{"gene":"NDFIP1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2001,"finding":"NDFIP1 (N4WBP5) binds Nedd4 family WW domains via two PPXY motifs in its amino terminus, is itself ubiquitinated, localizes to the Golgi complex, and its ectopic expression disrupts Golgi structure, suggesting a role as an adaptor for Nedd4-like proteins in ubiquitin-dependent protein sorting.","method":"Co-immunoprecipitation/WW-domain binding assays, subcellular fractionation, immunofluorescence, overexpression morphological analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays with multiple Nedd4 family members, direct localization with functional consequence (Golgi disruption), foundational mechanistic paper replicated by subsequent work","pmids":["11748237"],"is_preprint":false},{"year":2006,"finding":"Ndfip1 promotes the function of the E3 ubiquitin ligase Itch to ubiquitinate and reduce the half-life of JunB in T cells; absence of Ndfip1 leads to JunB accumulation, Th2 cytokine production, and inflammatory disease. T cell activation promotes Ndfip1 expression and its physical association with Itch.","method":"Ndfip1 knockout mouse, co-immunoprecipitation, JunB half-life measurement, T cell functional assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with defined cellular phenotype, co-IP confirming physical interaction, biochemical half-life assay, replicated by multiple subsequent studies","pmids":["17137798"],"is_preprint":false},{"year":2008,"finding":"Ndfip1 is sorted into exosomes and increases exosome secretion from cells; Ndfip1 expression recruits Nedd4, Nedd4-2, and Itch (which are otherwise absent) into exosomes, providing a mechanism for cargo-mediated removal of Nedd4 family proteins via the multivesicular body/exosome pathway.","method":"Exosome isolation and characterization, transfection overexpression, primary neuron exosome analysis, Western blot","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct exosome fractionation with functional consequence, single lab, two orthogonal approaches (transfected cells and primary neurons)","pmids":["18819914"],"is_preprint":false},{"year":2009,"finding":"Ndfip1 acts as an adaptor that binds DMT1 in response to iron or cobalt exposure in human neurons, recruits the E3 ligase Nedd4-2 to ubiquitinate DMT1, leading to DMT1 degradation, reduced metal entry, and protection from metal toxicity. Ndfip1 knockout mice accumulate iron in neurons.","method":"Co-immunoprecipitation, shRNAi knockdown, overexpression, ubiquitination assay, metal toxicity assay, Ndfip1−/− mouse brain analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, loss-of-function (shRNA and KO mouse), gain-of-function, ubiquitination assay, in vivo validation across multiple systems","pmids":["19706893"],"is_preprint":false},{"year":2010,"finding":"Ndfip1 and Ndfip2 associate with the EGF receptor and PTEN and control ubiquitination and protein abundance of PTEN, c-Cbl, and Src family kinases; depletion of Ndfip1 inhibits Akt activation in EGF-stimulated HeLa cells, stimulates Jnk activation, and enhances cell multiplication. Ndfip2 (but not Ndfip1) is phosphorylated by Src/Lyn and can scaffold Src phosphorylation of Ndfip1.","method":"siRNA depletion, co-immunoprecipitation, ubiquitination assay, EGF signaling pathway analysis, cell proliferation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, siRNA depletion with defined signaling readouts, single lab","pmids":["20534535"],"is_preprint":false},{"year":2010,"finding":"Ndfip1 knockout mice fed a low-iron diet show significantly higher DMT1 expression and activity in duodenal enterocytes, elevated serum iron and transferrin saturation, and increased liver/spleen iron stores, demonstrating that Ndfip1 is a critical in vivo regulator of DMT1 and systemic iron homeostasis.","method":"Ndfip1−/− mouse model, intestinal iron absorption assay, serum iron measurement, tissue iron quantification, Ndfip1−/−/Rag1−/− immunodeficient mice","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with defined molecular phenotype, epistasis with Rag1 KO to separate immune from iron phenotype, multiple orthogonal readouts","pmids":["20959604"],"is_preprint":false},{"year":2011,"finding":"TGF-β signaling transiently induces Ndfip1 expression during iTreg differentiation; once expressed, Ndfip1 promotes Itch-mediated ubiquitination and degradation of JunB, preventing IL-4 production and permitting sustained Foxp3 expression and iTreg cell differentiation.","method":"Ndfip1−/− T cell in vitro differentiation assays, cytokine measurement, JunB protein level analysis, TGF-β stimulation experiments","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined molecular mechanism (JunB accumulation), upstream signal identified (TGF-β), functional rescue experiments, high-impact journal","pmids":["22080920"],"is_preprint":false},{"year":2012,"finding":"Ndfip1 is required for the translocation of cytoplasmic Pten into neuronal nuclei after cerebral ischemia; Ndfip1 binds Pten, enhances its ubiquitination by Nedd4 E3 ligases, and increases the rate of Pten nuclear import as measured by FRAP. Ndfip1-deficient neurons fail to import Pten and suffer larger infarcts with suppressed pAkt activation.","method":"Ndfip1−/− mouse ischemia model, co-immunoprecipitation, ubiquitination assay, FRAP live imaging, infarct size measurement, pAkt Western blot","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — FRAP live imaging with functional validation, KO mouse with in vivo phenotype, co-IP and ubiquitination assay, multiple orthogonal methods in one study","pmids":["22213801"],"is_preprint":false},{"year":2012,"finding":"Ndfip1 negatively regulates RIG-I-dependent antiviral signaling by enhancing Smurf1 self-ubiquitination and facilitating Smurf1 interaction with MAVS, promoting K48-linked ubiquitination and proteasomal degradation of MAVS. Ndfip1 knockdown elevates MAVS levels and amplifies IFN-β and NF-κB signaling.","method":"Overexpression, siRNA knockdown, co-immunoprecipitation, luciferase reporter assays, IRF-3 phosphorylation assay, ubiquitination assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, gain- and loss-of-function with defined molecular readouts, single lab","pmids":["23087404"],"is_preprint":false},{"year":2012,"finding":"Endogenous Ndfip1 binds Nedd4-2 in the brain (with or without ischemia), as confirmed by immunoprecipitation. Ischemia-induced Itch upregulation promotes neuronal survival independently of Ndfip1.","method":"Co-immunoprecipitation from brain tissue, immunohistochemistry, ischemia model in rats","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous Co-IP from brain tissue confirmed interaction, in vivo ischemia model with defined molecular readouts, single lab","pmids":["22417925"],"is_preprint":false},{"year":2014,"finding":"Rab5 GTPase cooperates with Ndfip1 in Pten ubiquitination and nuclear trafficking; Rab5 colocalizes with Pten on endosomes, dominant-negative Rab5 reduces Pten ubiquitination and nuclear import, and Ndfip1 deletion abrogates nuclear trafficking of ubiquitinated Pten even in the presence of Rab5.","method":"Bimolecular fluorescence complementation (BiFC) for protein interaction imaging, dominant-negative Rab5 expression, Ndfip1 genomic deletion, co-immunoprecipitation, ubiquitination assay","journal":"Traffic","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BiFC live imaging, epistasis between Rab5 and Ndfip1, multiple