"18544533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NEDD4-1 directly binds and ubiquitylates activated FGFR1 via its WW3 domain interacting with a non-canonical non-PY motif on FGFR1, promoting FGFR1 endocytosis and attenuating downstream signaling (FRS2α, Akt, Erk1/2, PLCγ). FGFR1 lacking the Nedd4-1 recognition motif (FGFR1-Δ6) shows impaired endocytosis and sustained signaling; in neural stem cells promotes neuronal differentiation; in zebrafish disrupts anterior neuronal patterning.\",\n      \"method\": \"Direct binding assay, co-immunoprecipitation, in vitro/in vivo ubiquitination assay, receptor endocytosis assay, Nedd4-1 knockdown, FGFR1-Δ6 mutant in neural stem cells, zebrafish embryo expression\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro direct binding, in vitro ubiquitination, mutagenesis, cell-based endocytosis, and in vivo zebrafish validation in a single study\",\n      \"pmids\": [\"21765395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NEDD4 is the E3 ubiquitin ligase responsible for AMPA receptor (AMPAR/GluA1) ubiquitination in neurons; NEDD4 co-localizes and associates with AMPARs in synaptoneurosomes, ubiquitinates GluA1 at K868, reduces AMPAR surface expression, and suppresses excitatory synaptic transmission. Knockdown of NEDD4 abolishes AMPAR ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, synaptosome fractionation, ubiquitination assay with lysine-to-arginine mutants (K868R), siRNA knockdown, electrophysiology (mEPSC recording)\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, site-directed mutagenesis, siRNA KD, electrophysiological functional readout; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"21338354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NEDD4-1 is an E3 ubiquitin ligase for Mdm2; it interacts with Mdm2 via the Mdm2 RING domain and catalyzes K63-linked polyubiquitination of Mdm2 (distinct from K48-linked chains that Mdm2/MdmX forms), thereby stabilizing Mdm2. Nedd4-1 KO MEFs show increased p53 levels and activity, more robust DNA damage response, and increased G1 arrest.\",\n      \"method\": \"In vitro E3 ligase assay with recombinant proteins, Nedd4-1 KO MEF lysates, co-immunoprecipitation, ubiquitin linkage-specific analysis, Mdm2 half-life measurement (CHX chase), p53 reporter assays, cell cycle analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstituted ubiquitination assay with recombinant proteins, KO cells, multiple functional readouts; single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"24413081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NEDD4-1 acts as an E3 ubiquitin ligase for Ras; Ras signaling stimulates NEDD4-1 expression, but oncogenic Ras mutations or EGF signaling prevent NEDD4-1-mediated Ras ubiquitination, leading to NEDD4-1 accumulation and subsequent PTEN degradation. This defines a Ras–NEDD4-1–PTEN regulatory loop.\",\n      \"method\": \"In vivo ubiquitination assay, co-immunoprecipitation, expression analysis in human cancer samples and cancer cell lines, genetic rescue experiments\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based ubiquitination assays, co-IP, human cancer samples; single lab\",\n      \"pmids\": [\"24746824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NEDD4 ubiquitylates and destabilizes WW45 (SAV1) and LATS kinase (core Hippo pathway components), thereby suppressing Hippo signaling. MST1 protects WW45 but not LATS2 from NEDD4-mediated degradation. NEDD4 inactivation at high cell density enables contact inhibition via elevated Hippo activity. In Drosophila, NEDD4 promotes intestinal stem cell renewal by suppressing Hippo signaling.\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation, knockdown/overexpression with protein stability readouts, Drosophila genetic model (ISC proliferation)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro ubiquitination, reciprocal co-IP, cell-based and in vivo (Drosophila) validation; multiple orthogonal methods across two organisms\",\n      \"pmids\": [\"25692647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NEDD4 monoubiquitinates IRS-2, promoting its association with ubiquitin-binding protein Epsin1 and recruitment of IRS-2 to the membrane, thereby enhancing IGF-I receptor-induced IRS-2 tyrosine phosphorylation, downstream signaling, and cell proliferation. In zebrafish, NEDD4 overexpression accelerates embryonic growth through IRS-2.\",\n      \"method\": \"Co-immunoprecipitation, in vitro/in vivo ubiquitination assay, siRNA knockdown, membrane fractionation, IGF-I signaling assays, zebrafish embryo experiments\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple biochemical methods plus in vivo zebrafish validation; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25879670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Elevated NEDD4 is the E3 ubiquitin ligase responsible for HSF1 degradation induced by aggregated α-synuclein/proteotoxic stress; SIRT1-mediated deacetylation of HSF1 at K80 attenuates NEDD4-mediated HSF1 ubiquitination and stabilizes HSF1.