{"gene":"ZNRF1","run_date":"2026-06-11T09:02:07","timeline":{"discoveries":[{"year":2011,"finding":"ZNRF1, an E3 ubiquitin ligase, promotes Wallerian degeneration by targeting AKT for degradation via the ubiquitin-proteasome system. AKT degradation releases its inhibitory phosphorylation of GSK3B, allowing active GSK3B to phosphorylate CRMP2, which is required for microtubule reorganization in degenerating axons.","method":"Overexpression and knockdown experiments in neurons, AKT overexpression rescue assay, active GSK3B overexpression, CRMP2 phosphorylation assays, pharmacological GSK3B inhibition","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic and pharmacological experiments establishing a linear pathway, replicated across multiple conditions","pmids":["22057101"],"is_preprint":false},{"year":2015,"finding":"Oxidative stress activates ZNRF1 E3 ligase activity through EGFR-mediated phosphorylation at tyrosine 103. NADPH oxidase activity is required for EGFR-dependent phosphorylation of ZNRF1, which then drives AKT degradation via the ubiquitin-proteasome system, leading to neuronal apoptosis and Wallerian degeneration.","method":"Phosphorylation site mutagenesis (Y103), EGFR inhibition, NADPH oxidase inhibition, ubiquitin-proteasome assays, neuronal degeneration readouts","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — site-directed mutagenesis identifying Y103 phosphorylation, multiple orthogonal inhibitor experiments, single lab","pmids":["26572622"],"is_preprint":false},{"year":2017,"finding":"ZNRF1 physically interacts with caveolin-1 (CAV1) in response to LPS and mediates K-linked ubiquitination and proteasomal degradation of CAV1. This ZNRF1-CAV1 axis regulates AKT-GSK3β activity downstream of TLR4 activation to enhance pro-inflammatory cytokine production and inhibit IL-10.","method":"Co-immunoprecipitation, ubiquitination assays, ZNRF1 knockout mice (hematopoietic deletion), cytokine measurements, endotoxic shock model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo genetic knockout model, multiple cytokine readouts, single lab with multiple orthogonal methods","pmids":["28593998"],"is_preprint":false},{"year":2012,"finding":"ZNRF1 and ZNRF2 are N-myristoylated, localizing them to intracellular membranes where they interact with the Na+/K+ATPase α1 subunit via their UBZ domains. Their RING domains interact with E2 Ubc13/Uev1a to mediate Lys63-linked ubiquitination of the cytoplasmic loop of Na+/K+ATPase α1. Ouabain decreases ZNRF1 protein levels; ZNRF2 knockdown inhibits ouabain-induced decrease of Na+/K+ATPase surface levels.","method":"N-myristoylation assays, Co-immunoprecipitation (UBZ-Na+/K+ATPase interaction), in vitro ubiquitination assays with Lys63-linkage analysis, ZNRF2 knockdown, cell surface biotinylation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro ubiquitination assays, domain-mapping Co-IP, knockdown with defined cellular phenotype, multiple orthogonal methods in single study","pmids":["22797923"],"is_preprint":false},{"year":2018,"finding":"The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N reveals an unusually high-affinity interaction (Kd ~50 nM) between the ZNRF1 RING domain and Ube2N, compared to ~1 µM for Ube2D2. Mutational analyses confirmed the molecular basis of this affinity. Excess ZNRF1 (≥500 nM) inhibits Ube2N-mediated ubiquitination, suggesting a concentration-dependent autoregulatory mechanism.","method":"Crystal structure determination, isothermal titration calorimetry (Kd measurement), site-directed mutagenesis, in vitro ubiquitination assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with mutagenesis and in vitro ubiquitination assays, single lab but multiple orthogonal methods","pmids":["29626159"],"is_preprint":false},{"year":2021,"finding":"ZNRF1 mediates ligand-induced EGFR ubiquitination at distinct lysine residues from those targeted by CBL. ZNRF1 deletion inhibits endosome-to-lysosome sorting of EGFR, resulting in delayed receptor degradation and prolonged downstream signaling. Loss of ZNRF1 increases susceptibility to HSV-1 infection due to enhanced EGFR-dependent viral entry.","method":"ZNRF1 knockout cells, EGFR ubiquitination assays, endosomal trafficking assays, receptor degradation kinetics, lysine-site mapping comparing ZNRF1 vs. CBL, HSV-1 infection assay","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — knockout with defined trafficking phenotype, site-specific ubiquitination mapping, multiple orthogonal readouts, single lab","pmids":["33996800"],"is_preprint":false},{"year":2009,"finding":"ZNRF1 interacts with beta-tubulin type 2 (Tubb2) identified by yeast two-hybrid screening and confirmed by in vivo co-immunoprecipitation. ZNRF1 colocalizes with Tubb2, and both the RING finger domain and zinc finger domain are required for ZNRF1-induced morphological changes (neurite-like elongation).","method":"Yeast two-hybrid screening, in vivo co-immunoprecipitation, immunofluorescence colocalization, domain deletion analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid confirmed by Co-IP and colocalization, domain mutagenesis for morphological phenotype, single lab","pmids":["19737534"],"is_preprint":false},{"year":2023,"finding":"c-Src kinase, activated by TLR3 engagement, phosphorylates ZNRF1 at tyrosine 103, enabling ZNRF1 to mediate K63-linked ubiquitination of TLR3 at