assays, single lab","pmids":["24798731"],"is_preprint":false},{"year":2015,"finding":"Ndfip1 mediates ubiquitination of BRAT1 (a BRCA1-associated ATM activator), which is required for BRAT1 nuclear translocation during the DNA damage response. Without Ndfip1, BRAT1 fails to enter the nucleus and ATM phosphorylation is not maintained. Genotoxic stress increases both Ndfip1 and phospho-ATM levels.","method":"Co-immunoprecipitation, overexpression/knockdown, nuclear fractionation, genotoxic stress assays, brain injury model immunostaining","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, loss-of-function with defined nuclear trafficking phenotype, in vivo brain injury model, single lab","pmids":["25631046"],"is_preprint":false},{"year":2015,"finding":"Ndfip1 controls Pten nuclear compartmentalization during the cell cycle; Ndfip1 deletion abolishes nuclear Pten without affecting cytoplasmic Pten-PI3K/Akt activity, leading to dysregulated Plk1 and cyclin D1 levels and increased cell proliferation. Transgenic Ndfip1 overexpression in the developing brain increases nuclear Pten, lengthens neuronal progenitor cell cycle, and causes microencephaly.","method":"Ndfip1 genetic deletion, subcellular fractionation, cell proliferation assays, brain transgenic overexpression, cyclin D1/Plk1 Western blot","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO and transgenic overexpression with defined molecular and in vivo phenotypes, single lab","pmids":["25801959"],"is_preprint":false},{"year":2015,"finding":"Ndfip1 enhances Itch E3 ligase activity by recruiting the E2 ubiquitin-conjugating enzyme UbcH7 to Itch; the N-terminal region of Ndfip1 binds UbcH7 while its PY motif binds Itch, making Ndfip1 a scaffold/adaptor bridging E2 and E3. Ndfip1 facilitates Itch-mediated TAK1 ubiquitination and restrains proinflammatory cytokine production in airway inflammation.","method":"Co-immunoprecipitation, Ndfip1 mutant rescue experiments, ubiquitination assay, Ndfip1−/− and Itch−/− mouse airway inflammation model","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-mapping by mutant rescue, co-IP of endogenous proteins, functional rescue with full-length vs. mutant Ndfip1, KO mouse validation, multiple orthogonal methods","pmids":["25632008"],"is_preprint":false},{"year":2016,"finding":"Nedd4-2 and Ndfip1 limit IgE/FcεRI-mediated mast cell activation by ubiquitinating phosphorylated Syk tyrosine kinase, thereby attenuating downstream FcεRI signalosome activity. Loss of Nedd4-2 or Ndfip1 in mast cells results in exacerbated and prolonged IgE-mediated cutaneous anaphylaxis in vivo.","method":"Nedd4-2 and Ndfip1 knockout mast cells, ubiquitination assay for Syk, in vivo cutaneous anaphylaxis model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO cells and mice, direct substrate ubiquitination demonstrated, in vivo phenotype, high-impact journal","pmids":["27786273"],"is_preprint":false},{"year":2017,"finding":"Ndfip1 restricts mTORC1 signaling and glycolysis in Treg cells; Ndfip1-deficient Tregs show elevated mTORC1 activity and glycolysis. Ndfip1 restricts IL-4 production and mTORC1 signaling via distinct mechanisms (IL-4 deficiency does not prevent mTORC1 elevation). Ndfip1 deletion in Tregs causes autoinflammatory disease with loss of Foxp3 expression.","method":"Treg-specific Ndfip1 conditional knockout, metabolic profiling (Seahorse), proteomics, mTORC1 signaling assays, Foxp3 lineage tracing","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with cell-type-specific phenotype, metabolic profiling and proteomics as orthogonal methods, epistasis (IL-4 independence), high-impact journal","pmids":["28580955"],"is_preprint":false},{"year":2017,"finding":"Ndfip1 promotes degradation of RORγT in Th17 cells; Ndfip1-deficient Th17 cells accumulate RORγT and have increased frequency, enhanced IL-17 production, and greater colitis-inducing capacity in vivo.","method":"Ndfip1−/− mouse, Th17 cell differentiation assays, RORγT protein level analysis, in vivo transfer colitis model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined molecular (RORγT accumulation) and functional (colitis) phenotype, single lab","pmids":["28051111"],"is_preprint":false},{"year":2022,"finding":"NDFIP1 is required for hepcidin-induced ubiquitination and degradation of ferroportin (the iron export channel); siRNA depletion of NDFIP1 prevents BMP6- and exogenous hepcidin-induced ferroportin degradation in HepG2 cells, and AAV-mediated silencing of Ndfip1 in mouse liver increases hepatic ferroportin and circulating iron.","method":"siRNA screen of 88 ubiquitin pathway components, FPN-GFP inducible reporter cell line, AAV-mediated in vivo silencing, serum iron measurement","journal":"Haematologica","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic siRNA screen with functional readout, in vivo AAV silencing, two orthogonal systems (cell and mouse), single lab","pmids":["34320783"],"is_preprint":false},{"year":2023,"finding":"NDFIP1 limits cellular TAZ (WWTR1) accumulation by packaging it into exosomes; NDFIP1 interacts with TAZ via WW-domain recognition, and NDFIP1 knockout leads to TAZ accumulation without changes in mRNA or degradation rate. NDFIP1-mediated TAZ exosomal export suppresses NSCLC cell proliferation in vitro and in vivo.","method":"Co-immunoprecipitation, exosome isolation, NDFIP1 knockout, cell proliferation assays, xenograft mouse model","journal":"Protein & cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, KO with defined molecular readout (TAZ accumulation without mRNA change), in vivo xenograft, single lab","pmids":["36929005"],"is_preprint":false},{"year":2024,"finding":"NDFIP1 directly interacts with the TRESK (KCNK18) background potassium channel and negatively regulates its current; coexpression of NDFIP1 abolishes TRESK current in Xenopus oocytes, dependent on intact PPxY motifs (required for Nedd4 interaction) and partially reversed by dominant-negative Nedd4. NDFIP1 coexpression induces ubiquitination of TRESK.","method":"Xenopus oocyte two-electrode voltage clamp, co-immunoprecipitation, PPxY motif mutagenesis, dominant-negative Nedd4 overexpression, ubiquitination assay","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional electrophysiology with mutagenesis and biochemical validation, single lab, multiple orthogonal methods","pmids":["39201565"],"is_preprint":false},{"year":2013,"finding":"Ndfip1 is required for the normal development of pyramidal neuron dendrites and dendritic spines in the neocortex; conditional knockout neurons show stunted dendritic arbors, fewer spines, reduced postsynaptic density proteins (Arc, PSD-95), and altered PI3K/Akt signaling.","method":"Conditional neuron-specific Ndfip1 knockout mouse, electron microscopy, dendritic morphology analysis, PSD fractionation, Western blot for signaling components","journal":"Cerebral cortex","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined structural and biochemical phenotypes, single lab","pmids":["23897647"],"is_preprint":false},{"year":2023,"finding":"Ndfip1 acts as a negative regulator of spatial memory formation; spatial training decreases Ndfip1 expression and reduces its association with Nedd4-1, leading to decreased ubiquitination of Beclin 1 and PTEN in the hippocampus and increased levels of both proteins. Ndfip1 conditional heterozygous mice show enhanced spatial memory.","method":"Co-immunoprecipitation, ubiquitination assay