\",\n      \"method\": \"Ubiquitination assay in neuroblastoma cells, siRNA knockdown of NEDD4, co-immunoprecipitation, pharmacological SIRT1 activation, site-directed mutagenesis (K80 acetylation), mouse model and human tissue validation\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based ubiquitination assay, KD, mutagenesis, in vivo confirmation; single lab\",\n      \"pmids\": [\"26503960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"NEDD4 acts as the E3 ubiquitin ligase for the ErbB4 JM-a/CYT-1 intracellular domain (ICD); NEDD4 binds via its WW domains to three PY motifs in the CYT-1 C-terminus, catalyzes CYT-1 ICD ubiquitination, and promotes its proteasomal and lysosomal degradation. Mutation of PY motifs impairs NEDD4 binding, increases CYT-1 ICD stability, and (for one PY mutant) enhances nuclear translocation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro direct binding assay, in vitro ubiquitination assay, PY motif mutagenesis, proteasome/lysosome inhibitor experiments\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro direct binding, in vitro ubiquitination reconstitution, mutagenesis, cell-based stability assays; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19193720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NEDD4-1 and β-arrestin-1 cooperate to ubiquitylate and internalize NHE1 (Na+/H+ exchanger 1) at the plasma membrane. β-arrestin-1 bridges NEDD4-1 to the NHE1 C-terminus; truncation of NHE1 C-terminus to aa550 abolishes β-arrestin-1 binding and NHE1 ubiquitylation. Knockout of either Nedd4-1 or β-arrestin-1 in mouse fibroblasts increases NHE1 surface levels and transport activity.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, Nedd4-1 and β-arrestin-1 knockout mouse fibroblasts, ubiquitylation assay, surface biotinylation, ion transport assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse fibroblasts plus co-IP plus functional transport assay plus siRNA; multiple orthogonal methods\",\n      \"pmids\": [\"20855896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"NDFIP1 (Nedd4 family-interacting protein 1) recruits NEDD4 (and Nedd4-2 and Itch) into exosomes secreted from cells and primary neurons; without NDFIP1, these Nedd4 proteins are absent from exosomes. NDFIP1 increases overall exosome secretion.\",\n      \"method\": \"Exosome isolation, Western blot of exosomal fractions, co-transfection experiments, primary neuron exosome analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — biochemical fractionation with functional implication; single lab, replicated in both transfected cells and primary neurons\",\n      \"pmids\": [\"18819914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NEDD4-1 undergoes K29-linked auto-ubiquitination at K1279, which serves as a scaffold to recruit the deubiquitinase USP13. The resulting NEDD4-1–USP13 complex removes K48-linked polyubiquitin chains from VPS34 at K419, stabilizing VPS34 and promoting autophagy flux. Knockout of either NEDD4-1 or USP13 increases K48-linked ubiquitination and degradation of VPS34.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (linkage-specific), USP13/NEDD4-1 knockout cells, autophagosome formation assay, site-directed mutagenesis (K1279, K419)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, linkage-specific ubiquitination, KO cells, site-directed mutagenesis; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"32101753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NEDD4 ubiquitinates histone H3 on lysine 23/36/37 residues in a glucose-dependent manner; this H3 ubiquitination specifically recruits histone acetyltransferase GCN5 for subsequent H3 K9 acetylation at transcription start sites and enhancer regions, activating transcription of IL1α, IL1β, and GCLM, and promoting tumour sphere formation.\",\n      \"method\": \"ChIP-seq, microarray, in vitro ubiquitination assay with recombinant H3, site-directed mutagenesis of H3 lysines, GCN5 pulldown, tumour sphere formation assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of H3 ubiquitination, ChIP-seq genome-wide validation, mutagenesis, functional assays; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28300060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NEDD4 ubiquitylation activity is restrained by an evolutionarily conserved allosteric auto-inhibition mechanism: ubiquitylation of a conserved lysine on the HECT domain α1-helix promotes NEDD4 oligomerization (trimerization) that renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation and causes unrestrained FGFR1 ubiquitylation and constitutive IKS channel downregulation.