lysine 813, which promotes TLR3 sorting into multivesicular bodies/lysosomes and its degradation, terminating type I interferon signaling.","method":"ZNRF1 knockout mice and cells, phosphorylation site mapping (Y103), ubiquitination site mapping (K813 of TLR3), TLR3 lysosomal trafficking assays, SARS-CoV-2 and EMCV infection models","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout mouse model, site-specific mutagenesis of both ZNRF1 (Y103) and TLR3 (K813), in vivo infection models, multiple orthogonal methods","pmids":["37158982"],"is_preprint":false},{"year":2024,"finding":"In Drosophila, the ZNRF1/2 homologue detour interacts with HOPS complex subunits (VPS18/dor, VPS16A, VPS41/lt) and promotes their ubiquitination, regulating autophagosome-lysosome fusion. In mammalian cells, ablation of ZNRF1 or ZNRF2 increased basal autophagy; overexpression increased autophagic vesicle size.","method":"Drosophila genetic model (detour depletion/overexpression), mass spectrometry interactome, ubiquitination assays of HOPS subunits, mammalian ZNRF1/2 knockdown with autophagy flux assays","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — genetic model with defined phenotype, MS interactome, ubiquitination assays; mammalian data is knockdown only, Drosophila ortholog primary model","pmids":["38360932"],"is_preprint":false},{"year":2023,"finding":"LZTFL1 tumor suppressor inhibits kidney tumor cell proliferation by destabilizing AKT through a ZNRF1-mediated ubiquitin-proteasome pathway, placing LZTFL1 upstream of ZNRF1 in AKT regulation.","method":"Gain- and loss-of-function studies in kidney tumor cell lines, patient-derived xenograft model, AKT protein stability assays with ZNRF1","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — functional epistasis between LZTFL1 and ZNRF1 established in cell lines and PDX, but mechanistic detail of the LZTFL1-ZNRF1 interaction is limited in the abstract","pmids":["36966254"],"is_preprint":false},{"year":2024,"finding":"ZNRF1 interacts with lipocalin-2 (LCN2), an iron transport-related protein, and this interaction is disrupted by TFAM deficiency or ferroptosis. Overexpression of ZNRF1 maintains mitochondrial integrity and inhibits renal fibrosis.","method":"Co-immunoprecipitation (ZNRF1-LCN2 interaction), TFAM knockout mice, ferroptosis assays, ZNRF1 overexpression in CKD mouse model","journal":"European journal of pharmacology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP result for ZNRF1-LCN2, limited mechanistic detail on how ZNRF1 E3 activity relates to LCN2, single lab","pmids":["39349116"],"is_preprint":false},{"year":2025,"finding":"ZNRF1-dependent AKT degradation in neurons induces axon initial segment (AIS) shift and increases cell surface localization of voltage-gated sodium channel Nav1.2. ZNRF1 knockout mice exhibit enhanced short-term and contextual fear memory, linking ZNRF1-mediated AKT degradation to AIS plasticity and behavior.","method":"ZNRF1 knockout mice, AIS morphology analysis, Nav1.2 surface localization (fractionation/imaging), fear conditioning behavioral assays","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse with defined cellular phenotype (AIS shift, Nav1.2 relocalization) and behavioral readout, single lab, limited mechanistic detail on AKT-AIS link","pmids":["40331626"],"is_preprint":false},{"year":2025,"finding":"ZNRF1 in peripheral myeloid cells suppresses MHC class II surface expression on macrophages following EAE induction, thereby limiting antigen-specific T cell proliferation, Th1/Th17 polarization, and neuroinflammation.","method":"Myeloid-specific ZNRF1 knockout mice, EAE model, flow cytometry for MHC-II surface expression and T cell polarization, CNS immune cell infiltration analysis","journal":"Journal of neuroinflammation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific knockout with defined immunological phenotype, but the direct ubiquitination substrate mediating MHC-II regulation is not identified in the abstract","pmids":["41126242"],"is_preprint":false},{"year":2026,"finding":"ZNRF1 E3 ligase activity (requiring catalytically active C184) is required for terminal trafficking and surface exposure of FasL in macrophages. ZNRF1 deficiency weakens the Munc18-2 (Stxbp2)-Syntaxin-3 (Stx3) interaction; reconstitution with wild-type but not catalytically inactive ZNRF1 restores surface FasL, defining a ZNRF1-Munc18-2-Stx3 axis for lysosome-related organelle fusion.","method":"Myeloid-specific ZNRF1 knockout mice, confocal imaging of LAMP1 and FasL localization, biochemical co-immunoprecipitation (Stxbp2-Stx3 interaction), catalytic mutant (C184A) reconstitution, FasL surface flow cytometry, T cell killing assay","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Moderate — catalytic mutant rescue, Co-IP of SNARE complex, live imaging of organelle/cargo localization, knockout with functional killing assay, multiple orthogonal methods in single study","pmids":["41896526"],"is_preprint":false}],"current_model":"ZNRF1 is an N-myristoylated RING-domain E3 ubiquitin ligase that preferentially engages the E2 Ube2N (Kd ~50 nM) to catalyze K63-linked ubiquitination; it is activated by EGFR/Src-mediated phosphorylation at tyrosine 103 in response to oxidative stress or TLR engagement, and promotes proteasomal or lysosomal degradation of multiple substrates including AKT (driving GSK3B-CRMP2-dependent axonal degeneration), caveolin-1 (modulating TLR4 inflammatory signaling), EGFR (controlling endosome-to-lysosome trafficking), TLR3 (terminating interferon signaling via MVB sorting), and Na+/K+ATPase (via UBZ-domain docking); additionally, its E3 activity controls FasL surface delivery in macrophages through a Munc18-2–Syntaxin-3 docking axis and regulates autophagy through HOPS complex ubiquitination."