in hippocampal tissue, conditional heterozygous and KO mice, water maze behavioral testing","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous Co-IP with ubiquitination assay, genetic mouse models with behavioral phenotype, single lab","pmids":["37023120"],"is_preprint":false}],"current_model":"NDFIP1 is an endosomal/Golgi membrane-anchored adaptor protein that activates multiple HECT-type Nedd4-family E3 ubiquitin ligases (including Nedd4-1, Nedd4-2, and Itch) by bridging them to substrates and recruiting E2 enzymes (e.g., UbcH7); through this mechanism it drives ubiquitination and degradation or endosomal/nuclear trafficking of diverse substrates including DMT1 (regulating iron homeostasis), PTEN (promoting nuclear import to control cell proliferation and survival), JunB (limiting Th2/Th17 cytokine production), Syk (dampening mast cell activation), MAVS (restraining antiviral signaling), BRAT1 (enabling ATM activation after DNA damage), ferroportin (controlling iron export), TRESK potassium channel, and TAZ (via exosomal export); it also facilitates loading of Nedd4-family ligases into exosomes for intercellular removal."},"narrative":{"mechanistic_narrative":"NDFIP1 is a Golgi/endosomal membrane-anchored adaptor that activates HECT-type Nedd4-family E3 ubiquitin ligases, coupling them to substrates to control protein abundance and subcellular trafficking across diverse physiological settings [PMID:11748237, PMID:19706893]. It binds Nedd4-family WW domains through two N-terminal PPxY motifs and is itself ubiquitinated; mechanistically it functions as a scaffold that bridges substrate to ligase and additionally recruits the E2 conjugating enzyme UbcH7 to Itch via its N-terminal region, thereby enhancing ligase activity [PMID:11748237, PMID:25632008]. Through this adaptor activity NDFIP1 governs iron homeostasis at multiple nodes—directing Nedd4-2-mediated ubiquitination and degradation of the metal importer DMT1 to limit neuronal metal toxicity and to set systemic iron absorption, and enabling hepcidin-induced degradation of the iron exporter ferroportin [PMID:19706893, PMID:20959604, PMID:34320783]. In the immune system it restrains inflammation by promoting Itch-dependent degradation of JunB and RORγT and Itch-mediated TAK1 ubiquitination in T cells, by limiting mTORC1 activity and glycolysis to sustain Treg Foxp3 expression, and by directing Nedd4-2-mediated ubiquitination of phosphorylated Syk to dampen mast cell FcεRI signaling [PMID:17137798, PMID:22080920, PMID:25632008, PMID:28580955, PMID:28051111, PMID:27786273]. NDFIP1 also controls signaling protein localization, driving ubiquitination-dependent nuclear import of PTEN (in cooperation with Rab5) to regulate proliferation and ischemic neuronal survival, and of BRAT1 to maintain ATM activation after DNA damage [PMID:22213801, PMID:24798731, PMID:25801959, PMID:25631046]. Beyond degradative ubiquitination, NDFIP1 loads Nedd4-family ligases and substrates such as TAZ into exosomes for intercellular removal [PMID:18819914, PMID:36929005], and it negatively regulates the antiviral adaptor MAVS, the TRESK potassium channel, neuronal dendritic development, and spatial memory [PMID:23087404, PMID:39201565, PMID:23897647, PMID:37023120].","teleology":[{"year":2001,"claim":"Established NDFIP1 as a Golgi-localized adaptor that physically engages Nedd4-family ligases, defining its core molecular role in ubiquitin-dependent sorting.","evidence":"WW-domain binding/Co-IP, fractionation, and overexpression morphology in mammalian cells","pmids":["11748237"],"confidence":"High","gaps":["Substrates engaged through this adaptor not yet identified","Physiological consequence beyond Golgi disruption unknown"]},{"year":2006,"claim":"Defined a physiological substrate axis by showing NDFIP1 enables Itch-mediated JunB degradation in T cells, linking the adaptor to immune homeostasis.","evidence":"Ndfip1 knockout mouse, Co-IP, and JunB half-life assays with T cell functional readouts","pmids":["17137798"],"confidence":"High","gaps":["Whether NDFIP1 acts on other Itch substrates not addressed","Mechanism coupling T cell activation to Ndfip1 induction unclear"]},{"year":2008,"claim":"Revealed a non-degradative output: NDFIP1 routes Nedd4-family ligases into exosomes, suggesting cargo-mediated intercellular removal of ligases.","evidence":"Exosome fractionation from transfected cells and primary neurons with Western blot","pmids":["18819914"],"confidence":"Medium","gaps":["Functional consequence of ligase removal in recipient cells not shown","Single lab, ubiquitination dependence not dissected"]},{"year":2009,"claim":"Identified DMT1 as a NDFIP1/Nedd4-2 substrate, establishing a metal-sensing adaptor function that protects neurons from iron/cobalt toxicity.","evidence":"Reciprocal Co-IP, shRNA/overexpression, ubiquitination assay, and Ndfip1-/- mouse brain analysis","pmids":["19706893"],"confidence":"High","gaps":["How metal exposure triggers DMT1-NDFIP1 binding mechanistically unclear","Other metal transporters not tested"]},{"year":2010,"claim":"Extended NDFIP1 substrate scope to EGFR-pathway components (PTEN, c-Cbl, Src family) and connected it to Akt/Jnk signaling and proliferation control.","evidence":"siRNA depletion, Co-IP, ubiquitination assay, and EGF signaling/proliferation readouts in HeLa","pmids":["20534535"],"confidence":"Medium","gaps":["Direct vs indirect effects on each substrate not fully separated","Single lab"]},{"year":2010,"claim":"Demonstrated NDFIP1 as a critical in vivo regulator of systemic iron via duodenal DMT1, separating iron from immune phenotypes.","evidence":"Ndfip1-/- and Ndfip1-/-/Rag1-/- mice, iron absorption and serum/tissue iron measurements","pmids":["20959604"],"confidence":"High","gaps":["Ligase identity in enterocytes not confirmed in this study","Tissue-specific contributions not dissected"]},{"year":2012,"claim":"Showed NDFIP1 controls PTEN nuclear import through ubiquitination, defining a trafficking (rather than purely degradative) function with consequences for ischemic neuronal survival.","evidence":"Ndfip1-/- ischemia model, Co-IP, ubiquitination assay, FRAP live imaging, infarct measurement","pmids":["22213801"],"confidence":"High","gaps":["Nuclear import machinery downstream of ubiquitination unresolved","Which Nedd4 ligase predominates not pinned down"]},{"year":2012,"claim":"Broadened NDFIP1's regulatory range to innate antiviral signaling by linking it to Smurf1-mediated MAVS degradation.","evidence":"Overexpression/siRNA, Co-IP, luciferase reporters, IRF-3 phosphorylation and ubiquitination assays","pmids":["23087404"],"confidence":"Medium","gaps":["In vivo viral infection phenotype not shown","Single lab; Smurf1 vs other ligase specificity unclear"]},{"year":2012,"claim":"Confirmed endogenous NDFIP1-Nedd4-2 association in brain and distinguished an Ndfip1-independent Itch survival pathway during ischemia.","evidence":"Endogenous Co-IP from brain tissue, immunohistochemistry, rat ischemia model","pmids":["22417925"],"confidence":"Medium","gaps":["Functional consequence of the brain Nedd4-2 interaction not defined","Single lab"]},{"year":2013,"claim":"Established a developmental role: NDFIP1 is required for normal cortical dendrite and spine maturation linked to PI3K/Akt signaling.","evidence":"Neuron-specific conditional KO, EM, dendritic morphology, PSD fractionation, Western blot","pmids":["23897647"],"confidence":"Medium","gaps":["Specific