\",\n      \"method\": \"Biochemical and biophysical analysis (oligomerization assays), site-directed mutagenesis, in vitro ubiquitination assays, electrophysiology (IKS channel), TIRF microscopy, genetic experiments in yeast\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, mutagenesis, structural biophysics, electrophysiology, and genetic data across multiple substrates and organisms\",\n      \"pmids\": [\"28069708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NEDD4 catalytic activity is autoinhibited by WW domain linker regions that engage the HECT domain; allosteric activation by NDFIP1 (and engineered ubiquitin variants) relieves this autoinhibition. The WW domain linker also influences ubiquitin chain linkage (K48 vs K63) and degree of polyubiquitination on substrates.\",\n      \"method\": \"In vitro ubiquitination assays with recombinant NEDD4-1, linker deletion/phosphomimic mutants, NDFIP1 binding assays, substrate ubiquitination assays (PTEN, p62, WBP2)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with multiple mutants and substrates; single lab with rigorous biochemical dissection\",\n      \"pmids\": [\"31578285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"NEDD4 interacts constitutively with the adapter protein Grb10 (which couples to insulin and IGF-I receptors) via the C2 domain of NEDD4 and a region including the SH2/BPS domain of Grb10, both in yeast two-hybrid and by endogenous co-immunoprecipitation in mouse embryo fibroblasts; the interaction is Ca2+-independent.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation of endogenous proteins in MEFs, deletion mutagenesis of Grb10\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid confirmed by endogenous co-IP; single lab, no catalytic functional assay performed\",\n      \"pmids\": [\"10446181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NEDD4 and NEDD4-2 share the same preferred ubiquitin-conjugating enzyme (E2) specificity: UbcH5b is the most efficient E2 for both ligases in vitro, with Ube2e3 used less efficiently.\",\n      \"method\": \"In vitro ubiquitination assay with panel of E2 enzymes and recombinant NEDD4/NEDD4-2\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution; single lab, single study\",\n      \"pmids\": [\"16337426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NEDD4 and NEDD4L (NEDD4-2) ubiquitinate LGR5 receptor (and DVL2), targeting them for proteasomal and lysosomal degradation; loss of Nedd4/Nedd4l enhances ISC proliferation and sensitivity to R-spondin and accelerates tumour development in Apcmin mice, demonstrating that NEDD4/NEDD4L negatively regulate Wnt/β-catenin signaling through LGR5 degradation.\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation, conditional KO mouse models, organoid culture, Apcmin tumour model, protein stability assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro ubiquitination, KO mouse models, organoid/in vivo tumor phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"31867777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4 ubiquitinates VDAC2 and VDAC3 following erastin treatment, promoting their degradation; this suppresses erastin-induced ferroptosis. FOXM1-induced NEDD4 expression creates a negative feedback loop (FOXM1–NEDD4–VDAC2/3) that reduces ferroptosis sensitivity.\",\n      \"method\": \"siRNA knockdown of NEDD4, ubiquitination assay, co-immunoprecipitation, VDAC2/3 protein stability assay, ferroptosis assay (cell viability, lipid peroxidation)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, KD with functional ferroptosis readout; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31974380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NEDD4, activated downstream of FGFR3, interacts with PD-L1 and catalyzes Lys48-linked polyubiquitination of PD-L1, targeting it for proteasomal degradation. Phosphorylation of NEDD4 by FGFR3 activates its E3 activity toward PD-L1.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K48-linkage specific), NEDD4 KO bladder cancer cell lines, FGFR3 inhibition experiments, tissue microarray, in vivo tumor mouse models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, K48-specific ubiquitination assay, KO cells, in vivo tumor models; multiple orthogonal methods\",\n      \"pmids\": [\"34753771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4 controls spermatogonial progenitor cell (SPC) homeostasis by targeting the RNA-binding protein NANOS2 for ubiquitination/destabilization, promoting spermatogonial differentiation. NEDD4 is also required for stress granule (SG) clearance via the endosomal-lysosomal pathway during recovery from stress.\",\n      \"method\": \"Nedd4 conditional KO mice (spermatogonia), co-immunoprecipitation, ubiquitination assay, immunofluorescence (SG dynamics), lysosomal pathway inhibitor experiments\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO mice with cellular phenotype, co-IP, ubiquitination assay, multiple functional readouts\",\n      \"pmids\": [\"28585553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NEDD4-1 promotes glioma cell migration and invasion by physically interacting with CNrasGEF and promoting its polyubiquitination and proteasomal degradation, thereby removing a migration suppressor.