},"narrative":{"mechanistic_narrative":"ZNRF1 is an N-myristoylated RING-domain E3 ubiquitin ligase that operates on intracellular membranes to control receptor trafficking, kinase stability, and membrane-fusion events across neuronal and immune cells [PMID:22797923, PMID:22057101]. Membrane targeting via N-myristoylation positions ZNRF1 to engage substrates through its UBZ domain while its RING domain recruits the E2 enzyme Ube2N (Ubc13/Uev1a) with unusually high affinity (Kd ~50 nM) to assemble Lys63-linked ubiquitin chains [PMID:22797923, PMID:29626159]. A recurring activation mode involves phosphorylation at tyrosine 103 — by EGFR downstream of NADPH-oxidase-dependent oxidative stress, and by c-Src downstream of TLR3 engagement — which licenses its ligase activity [PMID:26572622, PMID:37158982]. Through this activity ZNRF1 drives degradation of AKT, releasing GSK3B to phosphorylate CRMP2 and execute Wallerian axonal degeneration [PMID:22057101, PMID:26572622], a substrate axis also exploited downstream of the tumor suppressor LZTFL1 and to remodel the axon initial segment and Nav1.2 surface localization [PMID:36966254, PMID:40331626]. ZNRF1 additionally ubiquitinates membrane and trafficking substrates to govern protein fate: caveolin-1 to tune TLR4 inflammatory cytokine output [PMID:28593998], EGFR at lysines distinct from those used by CBL to drive endosome-to-lysosome sorting and limit HSV-1 entry [PMID:33996800], and TLR3 at K813 to route it into multivesicular bodies and terminate interferon signaling [PMID:37158982]. In macrophages its catalytic activity (requiring C184) is required for terminal trafficking and surface exposure of FasL through a Munc18-2–Syntaxin-3 docking axis [PMID:41896526], and the conserved ZNRF1/2 module regulates autophagosome–lysosome fusion via ubiquitination of HOPS complex subunits [PMID:38360932].","teleology":[{"year":2009,"claim":"Establishing a physical partner and a structural requirement, this work showed ZNRF1 binds beta-tubulin and that both RING and zinc-finger domains are needed to drive neurite-like morphological change, hinting at a cytoskeletal/neuronal role.","evidence":"Yeast two-hybrid, Co-IP, colocalization, and domain-deletion analysis","pmids":["19737534"],"confidence":"Medium","gaps":["No ubiquitination substrate identified","Tubb2 not shown to be a ubiquitination target","Functional consequence limited to morphology"]},{"year":2011,"claim":"This defined ZNRF1's first substrate-level mechanism: it degrades AKT via the proteasome to derepress GSK3B and drive CRMP2 phosphorylation, establishing a linear pathway for Wallerian axonal degeneration.","evidence":"Overexpression/knockdown in neurons, AKT rescue, GSK3B/CRMP2 epistasis, pharmacological GSK3B inhibition","pmids":["22057101"],"confidence":"High","gaps":["Mechanism of substrate recognition of AKT not defined","Activation trigger for ZNRF1 not yet identified","Ubiquitin chain linkage not specified"]},{"year":2012,"claim":"Biochemical dissection established the catalytic and membrane-targeting machinery: N-myristoylation localizes ZNRF1 to membranes, the UBZ domain docks substrate (Na+/K+ATPase a1), and the RING engages Ubc13/Uev1a to build K63 chains.","evidence":"N-myristoylation assays, domain-mapping Co-IP, in vitro K63 ubiquitination, knockdown with surface biotinylation","pmids":["22797923"],"confidence":"High","gaps":["Functional consequence of Na+/K+ATPase ubiquitination on activity not fully resolved","ZNRF1 vs ZNRF2 division of labor unclear"]},{"year":2015,"claim":"This identified the activating signal for ZNRF1: oxidative stress drives EGFR-dependent phosphorylation at Y103, coupling NADPH oxidase signaling to AKT degradation and neuronal death.","evidence":"Y103 mutagenesis, EGFR and NADPH oxidase inhibition, ubiquitin-proteasome assays, degeneration readouts","pmids":["26572622"],"confidence":"High","gaps":["How Y103 phosphorylation alters ligase activity structurally not defined","Whether EGFR directly phosphorylates ZNRF1 not resolved"]},{"year":2017,"claim":"Extending ZNRF1 into innate immunity, this showed ZNRF1 ubiquitinates and degrades caveolin-1 downstream of TLR4 to amplify pro-inflammatory cytokine output via AKT-GSK3B.","evidence":"Reciprocal Co-IP, ubiquitination assays, hematopoietic ZNRF1 knockout mice, endotoxic shock model","pmids":["28593998"],"confidence":"High","gaps":["Ubiquitin linkage type on CAV1 stated only as K-linked","Site of CAV1 ubiquitination not mapped"]},{"year":2018,"claim":"Structural work explained ZNRF1's E2 selectivity, showing an unusually tight RING–Ube2N interface (Kd ~50 nM vs ~1 uM for Ube2D2) and a concentration-dependent autoinhibition by excess ZNRF1.","evidence":"Crystal structure, ITC, site-directed mutagenesis, in vitro ubiquitination","pmids":["29626159"],"confidence":"High","gaps":["Physiological relevance of autoinhibition not tested in cells","Full-length ZNRF1 structure not