substrate driving the dendritic phenotype not identified","Single lab"]},{"year":2014,"claim":"Placed PTEN ubiquitination/nuclear trafficking on the endosomal Rab5 pathway, integrating NDFIP1 with vesicular trafficking machinery.","evidence":"BiFC imaging, dominant-negative Rab5, Ndfip1 deletion, Co-IP, ubiquitination assay","pmids":["24798731"],"confidence":"Medium","gaps":["How Rab5 endosomes deliver PTEN to the nucleus mechanistically unclear","Single lab"]},{"year":2015,"claim":"Resolved the biochemical basis of ligase activation by mapping NDFIP1 as an E2 (UbcH7)-recruiting scaffold for Itch, with functional impact on airway inflammation.","evidence":"Co-IP, Ndfip1 domain-mutant rescue, ubiquitination assay, Ndfip1-/- and Itch-/- airway models","pmids":["25632008"],"confidence":"High","gaps":["Whether UbcH7 recruitment generalizes to other Nedd4-family ligases not tested","TAK1 ubiquitin linkage type not fully characterized"]},{"year":2015,"claim":"Showed NDFIP1 directs BRAT1 nuclear translocation to sustain ATM activation, extending its trafficking function to the DNA damage response.","evidence":"Co-IP, knockdown/overexpression, nuclear fractionation, genotoxic stress and brain injury models","pmids":["25631046"],"confidence":"Medium","gaps":["Ubiquitin linkage and ligase identity for BRAT1 not defined","Single lab"]},{"year":2015,"claim":"Demonstrated NDFIP1 sets nuclear PTEN levels to control cell-cycle regulators and proliferation, with dosage effects on brain size.","evidence":"Ndfip1 KO and brain transgenic overexpression, fractionation, proliferation assays, cyclin D1/Plk1 blots","pmids":["25801959"],"confidence":"Medium","gaps":["Mechanism linking nuclear PTEN to Plk1/cyclin D1 not detailed","Single lab"]},{"year":2016,"claim":"Identified phospho-Syk as a Nedd4-2/NDFIP1 substrate that dampens mast cell FcεRI signaling, defining an allergic-response regulatory node.","evidence":"Nedd4-2 and Ndfip1 KO mast cells, Syk ubiquitination assay, in vivo cutaneous anaphylaxis","pmids":["27786273"],"confidence":"High","gaps":["Whether NDFIP1 acts on other signalosome components unclear"]},{"year":2017,"claim":"Showed NDFIP1 restrains Treg mTORC1 and glycolysis via a mechanism distinct from its IL-4 control, linking the adaptor to immunometabolism and Foxp3 stability.","evidence":"Treg-specific conditional KO, Seahorse metabolic profiling, proteomics, mTORC1 assays, lineage tracing","pmids":["28580955"],"confidence":"High","gaps":["Substrate mediating mTORC1 restraint not identified","Single lab"]},{"year":2017,"claim":"Added RORγT as a NDFIP1-controlled substrate limiting Th17 expansion and colitogenic potential.","evidence":"Ndfip1-/- mouse, Th17 differentiation, RORγT level analysis, transfer colitis model","pmids":["28051111"],"confidence":"Medium","gaps":["Direct ubiquitination of RORγT not demonstrated","Ligase identity not defined"]},{"year":2023,"claim":"Revealed a degradation-independent output: NDFIP1 packages TAZ into exosomes via WW-domain recognition to limit TAZ accumulation and tumor cell proliferation.","evidence":"Co-IP, exosome isolation, NDFIP1 KO, proliferation assays, xenograft model","pmids":["36929005"],"confidence":"Medium","gaps":["Whether ubiquitination is required for TAZ exosomal loading unclear","Single lab"]},{"year":2023,"claim":"Defined NDFIP1 as a negative regulator of spatial memory through Nedd4-1-dependent ubiquitination of Beclin 1 and PTEN in hippocampus.","evidence":"Endogenous Co-IP, hippocampal ubiquitination assays, conditional het/KO mice, water maze","pmids":["37023120"],"confidence":"Medium","gaps":["Causal substrate driving memory phenotype not isolated","Single lab"]},{"year":2024,"claim":"Extended NDFIP1's targets to ion channels, showing PPxY/Nedd4-dependent ubiquitination and suppression of the TRESK potassium current.","evidence":"Xenopus oocyte voltage clamp, Co-IP, PPxY mutagenesis, dominant-negative Nedd4, ubiquitination assay","pmids":["39201565"],"confidence":"Medium","gaps":["Physiological relevance in native neurons not shown","Single lab; heterologous system"]},{"year":null,"claim":"How NDFIP1 selects among its many Nedd4-family ligases and dictates whether a substrate is degraded, trafficked to the nucleus, or exported via exosomes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model for substrate-versus-fate specificity","No structural data on the E2-adaptor-E3 ternary complex","Determinants routing cargo to exosomes vs proteasome unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[13,1]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,14,19]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[10]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[2,18]}],"pathway":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,13,14]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,13]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,6,14,15,16]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,5,17]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[7,10,11]}],"complexes":[],"partners":["NEDD4","NEDD4L","ITCH","PTEN","DMT1","SMURF1","UBE2L3","WWTR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BT67","full_name":"NEDD4 family-interacting protein 1","aliases":["Breast cancer-associated protein SGA-1M","NEDD4 WW domain-binding protein 5","Putative MAPK-activating protein PM13","Putative NF-kappa-B-activating protein 164","Putative NFKB and MAPK-activating protein"],"length_aa":221,"mass_kda":24.9,"function":"Activates HECT domain-containing E3 ubiquitin-protein ligases, including NEDD4 and ITCH, and consequently modulates the stability of their targets. As a result, controls many cellular processes. Prevents chronic T-helper cell-mediated inflammation by activating ITCH and thus controlling JUNB degradation (By similarity). Promotes pancreatic beta cell death through degradation of JUNB and inhibition of the unfolded protein response, leading to reduction of insulin secretion (PubMed:26319551). Restricts the production of pro-inflammatory cytokines in effector Th17 T-cells by promoting ITCH-mediated ubiquitination and degradation of RORC (By similarity). Together with NDFIP2, limits the cytokine signaling and expansion of effector Th2 T-cells by promoting degradation of JAK1, probably by ITCH- and NEDD4L-mediated ubiquitination (By similarity). Regulates peripheral T-cell tolerance to self and foreign antigens, forcing the exit of naive CD4+ T-cells from the cell cycle before they become effector T-cells (By similarity). Negatively regulates RLR-mediated antiviral response by promoting SMURF1-mediated ubiquitination and subsequent degradation of MAVS (PubMed:23087404). Negatively regulates KCNH2 potassium channel activity by decreasing its cell-surface expression and interfering with channel maturation through recruitment of NEDD4L to the Golgi apparatus where it mediates KCNH2 degradation (PubMed:26363003). In cortical neurons, mediates the ubiquitination of the divalent metal transporter SLC11A2/DMT1 by NEDD4L, leading to its down-regulation and protection of the cells from cobalt and iron toxicity (PubMed:19706893). Important for normal development of dendrites and dendritic spines in cortex (By similarity). Enhances the ubiquitination of BRAT1 mediated by: NEDD4, NEDD4L and ITCH and is required for the nuclear localization of ubiquitinated BRAT1 (PubMed:25631046). Enhances the ITCH-mediated ubiquitination of MAP3K7 by recruiting