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay, siRNA knockdown/overexpression with migration and invasion assays (Transwell, wound healing), glioma tissue expression analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus ubiquitination assay plus functional migration assays; single lab\",\n      \"pmids\": [\"24340059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NEDD4 ubiquitinates and targets LATS1 (a core Hippo pathway kinase) for degradation in hepatocellular carcinoma cells, promoting tumor progression.\",\n      \"method\": \"siRNA knockdown/overexpression, co-immunoprecipitation, Western blot for LATS1 protein levels, cell proliferation/invasion assays\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-IP and indirect protein level measurements; single lab, no in vitro ubiquitination assay performed\",\n      \"pmids\": [\"29480061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NDFIP1 and NDFIP2 are essential adaptor proteins that bridge NEDD4 (and NEDD4L) to aquaporin-2 (AQP2), enabling NEDD4/NEDD4L-mediated AQP2 ubiquitination and lysosomal degradation; PY motifs in NDFIP1/2 are required for this function.\",\n      \"method\": \"siRNA knockdown in mpkCCD cells, Membrane Yeast Two-Hybrid (NDFIP2-AQP2 interaction), HEK293T co-transfection ubiquitination assay, NDFIP1/2 PY-motif mutants, AQP2 abundance assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Y2H, co-expression ubiquitination, KD in relevant epithelial cells, mutagenesis; single lab with multiple methods\",\n      \"pmids\": [\"28931009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NEDD4 E3 ligase and β-arrestins regulate mGlu7 receptor ubiquitination and trafficking: upon agonist stimulation, β-arrestins recruit NEDD4 to mGlu7, facilitating NEDD4-mediated ubiquitination that promotes mGlu7 endocytosis and degradation by both proteasomal and lysosomal pathways; NEDD4 is also required for mGlu7-dependent MAPK signaling.\",\n      \"method\": \"Co-immunoprecipitation in HEK293 cells and rat neurons, ubiquitination assay, surface expression assay, endocytosis assay, MAPK signaling readout, siRNA knockdown\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP in neurons, ubiquitination assay, functional endocytosis and signaling readouts; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31373553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4 promotes oligodendrocyte differentiation and developmental myelination by stabilizing VHL via K63-linked ubiquitination; the Daam2-VHL-NEDD4 axis governs OPC differentiation during development and remyelination after white matter injury.\",\n      \"method\": \"Conditional KO mouse models (Nedd4 KO), proteomic analysis of Daam2-VHL complex, K63-linked ubiquitination assay, myelination/remyelination assays, human MS lesion analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO mice, proteomics, K63-specific ubiquitination assay, in vivo demyelination models, and human tissue validation\",\n      \"pmids\": [\"32792353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LRAD3, a member of the LDL receptor family, activates NEDD4 E3 ligase activity via its intracellular PPxY motifs binding to the WW domains of NEDD4; binding to the terminal PPxY motif of LRAD3 is required to promote NEDD4 auto-ubiquitination and subsequent proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination (auto-ubiquitination) assay, PPxY mutagenesis, proteasome inhibitor experiments\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro auto-ubiquitination assay, co-IP, site-directed mutagenesis; single lab\",\n      \"pmids\": [\"26854353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Lactate inhibits NEDD4-mediated ubiquitination of Caspase-11 by inducing K33 lactylation of NEDD4, which impairs the interaction between NEDD4 and Caspase-11, thereby allowing Caspase-11 accumulation and promoting non-canonical pyroptosis in macrophages during acetaminophen-induced liver injury.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, lactylation detection, site-directed mutagenesis (NEDD4 K33), macrophage pyroptosis assay, mouse APAP liver injury model\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, site-directed mutagenesis of lactylation site, in vivo mouse model; single lab\",\n      \"pmids\": [\"38385085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4 ubiquitinates and promotes proteasomal degradation of NLRP3 by interacting with adaptor protein SF3A2, which bridges NEDD4 to NLRP3; this inhibits NLRP3 inflammasome activation and macrophage pyroptosis.\",\n      \"method\": \"Co-immunoprecipitation (NEDD4-SF3A2-NLRP3), Western blot for NLRP3 levels after NEDD4 overexpression/knockdown, pyroptosis assay, mouse MI/R model\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP, protein level assays, functional pyroptosis readout in cells and mice; single lab\",\n      \"pmids\": [\"36732831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NEDD4-1 interacts with the PPTY motif of VDAC1 via its WW domain and catalyzes K48-linked ubiquitination of VDAC1, promoting its proteasomal degradation; hepatocyte-specific Nedd4-1 KO increases VDAC1 accumulation and oligomerization, worsening APAP-induced mitochondrial damage and liver injury.