determined"]},{"year":2021,"claim":"This established ZNRF1 as a trafficking regulator distinct from CBL, ubiquitinating EGFR at unique lysines to drive endosome-to-lysosome sorting; its loss prolongs signaling and increases HSV-1 entry.","evidence":"ZNRF1 knockout cells, lysine-site mapping vs CBL, endosomal trafficking and degradation kinetics, HSV-1 infection assay","pmids":["33996800"],"confidence":"High","gaps":["Specific EGFR lysines targeted not fully enumerated","Coordination with ESCRT machinery not defined"]},{"year":2023,"claim":"This unified the activation logic with a new substrate: TLR3 engagement activates c-Src to phosphorylate ZNRF1 at Y103, enabling K63-ubiquitination of TLR3 at K813 and its MVB/lysosomal degradation to terminate interferon signaling.","evidence":"ZNRF1 knockout mice, dual site mapping (Y103, TLR3 K813), trafficking assays, SARS-CoV-2 and EMCV infection models","pmids":["37158982"],"confidence":"High","gaps":["Whether the same Y103-phospho mechanism applies to all substrates not tested","Direct c-Src–ZNRF1 contact not structurally defined"]},{"year":2023,"claim":"This placed ZNRF1 in a tumor-suppressive axis, showing LZTFL1 destabilizes AKT through ZNRF1 to limit kidney tumor cell proliferation.","evidence":"Gain/loss of function in kidney tumor lines, PDX model, AKT stability assays","pmids":["36966254"],"confidence":"Medium","gaps":["LZTFL1-ZNRF1 physical interaction mechanism not detailed","Whether LZTFL1 acts as adaptor or activator unclear"]},{"year":2024,"claim":"Comparative genetics implicated the ZNRF1/2 module in autophagy, showing the Drosophila ortholog ubiquitinates HOPS subunits and regulates autophagosome–lysosome fusion, with mammalian knockdown altering autophagic flux.","evidence":"Drosophila detour genetics, MS interactome, HOPS subunit ubiquitination, mammalian knockdown autophagy flux","pmids":["38360932"],"confidence":"Medium","gaps":["Mammalian data are knockdown only","Functional consequence of HOPS ubiquitination on fusion not directly shown in mammals"]},{"year":2024,"claim":"A renal study reported ZNRF1 interacts with lipocalin-2 and preserves mitochondrial integrity and limits fibrosis, extending ZNRF1 to iron-handling and ferroptosis contexts.","evidence":"Co-IP (ZNRF1-LCN2), TFAM knockout mice, ferroptosis assays, ZNRF1 overexpression in CKD model","pmids":["39349116"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation","No link between ZNRF1 E3 activity and LCN2 fate established","Mechanism of mitochondrial protection unknown"]},{"year":2025,"claim":"This connected ZNRF1's AKT-degradation activity to neuronal excitability, showing it induces axon initial segment shift and Nav1.2 surface relocalization, with knockout mice showing enhanced fear memory.","evidence":"ZNRF1 knockout mice, AIS morphology, Nav1.2 surface localization, fear conditioning","pmids":["40331626"],"confidence":"Medium","gaps":["Molecular link between AKT degradation and AIS remodeling not defined","Whether Nav1.2 is a direct trafficking target unclear"]},{"year":2025,"claim":"This showed myeloid ZNRF1 suppresses macrophage MHC class II surface expression after EAE, dampening T-cell-driven neuroinflammation.","evidence":"Myeloid-specific ZNRF1 knockout mice, EAE model, flow cytometry for MHC-II and T cell polarization","pmids":["41126242"],"confidence":"Medium","gaps":["Direct ubiquitination substrate mediating MHC-II regulation not identified","Whether effect is trafficking- or stability-based unclear"]},{"year":2026,"claim":"This defined a catalytic-activity-dependent role in immune effector delivery, showing ZNRF1 (requiring C184) is needed for FasL surface exposure in macrophages via a Munc18-2–Syntaxin-3 docking axis controlling lysosome-related organelle fusion.","evidence":"Myeloid ZNRF1 knockout mice, C184A catalytic rescue, Stxbp2-Stx3 Co-IP, LAMP1/FasL imaging, FasL flow cytometry, T cell killing assay","pmids":["41896526"],"confidence":"High","gaps":["Direct ubiquitination substrate within the SNARE/SM machinery not identified","Whether Munc18-2 or Stx3 is the ubiquitinated target unresolved"]},{"year":null,"claim":"How a single membrane-anchored E3 selects among its diverse substrates (AKT, CAV1, EGFR, TLR3, Na+/K+ATPase) and whether Y103 phosphorylation is the universal switch governing this selectivity remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified substrate-recognition model across the substrate repertoire","Substrate-specific adaptor proteins largely uncharacterized","Linkage between activation state and substrate choice not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,3,4,13]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,3,5,7]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[3,4,7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5,7]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[5,7,13]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,7,12,13]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,3,5,7]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[5,7,13]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[8]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,1,11]}],"complexes":[],"partners":["UBE2N","CAV1","EGFR","TLR3","ATP1A1","TUBB2","STXBP2","STX3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8ND25","full_name":"E3 ubiquitin-protein ligase ZNRF1","aliases":["Nerve