E2 ubiquitin-conjugating enzyme UBE2L3 to ITCH (By similarity). Modulates EGFR signaling through multiple pathways. In particular, may regulate the ratio of AKT1-to-MAPK8 signaling in response to EGF, acting on AKT1 probably through PTEN destabilization and on MAPK8 through ITCH-dependent MAP2K4 inactivation. As a result, may control cell growth rate (PubMed:20534535). Inhibits cell proliferation by promoting PTEN nuclear localization and changing its signaling specificity (PubMed:25801959)","subcellular_location":"Endosome membrane; Golgi apparatus membrane; Synapse, synaptosome; Cell projection, dendrite; Secreted","url":"https://www.uniprot.org/uniprotkb/Q9BT67/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NDFIP1","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/NDFIP1","total_profiled":1310},"omim":[{"mim_id":"612051","title":"BRAIN-EXPRESSED, ASSOCIATED WITH NEDD4, 1; BEAN1","url":"https://www.omim.org/entry/612051"},{"mim_id":"612050","title":"NEDD4 FAMILY-INTERACTING PROTEIN 1; NDFIP1","url":"https://www.omim.org/entry/612050"},{"mim_id":"600523","title":"SOLUTE CARRIER FAMILY 11 (PROTON-COUPLED DIVALENT METAL ION TRANSPORTER), MEMBER 2; SLC11A2","url":"https://www.omim.org/entry/600523"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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\"Co-immunoprecipitation/WW-domain binding assays, subcellular fractionation, immunofluorescence, overexpression morphological analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays with multiple Nedd4 family members, direct localization with functional consequence (Golgi disruption), foundational mechanistic paper replicated by subsequent work\",\n      \"pmids\": [\"11748237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Ndfip1 promotes the function of the E3 ubiquitin ligase Itch to ubiquitinate and reduce the half-life of JunB in T cells; absence of Ndfip1 leads to JunB accumulation, Th2 cytokine production, and inflammatory disease. T cell activation promotes Ndfip1 expression and its physical association with Itch.\",\n      \"method\": \"Ndfip1 knockout mouse, co-immunoprecipitation, JunB half-life measurement, T cell functional assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with defined cellular phenotype, co-IP confirming physical interaction, biochemical half-life assay, replicated by multiple subsequent studies\",\n      \"pmids\": [\"17137798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ndfip1 is sorted into exosomes and increases exosome secretion from cells; Ndfip1 expression recruits Nedd4, Nedd4-2, and Itch (which are otherwise absent) into exosomes, providing a mechanism for cargo-mediated removal of Nedd4 family proteins via the multivesicular body/exosome pathway.\",\n      \"method\": \"Exosome isolation and characterization, transfection overexpression, primary neuron exosome analysis, Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct exosome fractionation with functional consequence, single lab, two orthogonal approaches (transfected cells and primary neurons)\",\n      \"pmids\": [\"18819914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Ndfip1 acts as an adaptor that binds DMT1 in response to iron or cobalt exposure in human neurons, recruits the E3 ligase Nedd4-2 to ubiquitinate DMT1, leading to DMT1 degradation, reduced metal entry, and protection from metal toxicity. Ndfip1 knockout mice accumulate iron in neurons.\",\n      \"method\": \"Co-immunoprecipitation, shRNAi knockdown, overexpression, ubiquitination assay, metal toxicity assay, Ndfip1−/− mouse brain analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, loss-of-function (shRNA and KO mouse), gain-of-function, ubiquitination assay, in vivo validation across multiple systems\",\n      \"pmids\": [\"19706893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Ndfip1 and Ndfip2 associate with the EGF receptor and PTEN and control ubiquitination and protein abundance of PTEN, c-Cbl, and Src family kinases; depletion of Ndfip1 inhibits Akt activation in EGF-stimulated HeLa cells, stimulates Jnk activation, and enhances cell multiplication. Ndfip2 (but not Ndfip1) is phosphorylated by Src/Lyn and can scaffold Src phosphorylation of Ndfip1.\",\n      \"method\": \"siRNA depletion, co-immunoprecipitation, ubiquitination assay, EGF signaling pathway analysis, cell proliferation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, siRNA depletion with defined signaling readouts, single lab\",\n      \"pmids\": [\"20534535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Ndfip1 knockout mice fed a low-iron diet show significantly higher DMT1 expression and activity in duodenal enterocytes, elevated serum iron and transferrin saturation, and increased liver/spleen iron stores, demonstrating that Ndfip1 is a critical in vivo regulator of DMT1 and systemic iron homeostasis.\",\n      \"method\": \"Ndfip1−/− mouse model, intestinal iron absorption assay, serum iron measurement, tissue iron quantification, Ndfip1−/−/Rag1−/− immunodeficient mice\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with defined molecular phenotype, epistasis with Rag1 KO to separate immune from iron phenotype, multiple orthogonal readouts\",\n      \"pmids\": [\"20959604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TGF-β signaling transiently induces Ndfip1 expression during iTreg differentiation; once expressed, Ndfip1 promotes Itch-mediated ubiquitination and degradation of JunB, preventing IL-4 production and permitting sustained Foxp3 expression and iTreg cell differentiation.\",\n      \"method\": \"Ndfip1−/− T cell in vitro differentiation assays, cytokine measurement, JunB protein level analysis, TGF-β stimulation experiments\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined molecular mechanism (JunB accumulation), upstream signal identified (TGF-β), functional rescue experiments, high-impact journal\",\n      \"pmids\": [\"22080920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ndfip1 is required for the translocation of cytoplasmic Pten into neuronal nuclei after cerebral ischemia; Ndfip1 binds Pten, enhances its ubiquitination by Nedd4 E3 ligases, and increases the rate of Pten nuclear import as measured by FRAP. Ndfip1-deficient neurons fail to import Pten and suffer larger infarcts with suppressed pAkt activation.\",\n      \"method\": \"Ndfip1−/− mouse ischemia model, co-immunoprecipitation, ubiquitination assay, FRAP live imaging, infarct size measurement, pAkt Western blot\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — FRAP live imaging with functional validation, KO mouse with in vivo phenotype, co-IP and ubiquitination assay, multiple orthogonal methods in one study\",\n      \"pmids\": [\"22213801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ndfip1 negatively regulates RIG-I-dependent antiviral signaling by enhancing Smurf1 self-ubiquitination and facilitating Smurf1 interaction with MAVS, promoting K48-linked ubiquitination and proteasomal degradation of MAVS. Ndfip1 knockdown elevates MAVS levels and amplifies IFN-β and NF-κB signaling.\",\n      \"method\": \"Overexpression, siRNA knockdown, co-immunoprecipitation, luciferase reporter assays, IRF-3 phosphorylation assay, ubiquitination assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, gain- and loss-of-function with defined molecular readouts, single lab\",\n      \"pmids\": [\"23087404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Endogenous Ndfip1 binds Nedd4-2 in the brain (with or without ischemia), as confirmed by immunoprecipitation. Ischemia-induced Itch upregulation promotes neuronal survival independently of Ndfip1.