\",\n      \"method\": \"Co-immunoprecipitation, in vivo/in vitro ubiquitination assay (K48-linkage specific), hepatocyte-specific KO and overexpression mouse models, VDAC1 knockdown rescue experiments\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, K48-specific ubiquitination assay, hepatocyte KO mouse, overexpression, and genetic rescue; multiple orthogonal methods\",\n      \"pmids\": [\"37139424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NEDD4 is the E3 ubiquitin ligase for E-cadherin; N4BP3 acts as an adaptor that promotes NEDD4-mediated K48/K63-linked polyubiquitination and degradation of E-cadherin, driving breast cancer metastasis via EMT.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, in vivo metastasis assay, N4BP3 depletion experiments\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, in vivo metastasis model; single lab\",\n      \"pmids\": [\"36162713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4 promotes TGFBR1 signaling by binding directly to TGF-β type I receptor (TGFBR1) and forming K27-linked ubiquitin at lysine 391, thereby enhancing TGF-β signal transduction and tumor cell migration in hepatocellular carcinoma.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay (K27-specific), NEDD4 knockdown with HCC migration/invasion assays, PDX model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, linkage-specific ubiquitination, in vivo PDX model; single lab\",\n      \"pmids\": [\"35501461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NEDD4 interacts with and ubiquitinates DMT1 (divalent metal transporter 1) at lysine residues 6, 69, and 277, facilitating DMT1 degradation and reducing iron accumulation/ferroptosis after intracerebral hemorrhage; NEDD4 overexpression alleviated ferroptosis and improved neurological outcomes.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (site-directed mutagenesis of K6/K69/K277), lentiviral Nedd4 overexpression, ferroptosis markers (GPX4, MDA, iron content), mouse ICH model\",\n      \"journal\": \"CNS neuroscience & therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, site-directed mutagenesis of ubiquitination sites, in vivo mouse model; single lab\",\n      \"pmids\": [\"38634270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NEDD4 catalyzes K48-linked ubiquitination of TAK1 at sites in TAK1, promoting its proteasomal degradation; THBS1 protects TAK1 from NEDD4-mediated ubiquitination by interacting with TAK1, and THBS1 silencing upregulates NEDD4 expression, leading to increased TAK1 degradation and necroptosis in trophoblasts.\",\n      \"method\": \"Co-immunoprecipitation, K48-specific ubiquitination assay, NEDD4 knockdown/overexpression, THBS1 silencing, mouse PE model\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, K48-specific ubiquitination assay, in vivo mouse model; single lab\",\n      \"pmids\": [\"38569496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4 binds to and mediates ubiquitination and proteasomal degradation of CSF1R in macrophages; IFN-I signaling increases NEDD4-mediated CSF1R ubiquitination, and USP18 impairs this by interrupting NEDD4 binding to CSF1R.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, USP18 KO macrophage experiments, CSF1R protein stability assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, genetic KO; single lab with multiple methods\",\n      \"pmids\": [\"38100351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NEDD4 triggers ubiquitination and proteasomal degradation of the transcription factor FOXA1; FOXA1 transcriptionally activates miR-340-5p which suppresses ATF1, creating a regulatory axis downstream of NEDD4 in colon cancer progression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, chromatin immunoprecipitation, luciferase reporter assay, in vivo xenograft model\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, ChIP, luciferase, in vivo model; single lab\",\n      \"pmids\": [\"33530829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"YOD1 (deubiquitinase) binds to and deubiquitinates NEDD4; YOD1 removes K63-linked polyubiquitin chains from NEDD4 and suppresses NEDD4-induced cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, K63-specific ubiquitin chain analysis, cell proliferation assay with YOD1/NEDD4 overexpression\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP, deubiquitination assay, functional proliferation readout; single lab\",\n      \"pmids\": [\"31916733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4-1 ubiquitinates SR-BI at K500 and K508, promoting its degradation; hepatocyte lipocalin-2 (Lcn2) blocks this Nedd4-1-mediated SR-BI ubiquitination, improving HDL metabolism and reducing atherosclerosis in mice.