injury-induced gene 283 protein","RING-type E3 ubiquitin transferase ZNRF1","Zinc/RING finger protein 1"],"length_aa":227,"mass_kda":23.8,"function":"E3 ubiquitin-protein ligase that plays a role in different processes including cell differentiation, receptor recycling or regulation of inflammation (PubMed:28593998, PubMed:33996800, PubMed:37158982). Mediates the ubiquitination of AKT1 and GLUL, thereby playing a role in neuron cells differentiation. Plays a role in the establishment and maintenance of neuronal transmission and plasticity. Regulates Schwann cells differentiation by mediating ubiquitination of GLUL. Promotes neurodegeneration by mediating 'Lys-48'-linked polyubiquitination and subsequent degradation of AKT1 in axons: degradation of AKT1 prevents AKT1-mediated phosphorylation of GSK3B, leading to GSK3B activation and phosphorylation of DPYSL2/CRMP2 followed by destabilization of microtubule assembly in axons. Ubiquitinates the Na(+)/K(+) ATPase alpha-1 subunit/ATP1A1 and thereby influences its endocytosis and/or degradation (PubMed:22797923). Controls ligand-induced EGFR signaling via mediating receptor ubiquitination and recruitment of the ESCRT machinery (PubMed:33996800). Acts as a negative feedback mechanism controlling TLR3 trafficking by mediating TLR3 'Lys-63'-linked polyubiquitination to reduce type I IFN production (PubMed:37158982). Modulates inflammation by promoting caveolin-1/CAV1 ubiquitination and degradation to regulate TLR4-activated immune response (PubMed:28593998)","subcellular_location":"Endosome; Lysosome; Membrane; Cytoplasmic vesicle, secretory vesicle, synaptic vesicle membrane","url":"https://www.uniprot.org/uniprotkb/Q8ND25/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ZNRF1","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/ZNRF1","total_profiled":1310},"omim":[{"mim_id":"612061","title":"ZINC FINGER AND RING FINGER PROTEIN 2; ZNRF2","url":"https://www.omim.org/entry/612061"},{"mim_id":"612060","title":"ZINC FINGER AND RING FINGER PROTEIN 1; ZNRF1","url":"https://www.omim.org/entry/612060"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Primary cilium tip","reliability":"Additional"},{"location":"Primary cilium transition zone","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ZNRF1"},"hgnc":{"alias_symbol":["nin283","FLJ14846","DKFZp434E229"],"prev_symbol":[]},"alphafold":{"accession":"Q8ND25","domains":[{"cath_id":"-","chopping":"142-170","consensus_level":"medium","plddt":90.9431,"start":142,"end":170},{"cath_id":"3.30.40.10","chopping":"172-227","consensus_level":"medium","plddt":94.6957,"start":172,"end":227}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8ND25","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8ND25-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8ND25-F1-predicted_aligned_error_v6.png","plddt_mean":67.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ZNRF1","jax_strain_url":"https://www.jax.org/strain/search?query=ZNRF1"},"sequence":{"accession":"Q8ND25","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8ND25.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8ND25/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8ND25"}},"corpus_meta":[{"pmid":"22057101","id":"PMC_22057101","title":"ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B-dependent CRMP2 phosphorylation.","date":"2011","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/22057101","citation_count":132,"is_preprint":false},{"pmid":"28593998","id":"PMC_28593998","title":"The ubiquitin ligase ZNRF1 promotes caveolin-1 ubiquitination and degradation to modulate inflammation.","date":"2017","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/28593998","citation_count":53,"is_preprint":false},{"pmid":"26572622","id":"PMC_26572622","title":"Oxidative stress-dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration.","date":"2015","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/26572622","citation_count":46,"is_preprint":false},{"pmid":"22797923","id":"PMC_22797923","title":"ZNRF2 is released from membranes by growth factors and, together with ZNRF1, regulates the Na+/K+ATPase.","date":"2012","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/22797923","citation_count":26,"is_preprint":false},{"pmid":"33996800","id":"PMC_33996800","title":"ZNRF1 Mediates Epidermal Growth Factor Receptor Ubiquitination to Control Receptor Lysosomal Trafficking and Degradation.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33996800","citation_count":24,"is_preprint":false},{"pmid":"19737534","id":"PMC_19737534","title":"ZNRF1 interacts with tubulin and regulates cell morphogenesis.","date":"2009","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/19737534","citation_count":14,"is_preprint":false},{"pmid":"27195063","id":"PMC_27195063","title":"NADPH oxidases promote apoptosis by activating ZNRF1 ubiquitin ligase in neurons treated with an exogenously applied oxidant.","date":"2016","source":"Communicative & integrative biology","url":"https://pubmed.ncbi.nlm.nih.gov/27195063","citation_count":13,"is_preprint":false},{"pmid":"30118738","id":"PMC_30118738","title":"Regulation of neuronal/axonal degeneration by ZNRF1 ubiquitin