\",\n      \"method\": \"Co-immunoprecipitation from brain tissue, immunohistochemistry, ischemia model in rats\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous Co-IP from brain tissue confirmed interaction, in vivo ischemia model with defined molecular readouts, single lab\",\n      \"pmids\": [\"22417925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Rab5 GTPase cooperates with Ndfip1 in Pten ubiquitination and nuclear trafficking; Rab5 colocalizes with Pten on endosomes, dominant-negative Rab5 reduces Pten ubiquitination and nuclear import, and Ndfip1 deletion abrogates nuclear trafficking of ubiquitinated Pten even in the presence of Rab5.\",\n      \"method\": \"Bimolecular fluorescence complementation (BiFC) for protein interaction imaging, dominant-negative Rab5 expression, Ndfip1 genomic deletion, co-immunoprecipitation, ubiquitination assay\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BiFC live imaging, epistasis between Rab5 and Ndfip1, multiple assays, single lab\",\n      \"pmids\": [\"24798731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ndfip1 mediates ubiquitination of BRAT1 (a BRCA1-associated ATM activator), which is required for BRAT1 nuclear translocation during the DNA damage response. Without Ndfip1, BRAT1 fails to enter the nucleus and ATM phosphorylation is not maintained. Genotoxic stress increases both Ndfip1 and phospho-ATM levels.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown, nuclear fractionation, genotoxic stress assays, brain injury model immunostaining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, loss-of-function with defined nuclear trafficking phenotype, in vivo brain injury model, single lab\",\n      \"pmids\": [\"25631046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ndfip1 controls Pten nuclear compartmentalization during the cell cycle; Ndfip1 deletion abolishes nuclear Pten without affecting cytoplasmic Pten-PI3K/Akt activity, leading to dysregulated Plk1 and cyclin D1 levels and increased cell proliferation. Transgenic Ndfip1 overexpression in the developing brain increases nuclear Pten, lengthens neuronal progenitor cell cycle, and causes microencephaly.\",\n      \"method\": \"Ndfip1 genetic deletion, subcellular fractionation, cell proliferation assays, brain transgenic overexpression, cyclin D1/Plk1 Western blot\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO and transgenic overexpression with defined molecular and in vivo phenotypes, single lab\",\n      \"pmids\": [\"25801959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ndfip1 enhances Itch E3 ligase activity by recruiting the E2 ubiquitin-conjugating enzyme UbcH7 to Itch; the N-terminal region of Ndfip1 binds UbcH7 while its PY motif binds Itch, making Ndfip1 a scaffold/adaptor bridging E2 and E3. Ndfip1 facilitates Itch-mediated TAK1 ubiquitination and restrains proinflammatory cytokine production in airway inflammation.\",\n      \"method\": \"Co-immunoprecipitation, Ndfip1 mutant rescue experiments, ubiquitination assay, Ndfip1−/− and Itch−/− mouse airway inflammation model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-mapping by mutant rescue, co-IP of endogenous proteins, functional rescue with full-length vs. mutant Ndfip1, KO mouse validation, multiple orthogonal methods\",\n      \"pmids\": [\"25632008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Nedd4-2 and Ndfip1 limit IgE/FcεRI-mediated mast cell activation by ubiquitinating phosphorylated Syk tyrosine kinase, thereby attenuating downstream FcεRI signalosome activity. Loss of Nedd4-2 or Ndfip1 in mast cells results in exacerbated and prolonged IgE-mediated cutaneous anaphylaxis in vivo.\",\n      \"method\": \"Nedd4-2 and Ndfip1 knockout mast cells, ubiquitination assay for Syk, in vivo cutaneous anaphylaxis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO cells and mice, direct substrate ubiquitination demonstrated, in vivo phenotype, high-impact journal\",\n      \"pmids\": [\"27786273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ndfip1 restricts mTORC1 signaling and glycolysis in Treg cells; Ndfip1-deficient Tregs show elevated mTORC1 activity and glycolysis. Ndfip1 restricts IL-4 production and mTORC1 signaling via distinct mechanisms (IL-4 deficiency does not prevent mTORC1 elevation). Ndfip1 deletion in Tregs causes autoinflammatory disease with loss of Foxp3 expression.\",\n      \"method\": \"Treg-specific Ndfip1 conditional knockout, metabolic profiling (Seahorse), proteomics, mTORC1 signaling assays, Foxp3 lineage tracing\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with cell-type-specific phenotype, metabolic profiling and proteomics as orthogonal methods, epistasis (IL-4 independence), high-impact journal\",\n      \"pmids\": [\"28580955\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ndfip1 promotes degradation of RORγT in Th17 cells; Ndfip1-deficient Th17 cells accumulate RORγT and have increased frequency, enhanced IL-17 production, and greater colitis-inducing capacity in vivo.\",\n      \"method\": \"Ndfip1−/− mouse, Th17 cell differentiation assays, RORγT protein level analysis, in vivo transfer colitis model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined molecular (RORγT accumulation) and functional (colitis) phenotype, single lab\",\n      \"pmids\": [\"28051111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NDFIP1 is required for hepcidin-induced ubiquitination and degradation of ferroportin (the iron export channel); siRNA depletion of NDFIP1 prevents BMP6- and exogenous hepcidin-induced ferroportin degradation in HepG2 cells, and AAV-mediated silencing of Ndfip1 in mouse liver increases hepatic ferroportin and circulating iron.\",\n      \"method\": \"siRNA screen of 88 ubiquitin pathway components, FPN-GFP inducible reporter cell line, AAV-mediated in vivo silencing, serum iron measurement\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic siRNA screen with functional readout, in vivo AAV silencing, two orthogonal systems (cell and mouse), single lab\",\n      \"pmids\": [\"34320783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NDFIP1 limits cellular TAZ (WWTR1) accumulation by packaging it into exosomes; NDFIP1 interacts with TAZ via WW-domain recognition, and NDFIP1 knockout leads to TAZ accumulation without changes in mRNA or degradation rate. NDFIP1-mediated TAZ exosomal export suppresses NSCLC cell proliferation in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, exosome isolation, NDFIP1 knockout, cell proliferation assays, xenograft mouse model\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, KO with defined molecular readout (TAZ accumulation without mRNA change), in vivo xenograft, single lab\",\n      \"pmids\": [\"36929005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NDFIP1 directly interacts with the TRESK (KCNK18) background potassium channel and negatively regulates its current; coexpression of NDFIP1 abolishes TRESK current in Xenopus oocytes, dependent on intact PPxY motifs (required for Nedd4 interaction) and partially reversed by dominant-negative Nedd4. NDFIP1 coexpression induces ubiquitination of TRESK.