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (site-directed mutagenesis K500/K508), hepatocyte-specific Lcn2 KO and overexpression, Nedd4-1 KO mice, SR-BI K500A/K508A knock-in mice\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, site-directed mutagenesis of ubiquitination sites, multiple conditional KO mouse models including knock-in; strong in vivo validation\",\n      \"pmids\": [\"37863040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GSK-3β phosphorylates PGC-1α at Thr295, creating a recognition motif for NEDD4-1, which then catalyzes ubiquitin-dependent proteasomal degradation of PGC-1α; mutation of Thr295 negates shikonin's suppressive effects on mitochondrial biogenesis and TNBC metastasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, phosphorylation site mutagenesis (Thr295), LC-MS/MS identification of phosphorylation, in vivo TNBC metastasis model\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation-dependent co-IP, ubiquitination assay, mutagenesis, in vivo model; single lab\",\n      \"pmids\": [\"38937832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Adaptor protein Numb binds NEDD4-1 and regulates its E3 ligase activity and its association with PTEN, thereby controlling PTEN ubiquitination; Numb regulates cell proliferation and glucose metabolism in a PTEN-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation (Numb-NEDD4-1 interaction), ubiquitination assay for PTEN, Numb knockdown with PTEN stability readout, cell proliferation and glucose metabolism assays\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP, ubiquitination assay, knockdown with functional readout; single lab\",\n      \"pmids\": [\"28437168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NEDD4 TV3 (a specific transcript variant) activates the NF-κB pathway through its association with the adaptor protein RIP, contributing to inflammation in keloid formation.\",\n      \"method\": \"Forced expression and selective knockdown of NEDD4 TV3, NF-κB reporter assay, co-immunoprecipitation with RIP, in vivo keloid mouse model\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP of NEDD4 TV3 with RIP, functional NF-κB reporter, in vivo mouse model; single lab\",\n      \"pmids\": [\"30273597\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NEDD4 (NEDD4-1) is a HECT-domain E3 ubiquitin ligase that recognizes substrates primarily via WW domain–PY motif (PPxY) interactions; it ubiquitinates a broad array of membrane proteins (ENaC, FGFR1, AMPAR/GluA1, β2AR, mGlu7, NHE1, VDAC1/2/3, LGR5, PD-L1, SR-BI, DMT1), nuclear substrates (histone H3, HSF1, FOXA1), and signaling proteins (Mdm2, PTEN, Ras, WW45/LATS, VPS34, Caspase-11, NLRP3, TAK1, CSF1R, TGFBR1), using K48-, K63-, K27-, or K29-linked ubiquitin chains to direct substrates to proteasomal or lysosomal degradation or to scaffold non-degradative signaling; its own HECT domain is subject to intramolecular autoinhibition by WW domain linkers, and allosteric activation is achieved through NDFIP1 binding, PPxY-containing adaptors (LRAD3), or phosphorylation (by GRK2 or FGFR3), while K29-linked auto-ubiquitination recruits the deubiquitinase USP13 to form a complex that stabilizes VPS34 and promotes autophagy, revealing both degradative and non-degradative scaffolding functions of the ligase.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NEDD4 (NEDD4-1) is a HECT-domain E3 ubiquitin ligase that controls the abundance and trafficking of membrane receptors, ion channels, and signaling proteins, governing diverse processes from epithelial Na+ transport and synaptic transmission to growth-factor signaling, tumor suppression, and cell death [#2, #8, #11]. Substrate selection is driven principally by recognition of PY (PPxY) motifs through its WW domains, as established for ENaC subunits, ErbB4 CYT-1, and VDAC1 [#2, #14, #35], while its C2 domain mediates Ca2+-dependent membrane localization and constitutive adaptor binding (Grb10) [#2, #21]. The ligase assembles ubiquitin chains of multiple linkages to dictate substrate fate: K48-linked chains target PD-L1, VDAC1, TAK1, and Mdm2 for proteasomal degradation [#25, #35, #39], whereas K63- and K27-linked chains can be non-degradative, stabilizing Mdm2 and VHL or potentiating TGFBR1 signaling [#9, #31, #37]. Beyond catalyzing degradation, NEDD4 acts as a non-degradative scaffold: K29-linked auto-ubiquitination at K1279 recruits the deubiquitinase USP13 to protect VPS34 from K48 chains and promote autophagy [#17]. NEDD4 ubiquitinates numerous receptors for endocytic downregulation, frequently using arrestin or NDFIP adaptors to bridge substrate recognition—β-arrestin recruits NEDD4 to the agonist-activated β2-adrenergic and mGlu7 receptors, and to NHE1, while NDFIP1/2 bridge it to aquaporin-2 [#6, #30, #15, #29]. Its activity is tightly autoregulated: WW-domain linker regions and an oligomerization-promoting HECT α1 lysine impose intramolecular autoinhibition that is relieved by NDFIP1, PPxY adaptors (LRAD3), or substrate-directed phosphorylation by GRK2 and FGFR3 [#19, #20, #5, #32, #25]. NEDD4 also ubiquitinates nuclear substrates including histone H3, where glucose-dependent H3 ubiquitination recruits GCN5 to activate transcription [#18]. NEDD4 is reported as an E3 ligase for PTEN, but two independent Nedd4-1 knockout mouse lines found no effect on PTEN interaction, stability, or downstream Akt activity, leaving the physiological relevance of this substrate unresolved [#0, #1].