ligase.","date":"2018","source":"Neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/30118738","citation_count":11,"is_preprint":false},{"pmid":"29626159","id":"PMC_29626159","title":"Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications.","date":"2018","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/29626159","citation_count":11,"is_preprint":false},{"pmid":"37158982","id":"PMC_37158982","title":"The Src-ZNRF1 axis controls TLR3 trafficking and interferon responses to limit lung barrier damage.","date":"2023","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37158982","citation_count":6,"is_preprint":false},{"pmid":"38360932","id":"PMC_38360932","title":"The Drosophila ZNRF1/2 homologue, detour, interacts with HOPS complex and regulates autophagy.","date":"2024","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/38360932","citation_count":6,"is_preprint":false},{"pmid":"36966254","id":"PMC_36966254","title":"LZTFL1 inhibits kidney tumor cell growth by destabilizing AKT through ZNRF1-mediated ubiquitin proteosome pathway.","date":"2023","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/36966254","citation_count":4,"is_preprint":false},{"pmid":"39349116","id":"PMC_39349116","title":"Myricanol represses renal fibrosis by activating TFAM and ZNRF1 to inhibit tubular epithelial cells ferroptosis.","date":"2024","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/39349116","citation_count":4,"is_preprint":false},{"pmid":"41126242","id":"PMC_41126242","title":"Myeloid ZNRF1 suppresses autoimmune demyelination and neuroinflammation by regulating MHC-II-mediated T cell activation.","date":"2025","source":"Journal of neuroinflammation","url":"https://pubmed.ncbi.nlm.nih.gov/41126242","citation_count":1,"is_preprint":false},{"pmid":"40233431","id":"PMC_40233431","title":"The essential role of E3 ubiquitin ligases in the pathogenesis of neurodevelopmental and psychiatric disorders: Cul3, Cul4, Ube3a, and ZNRF1.","date":"2025","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/40233431","citation_count":1,"is_preprint":false},{"pmid":"40331626","id":"PMC_40331626","title":"ZNRF1-dependent regulation of AKT activity modulates Nav subcellular localization and AIS position in neurons to regulate fear-related behaviour.","date":"2025","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40331626","citation_count":0,"is_preprint":false},{"pmid":"41896526","id":"PMC_41896526","title":"ZNRF1 deficiency disrupts Fas ligand trafficking and immune balance.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41896526","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9348,"output_tokens":4023,"usd":0.044194,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11808,"output_tokens":4942,"usd":0.091295,"stage2_stop_reason":"end_turn"},"total_usd":0.135489,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"ZNRF1, an E3 ubiquitin ligase, promotes Wallerian degeneration by targeting AKT for degradation via the ubiquitin-proteasome system. AKT degradation releases its inhibitory phosphorylation of GSK3B, allowing active GSK3B to phosphorylate CRMP2, which is required for microtubule reorganization in degenerating axons.\",\n      \"method\": \"Overexpression and knockdown experiments in neurons, AKT overexpression rescue assay, active GSK3B overexpression, CRMP2 phosphorylation assays, pharmacological GSK3B inhibition\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic and pharmacological experiments establishing a linear pathway, replicated across multiple conditions\",\n      \"pmids\": [\"22057101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Oxidative stress activates ZNRF1 E3 ligase activity through EGFR-mediated phosphorylation at tyrosine 103. NADPH oxidase activity is required for EGFR-dependent phosphorylation of ZNRF1, which then drives AKT degradation via the ubiquitin-proteasome system, leading to neuronal apoptosis and Wallerian degeneration.\",\n      \"method\": \"Phosphorylation site mutagenesis (Y103), EGFR inhibition, NADPH oxidase inhibition, ubiquitin-proteasome assays, neuronal degeneration readouts\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — site-directed mutagenesis identifying Y103 phosphorylation, multiple orthogonal inhibitor experiments, single lab\",\n      \"pmids\": [\"26572622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ZNRF1 physically interacts with caveolin-1 (CAV1) in response to LPS and mediates K-linked ubiquitination and proteasomal degradation of CAV1. This ZNRF1-CAV1 axis regulates AKT-GSK3β activity downstream of TLR4 activation to enhance pro-inflammatory cytokine production and inhibit IL-10.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, ZNRF1 knockout mice (hematopoietic deletion), cytokine measurements, endotoxic shock model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo genetic knockout model, multiple cytokine readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28593998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ZNRF1 and ZNRF2 are N-myristoylated, localizing them to intracellular membranes where they interact with the Na+/K+ATPase α1 subunit via their UBZ domains. Their RING domains interact with E2 Ubc13/Uev1a to mediate Lys63-linked ubiquitination of the cytoplasmic loop of Na+/K+ATPase α1. Ouabain decreases ZNRF1 protein levels; ZNRF2 knockdown inhibits ouabain-induced decrease of Na+/K+ATPase surface levels.