\",\n      \"method\": \"Xenopus oocyte two-electrode voltage clamp, co-immunoprecipitation, PPxY motif mutagenesis, dominant-negative Nedd4 overexpression, ubiquitination assay\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional electrophysiology with mutagenesis and biochemical validation, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39201565\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Ndfip1 is required for the normal development of pyramidal neuron dendrites and dendritic spines in the neocortex; conditional knockout neurons show stunted dendritic arbors, fewer spines, reduced postsynaptic density proteins (Arc, PSD-95), and altered PI3K/Akt signaling.\",\n      \"method\": \"Conditional neuron-specific Ndfip1 knockout mouse, electron microscopy, dendritic morphology analysis, PSD fractionation, Western blot for signaling components\",\n      \"journal\": \"Cerebral cortex\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined structural and biochemical phenotypes, single lab\",\n      \"pmids\": [\"23897647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Ndfip1 acts as a negative regulator of spatial memory formation; spatial training decreases Ndfip1 expression and reduces its association with Nedd4-1, leading to decreased ubiquitination of Beclin 1 and PTEN in the hippocampus and increased levels of both proteins. Ndfip1 conditional heterozygous mice show enhanced spatial memory.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay in hippocampal tissue, conditional heterozygous and KO mice, water maze behavioral testing\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous Co-IP with ubiquitination assay, genetic mouse models with behavioral phenotype, single lab\",\n      \"pmids\": [\"37023120\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NDFIP1 is an endosomal/Golgi membrane-anchored adaptor protein that activates multiple HECT-type Nedd4-family E3 ubiquitin ligases (including Nedd4-1, Nedd4-2, and Itch) by bridging them to substrates and recruiting E2 enzymes (e.g., UbcH7); through this mechanism it drives ubiquitination and degradation or endosomal/nuclear trafficking of diverse substrates including DMT1 (regulating iron homeostasis), PTEN (promoting nuclear import to control cell proliferation and survival), JunB (limiting Th2/Th17 cytokine production), Syk (dampening mast cell activation), MAVS (restraining antiviral signaling), BRAT1 (enabling ATM activation after DNA damage), ferroportin (controlling iron export), TRESK potassium channel, and TAZ (via exosomal export); it also facilitates loading of Nedd4-family ligases into exosomes for intercellular removal.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NDFIP1 is a Golgi/endosomal membrane-anchored adaptor that activates HECT-type Nedd4-family E3 ubiquitin ligases, coupling them to substrates to control protein abundance and subcellular trafficking across diverse physiological settings [#0, #3]. It binds Nedd4-family WW domains through two N-terminal PPxY motifs and is itself ubiquitinated; mechanistically it functions as a scaffold that bridges substrate to ligase and additionally recruits the E2 conjugating enzyme UbcH7 to Itch via its N-terminal region, thereby enhancing ligase activity [#0, #13]. Through this adaptor activity NDFIP1 governs iron homeostasis at multiple nodes—directing Nedd4-2-mediated ubiquitination and degradation of the metal importer DMT1 to limit neuronal metal toxicity and to set systemic iron absorption, and enabling hepcidin-induced degradation of the iron exporter ferroportin [#3, #5, #17]. In the immune system it restrains inflammation by promoting Itch-dependent degradation of JunB and RORγT and Itch-mediated TAK1 ubiquitination in T cells, by limiting mTORC1 activity and glycolysis to sustain Treg Foxp3 expression, and by directing Nedd4-2-mediated ubiquitination of phosphorylated Syk to dampen mast cell FcεRI signaling [#1, #6, #13, #15, #16, #14]. NDFIP1 also controls signaling protein localization, driving ubiquitination-dependent nuclear import of PTEN (in cooperation with Rab5) to regulate proliferation and ischemic neuronal survival, and of BRAT1 to maintain ATM activation after DNA damage [#7, #10, #12, #11]. Beyond degradative ubiquitination, NDFIP1 loads Nedd4-family ligases and substrates such as TAZ into exosomes for intercellular removal [#2, #18], and it negatively regulates the antiviral adaptor MAVS, the TRESK potassium channel, neuronal dendritic development, and spatial memory [#8, #19, #20, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established NDFIP1 as a Golgi-localized adaptor that physically engages Nedd4-family ligases, defining its core molecular role in ubiquitin-dependent sorting.\",\n      \"evidence\": \"WW-domain binding/Co-IP, fractionation, and overexpression morphology in mammalian cells\",\n      \"pmids\": [\"11748237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrates engaged through this adaptor not yet identified\", \"Physiological consequence beyond Golgi disruption unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined a physiological substrate axis by showing NDFIP1 enables Itch-mediated JunB degradation in T cells, linking the adaptor to immune homeostasis.\",\n      \"evidence\": \"Ndfip1 knockout mouse, Co-IP, and JunB half-life assays with T cell functional readouts\",\n      \"pmids\": [\"17137798\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NDFIP1 acts on other Itch substrates not addressed\", \"Mechanism coupling T cell activation to Ndfip1 induction unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed a non-degradative output: NDFIP1 routes Nedd4-family ligases into exosomes, suggesting cargo-mediated intercellular removal of ligases.\",\n      \"evidence\": \"Exosome fractionation from transfected cells and primary neurons with Western blot\",\n      \"pmids\": [\"18819914\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of ligase removal in recipient cells not shown\", \"Single lab, ubiquitination dependence not dissected\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified DMT1 as a NDFIP1/Nedd4-2 substrate, establishing a metal-sensing adaptor function that protects neurons from iron/cobalt toxicity.\",\n      \"evidence\": \"Reciprocal Co-IP, shRNA/overexpression, ubiquitination assay, and Ndfip1-/- mouse brain analysis\",\n      \"pmids\": [\"19706893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How metal exposure triggers DMT1-NDFIP1 binding mechanistically unclear\", \"Other metal transporters not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended NDFIP1 substrate scope to EGFR-pathway components (PTEN, c-Cbl, Src family) and connected it to Akt/Jnk signaling and proliferation control.\",\n      \"evidence\": \"siRNA depletion, Co-IP, ubiquitination assay, and EGF signaling/proliferation readouts in HeLa\",\n      \"pmids\": [\"20534535\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect effects on each substrate not fully separated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated NDFIP1 as a critical in vivo regulator of systemic iron via duodenal DMT1, separating iron from immune phenotypes.\",\n      \"evidence\": \"Ndfip1-/- and Ndfip1-/-/Rag1-/- mice, iron absorption and serum/tissue iron measurements\",\n      \"pmids\": [\"20959604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligase identity in enterocytes not confirmed in this study\", \"Tissue-specific contributions not dissected\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed NDFIP1 controls PTEN nuclear import through ubiquitination, defining a trafficking (rather than purely degradative) function with consequences for ischemic neuronal survival.