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established the core substrate-recognition logic of NEDD4 by showing WW-domain engagement of PY motifs drives ubiquitination and surface downregulation of a physiological ion channel.\",\n      \"evidence\": \"Co-IP, electrophysiology, PY-motif mutagenesis (Liddle's mutations), and C2-domain localization in polarized MDCK cells for ENaC\",\n      \"pmids\": [\"10720933\", \"10882424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve ubiquitin chain linkage type on ENaC\", \"Regulation of NEDD4 activity itself not addressed\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed that kinase phosphorylation can regulate NEDD4-family channel control, defining a signaling input to the ENaC pathway.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, and Na+ current measurement of SGK acting on NEDD4/NEDD4-2\",\n      \"pmids\": [\"11696533\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SGK phosphorylates NEDD4-2 but not NEDD4, leaving NEDD4 regulation here open\", \"Structural basis of phospho-dependent affinity change not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Proposed PTEN as a NEDD4 substrate linking the ligase to PI3K/Akt and transformation, the first oncogenic role assigned.\",\n      \"evidence\": \"Biochemical E3 purification, in vitro and cell ubiquitination, and xenograft model\",\n      \"pmids\": [\"17218260\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance contested by subsequent KO data\", \"In vivo requirement not tested with genetic loss-of-function in this study\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Directly challenged the PTEN-substrate model using genetic loss-of-function, establishing that NEDD4 substrate assignments require in vivo validation.\",\n      \"evidence\": \"Two independent Nedd4-1 KO mouse strains, co-IP attempts, and PTEN stability/Akt readouts in MEFs\",\n      \"pmids\": [\"18562292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not exclude context- or tissue-specific PTEN regulation\", \"Cannot rule out redundancy with NEDD4L\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined arrestin-bridged recruitment as a mechanism for NEDD4 targeting of GPCRs, generalizing beyond direct PY recognition.\",\n      \"evidence\": \"β-arrestin2 knockdown, dominant-negative NEDD4, complex co-IP, and lysosomal trafficking assay for β2AR\",\n      \"pmids\": [\"18544533\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain linkage on β2AR not determined\", \"Structural basis of arrestin-NEDD4 bridging not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed NEDD4 attenuates receptor tyrosine kinase signaling via endocytic downregulation, and that recognition can occur through non-canonical non-PY motifs.\",\n      \"evidence\": \"Direct binding, in vitro/in vivo ubiquitination, endocytosis assay, FGFR1-Δ6 mutant, and zebrafish neuronal patterning for FGFR1\",\n      \"pmids\": [\"21765395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of non-PY recognition across substrates unclear\", \"Chain linkage directing FGFR1 endocytosis not specified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended NEDD4 control to neuronal AMPA receptors, linking the ligase to synaptic strength.\",\n      \"evidence\": \"Synaptosome co-IP, GluA1 K868R mutagenesis, siRNA knockdown, and mEPSC recording\",\n      \"pmids\": [\"21338354\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signal triggering NEDD4-AMPAR engagement not defined\", \"In vivo behavioral consequence not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed linkage-specific non-degradative ubiquitination by showing NEDD4 stabilizes Mdm2 via K63 chains, distinct from degradative K48 control.\",\n      \"evidence\": \"Reconstituted in vitro ligase assay, Nedd4-1 KO MEFs, linkage analysis, CHX chase, and p53/cell-cycle readouts\",\n      \"pmids\": [\"24413081\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How K63 stabilization opposes Mdm2 autoubiquitination mechanistically unclear\", \"Physiological p53 contexts not fully mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed NEDD4 as a negative regulator of the Hippo tumor-suppressor pathway and a positive regulator of growth signaling, defining opposing pro-proliferative roles.\",\n      \"evidence\": \"In vitro ubiquitination, co-IP, protein stability, Drosophila ISC models for WW45/LATS, and IRS-2 monoubiquitination/Epsin1 recruitment with zebrafish growth assays\",\n      \"pmids\": [\"25692647\", \"25879670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Switch between degradative and signaling-promoting outputs not unified mechanistically\", \"Cross-talk with autoinhibition state not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Uncovered NEDD4 as a non-degradative scaffold in autophagy, where self-K29-ubiquitination recruits a deubiquitinase to protect a third protein.