\",\n      \"method\": \"N-myristoylation assays, Co-immunoprecipitation (UBZ-Na+/K+ATPase interaction), in vitro ubiquitination assays with Lys63-linkage analysis, ZNRF2 knockdown, cell surface biotinylation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro ubiquitination assays, domain-mapping Co-IP, knockdown with defined cellular phenotype, multiple orthogonal methods in single study\",\n      \"pmids\": [\"22797923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N reveals an unusually high-affinity interaction (Kd ~50 nM) between the ZNRF1 RING domain and Ube2N, compared to ~1 µM for Ube2D2. Mutational analyses confirmed the molecular basis of this affinity. Excess ZNRF1 (≥500 nM) inhibits Ube2N-mediated ubiquitination, suggesting a concentration-dependent autoregulatory mechanism.\",\n      \"method\": \"Crystal structure determination, isothermal titration calorimetry (Kd measurement), site-directed mutagenesis, in vitro ubiquitination assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with mutagenesis and in vitro ubiquitination assays, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"29626159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ZNRF1 mediates ligand-induced EGFR ubiquitination at distinct lysine residues from those targeted by CBL. ZNRF1 deletion inhibits endosome-to-lysosome sorting of EGFR, resulting in delayed receptor degradation and prolonged downstream signaling. Loss of ZNRF1 increases susceptibility to HSV-1 infection due to enhanced EGFR-dependent viral entry.\",\n      \"method\": \"ZNRF1 knockout cells, EGFR ubiquitination assays, endosomal trafficking assays, receptor degradation kinetics, lysine-site mapping comparing ZNRF1 vs. CBL, HSV-1 infection assay\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout with defined trafficking phenotype, site-specific ubiquitination mapping, multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"33996800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ZNRF1 interacts with beta-tubulin type 2 (Tubb2) identified by yeast two-hybrid screening and confirmed by in vivo co-immunoprecipitation. ZNRF1 colocalizes with Tubb2, and both the RING finger domain and zinc finger domain are required for ZNRF1-induced morphological changes (neurite-like elongation).\",\n      \"method\": \"Yeast two-hybrid screening, in vivo co-immunoprecipitation, immunofluorescence colocalization, domain deletion analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid confirmed by Co-IP and colocalization, domain mutagenesis for morphological phenotype, single lab\",\n      \"pmids\": [\"19737534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"c-Src kinase, activated by TLR3 engagement, phosphorylates ZNRF1 at tyrosine 103, enabling ZNRF1 to mediate K63-linked ubiquitination of TLR3 at lysine 813, which promotes TLR3 sorting into multivesicular bodies/lysosomes and its degradation, terminating type I interferon signaling.\",\n      \"method\": \"ZNRF1 knockout mice and cells, phosphorylation site mapping (Y103), ubiquitination site mapping (K813 of TLR3), TLR3 lysosomal trafficking assays, SARS-CoV-2 and EMCV infection models\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout mouse model, site-specific mutagenesis of both ZNRF1 (Y103) and TLR3 (K813), in vivo infection models, multiple orthogonal methods\",\n      \"pmids\": [\"37158982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In Drosophila, the ZNRF1/2 homologue detour interacts with HOPS complex subunits (VPS18/dor, VPS16A, VPS41/lt) and promotes their ubiquitination, regulating autophagosome-lysosome fusion. In mammalian cells, ablation of ZNRF1 or ZNRF2 increased basal autophagy; overexpression increased autophagic vesicle size.\",\n      \"method\": \"Drosophila genetic model (detour depletion/overexpression), mass spectrometry interactome, ubiquitination assays of HOPS subunits, mammalian ZNRF1/2 knockdown with autophagy flux assays\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — genetic model with defined phenotype, MS interactome, ubiquitination assays; mammalian data is knockdown only, Drosophila ortholog primary model\",\n      \"pmids\": [\"38360932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LZTFL1 tumor suppressor inhibits kidney tumor cell proliferation by destabilizing AKT through a ZNRF1-mediated ubiquitin-proteasome pathway, placing LZTFL1 upstream of ZNRF1 in AKT regulation.\",\n      \"method\": \"Gain- and loss-of-function studies in kidney tumor cell lines, patient-derived xenograft model, AKT protein stability assays with ZNRF1\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — functional epistasis between LZTFL1 and ZNRF1 established in cell lines and PDX, but mechanistic detail of the LZTFL1-ZNRF1 interaction is limited in the abstract\",\n      \"pmids\": [\"36966254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZNRF1 interacts with lipocalin-2 (LCN2), an iron transport-related protein, and this interaction is disrupted by TFAM deficiency or ferroptosis. Overexpression of ZNRF1 maintains mitochondrial integrity and inhibits renal fibrosis.\",\n      \"method\": \"Co-immunoprecipitation (ZNRF1-LCN2 interaction), TFAM knockout mice, ferroptosis assays, ZNRF1 overexpression in CKD mouse model\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP result for ZNRF1-LCN2, limited mechanistic detail on how ZNRF1 E3 activity relates to LCN2, single lab\",\n      \"pmids\": [\"39349116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZNRF1-dependent AKT degradation in neurons induces axon initial segment (AIS) shift and increases cell surface localization of voltage-gated sodium channel Nav1.2. ZNRF1 knockout mice exhibit enhanced short-term and contextual fear memory, linking ZNRF1-mediated AKT degradation to AIS plasticity and behavior.