\",\n      \"evidence\": \"Ndfip1-/- ischemia model, Co-IP, ubiquitination assay, FRAP live imaging, infarct measurement\",\n      \"pmids\": [\"22213801\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear import machinery downstream of ubiquitination unresolved\", \"Which Nedd4 ligase predominates not pinned down\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Broadened NDFIP1's regulatory range to innate antiviral signaling by linking it to Smurf1-mediated MAVS degradation.\",\n      \"evidence\": \"Overexpression/siRNA, Co-IP, luciferase reporters, IRF-3 phosphorylation and ubiquitination assays\",\n      \"pmids\": [\"23087404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo viral infection phenotype not shown\", \"Single lab; Smurf1 vs other ligase specificity unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Confirmed endogenous NDFIP1-Nedd4-2 association in brain and distinguished an Ndfip1-independent Itch survival pathway during ischemia.\",\n      \"evidence\": \"Endogenous Co-IP from brain tissue, immunohistochemistry, rat ischemia model\",\n      \"pmids\": [\"22417925\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the brain Nedd4-2 interaction not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established a developmental role: NDFIP1 is required for normal cortical dendrite and spine maturation linked to PI3K/Akt signaling.\",\n      \"evidence\": \"Neuron-specific conditional KO, EM, dendritic morphology, PSD fractionation, Western blot\",\n      \"pmids\": [\"23897647\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific substrate driving the dendritic phenotype not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed PTEN ubiquitination/nuclear trafficking on the endosomal Rab5 pathway, integrating NDFIP1 with vesicular trafficking machinery.\",\n      \"evidence\": \"BiFC imaging, dominant-negative Rab5, Ndfip1 deletion, Co-IP, ubiquitination assay\",\n      \"pmids\": [\"24798731\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How Rab5 endosomes deliver PTEN to the nucleus mechanistically unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the biochemical basis of ligase activation by mapping NDFIP1 as an E2 (UbcH7)-recruiting scaffold for Itch, with functional impact on airway inflammation.\",\n      \"evidence\": \"Co-IP, Ndfip1 domain-mutant rescue, ubiquitination assay, Ndfip1-/- and Itch-/- airway models\",\n      \"pmids\": [\"25632008\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether UbcH7 recruitment generalizes to other Nedd4-family ligases not tested\", \"TAK1 ubiquitin linkage type not fully characterized\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed NDFIP1 directs BRAT1 nuclear translocation to sustain ATM activation, extending its trafficking function to the DNA damage response.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression, nuclear fractionation, genotoxic stress and brain injury models\",\n      \"pmids\": [\"25631046\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin linkage and ligase identity for BRAT1 not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated NDFIP1 sets nuclear PTEN levels to control cell-cycle regulators and proliferation, with dosage effects on brain size.\",\n      \"evidence\": \"Ndfip1 KO and brain transgenic overexpression, fractionation, proliferation assays, cyclin D1/Plk1 blots\",\n      \"pmids\": [\"25801959\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking nuclear PTEN to Plk1/cyclin D1 not detailed\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified phospho-Syk as a Nedd4-2/NDFIP1 substrate that dampens mast cell FcεRI signaling, defining an allergic-response regulatory node.\",\n      \"evidence\": \"Nedd4-2 and Ndfip1 KO mast cells, Syk ubiquitination assay, in vivo cutaneous anaphylaxis\",\n      \"pmids\": [\"27786273\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NDFIP1 acts on other signalosome components unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed NDFIP1 restrains Treg mTORC1 and glycolysis via a mechanism distinct from its IL-4 control, linking the adaptor to immunometabolism and Foxp3 stability.\",\n      \"evidence\": \"Treg-specific conditional KO, Seahorse metabolic profiling, proteomics, mTORC1 assays, lineage tracing\",\n      \"pmids\": [\"28580955\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate mediating mTORC1 restraint not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Added RORγT as a NDFIP1-controlled substrate limiting Th17 expansion and colitogenic potential.\",\n      \"evidence\": \"Ndfip1-/- mouse, Th17 differentiation, RORγT level analysis, transfer colitis model\",\n      \"pmids\": [\"28051111\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination of RORγT not demonstrated\", \"Ligase identity not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a degradation-independent output: NDFIP1 packages TAZ into exosomes via WW-domain recognition to limit TAZ accumulation and tumor cell proliferation.\",\n      \"evidence\": \"Co-IP, exosome isolation, NDFIP1 KO, proliferation assays, xenograft model\",\n      \"pmids\": [\"36929005\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ubiquitination is required for TAZ exosomal loading unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined NDFIP1 as a negative regulator of spatial memory through Nedd4-1-dependent ubiquitination of Beclin 1 and PTEN in hippocampus.\",\n      \"evidence\": \"Endogenous Co-IP, hippocampal ubiquitination assays, conditional het/KO mice, water maze\",\n      \"pmids\": [\"37023120\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal substrate driving memory phenotype not isolated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended NDFIP1's targets to ion channels, showing PPxY/Nedd4-dependent ubiquitination and suppression of the TRESK potassium current.\",\n      \"evidence\": \"Xenopus oocyte voltage clamp, Co-IP, PPxY mutagenesis, dominant-negative Nedd4, ubiquitination assay\",\n      \"pmids\": [\"39201565\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance in native neurons not shown\", \"Single lab; heterologous system\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NDFIP1 selects among its many Nedd4-family ligases and dictates whether a substrate is degraded, trafficked to the nucleus, or exported via exosomes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model for substrate-versus-fate specificity\", \"No structural data on the E2-adaptor-E3 ternary complex\", \"Determinants routing cargo to exosomes vs proteasome unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [13, 1]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 14, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [2, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 13, 14]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 13]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 6, 14, 15, 16]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 5, 17]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [7, 10, 11]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NEDD4\", \"NEDD4L\", \"ITCH\", \"PTEN\", \"DMT1\", \"SMURF1\", \"UBE2L3\", \"WWTR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}