\",\n      \"evidence\": \"Linkage-specific ubiquitination, NEDD4-1/USP13 KO cells, K1279/K419 mutagenesis, and autophagosome assays for the VPS34 complex\",\n      \"pmids\": [\"32101753\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger inducing K29 auto-ubiquitination unknown\", \"Structural basis of USP13 recruitment by K29 chains not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified a nuclear/chromatin function for NEDD4, coupling glucose-dependent histone H3 ubiquitination to GCN5 recruitment and transcriptional activation.\",\n      \"evidence\": \"In vitro H3 ubiquitination, ChIP-seq, H3 lysine mutagenesis, GCN5 pulldown, and tumorsphere assays\",\n      \"pmids\": [\"28300060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a cytoplasmic membrane ligase accesses chromatin not resolved\", \"Metabolic signal linking glucose to NEDD4 activity unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the molecular basis of NEDD4 autoinhibition and its allosteric relief, explaining how the ligase is held inactive and switched on.\",\n      \"evidence\": \"Oligomerization biophysics and HECT α1 lysine mutagenesis (28069708); WW-linker deletion/phosphomimic mutants and NDFIP1 activation across PTEN/p62/WBP2 (31578285)\",\n      \"pmids\": [\"28069708\", \"31578285\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo prevalence of the oligomeric inactive state unknown\", \"How phosphorylation by GRK2/FGFR3 intersects with linker autoinhibition not fully mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established physiological roles of NEDD4 in development and cell-fate via genetic models, including spermatogonial homeostasis and myelination.\",\n      \"evidence\": \"Conditional Nedd4 KO mice with NANOS2 ubiquitination and stress-granule clearance (28585553); Daam2-VHL-NEDD4 K63 axis in oligodendrocyte differentiation with human MS lesion analysis (32792353)\",\n      \"pmids\": [\"28585553\", \"32792353\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct WW-PY recognition of these substrates not always demonstrated\", \"Linkage-to-outcome rules across these substrates not unified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked NEDD4 to immune checkpoint and cell-death control, showing FGFR3-activated NEDD4 degrades PD-L1 and connecting it to ferroptosis regulation.\",\n      \"evidence\": \"K48-specific ubiquitination, NEDD4 KO cancer cells, in vivo tumor models for PD-L1 (34753771); VDAC2/3 ubiquitination in the FOXM1-NEDD4 ferroptosis loop (31974380)\",\n      \"pmids\": [\"34753771\", \"31974380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"FGFR3-dependent phospho-activation mechanism on NEDD4 not structurally defined\", \"Selectivity among VDAC paralogs not fully explained\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated post-translational modification of NEDD4 itself (lactylation) as a substrate-selective regulatory switch, expanding the modes controlling its activity.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, NEDD4 K33 lactylation detection/mutagenesis, and APAP liver injury model for Caspase-11\",\n      \"pmids\": [\"38385085\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single substrate context for lactylation\", \"Whether lactylation affects other substrates not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NEDD4 integrates its autoinhibition state, adaptor repertoire, and diverse post-translational modifications to select among its many substrates and chain linkages in a given cellular context remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model predicting substrate choice or chain-linkage output\", \"Tissue-specific adaptor and modification landscapes uncharacterized\", \"Physiological vs. overexpression-based substrate assignments not consistently distinguished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 9, 17, 25, 35]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [2, 14, 18, 25, 35]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 9, 25, 35]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [19, 20]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 6, 15]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [16, 26]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 9, 25, 35]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 7, 11, 30, 37]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [2, 15, 29, 38]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [25, 33, 34, 40]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [24, 33, 34, 38, 39]}\n    ],\n    \"complexes\": [\n      \"NEDD4-USP13 complex\"\n    ],\n    \"partners\": [\n      \"NDFIP1\",\n      \"ARRB2\",\n      \"USP13\",\n      \"GRK2\",\n      \"SGK\",\n      \"GRB10\",\n      \"LRAD3\",\n      \"YOD1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}