\",\n      \"method\": \"ZNRF1 knockout mice, AIS morphology analysis, Nav1.2 surface localization (fractionation/imaging), fear conditioning behavioral assays\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with defined cellular phenotype (AIS shift, Nav1.2 relocalization) and behavioral readout, single lab, limited mechanistic detail on AKT-AIS link\",\n      \"pmids\": [\"40331626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZNRF1 in peripheral myeloid cells suppresses MHC class II surface expression on macrophages following EAE induction, thereby limiting antigen-specific T cell proliferation, Th1/Th17 polarization, and neuroinflammation.\",\n      \"method\": \"Myeloid-specific ZNRF1 knockout mice, EAE model, flow cytometry for MHC-II surface expression and T cell polarization, CNS immune cell infiltration analysis\",\n      \"journal\": \"Journal of neuroinflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific knockout with defined immunological phenotype, but the direct ubiquitination substrate mediating MHC-II regulation is not identified in the abstract\",\n      \"pmids\": [\"41126242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ZNRF1 E3 ligase activity (requiring catalytically active C184) is required for terminal trafficking and surface exposure of FasL in macrophages. ZNRF1 deficiency weakens the Munc18-2 (Stxbp2)-Syntaxin-3 (Stx3) interaction; reconstitution with wild-type but not catalytically inactive ZNRF1 restores surface FasL, defining a ZNRF1-Munc18-2-Stx3 axis for lysosome-related organelle fusion.\",\n      \"method\": \"Myeloid-specific ZNRF1 knockout mice, confocal imaging of LAMP1 and FasL localization, biochemical co-immunoprecipitation (Stxbp2-Stx3 interaction), catalytic mutant (C184A) reconstitution, FasL surface flow cytometry, T cell killing assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic mutant rescue, Co-IP of SNARE complex, live imaging of organelle/cargo localization, knockout with functional killing assay, multiple orthogonal methods in single study\",\n      \"pmids\": [\"41896526\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ZNRF1 is an N-myristoylated RING-domain E3 ubiquitin ligase that preferentially engages the E2 Ube2N (Kd ~50 nM) to catalyze K63-linked ubiquitination; it is activated by EGFR/Src-mediated phosphorylation at tyrosine 103 in response to oxidative stress or TLR engagement, and promotes proteasomal or lysosomal degradation of multiple substrates including AKT (driving GSK3B-CRMP2-dependent axonal degeneration), caveolin-1 (modulating TLR4 inflammatory signaling), EGFR (controlling endosome-to-lysosome trafficking), TLR3 (terminating interferon signaling via MVB sorting), and Na+/K+ATPase (via UBZ-domain docking); additionally, its E3 activity controls FasL surface delivery in macrophages through a Munc18-2–Syntaxin-3 docking axis and regulates autophagy through HOPS complex ubiquitination.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ZNRF1 is an N-myristoylated RING-domain E3 ubiquitin ligase that operates on intracellular membranes to control receptor trafficking, kinase stability, and membrane-fusion events across neuronal and immune cells [#3, #0]. Membrane targeting via N-myristoylation positions ZNRF1 to engage substrates through its UBZ domain while its RING domain recruits the E2 enzyme Ube2N (Ubc13/Uev1a) with unusually high affinity (Kd ~50 nM) to assemble Lys63-linked ubiquitin chains [#3, #4]. A recurring activation mode involves phosphorylation at tyrosine 103 — by EGFR downstream of NADPH-oxidase-dependent oxidative stress, and by c-Src downstream of TLR3 engagement — which licenses its ligase activity [#1, #7]. Through this activity ZNRF1 drives degradation of AKT, releasing GSK3B to phosphorylate CRMP2 and execute Wallerian axonal degeneration [#0, #1], a substrate axis also exploited downstream of the tumor suppressor LZTFL1 and to remodel the axon initial segment and Nav1.2 surface localization [#9, #11]. ZNRF1 additionally ubiquitinates membrane and trafficking substrates to govern protein fate: caveolin-1 to tune TLR4 inflammatory cytokine output [#2], EGFR at lysines distinct from those used by CBL to drive endosome-to-lysosome sorting and limit HSV-1 entry [#5], and TLR3 at K813 to route it into multivesicular bodies and terminate interferon signaling [#7]. In macrophages its catalytic activity (requiring C184) is required for terminal trafficking and surface exposure of FasL through a Munc18-2–Syntaxin-3 docking axis [#13], and the conserved ZNRF1/2 module regulates autophagosome–lysosome fusion via ubiquitination of HOPS complex subunits [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing a physical partner and a structural requirement, this work showed ZNRF1 binds beta-tubulin and that both RING and zinc-finger domains are needed to drive neurite-like morphological change, hinting at a cytoskeletal/neuronal role.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, colocalization, and domain-deletion analysis\",\n      \"pmids\": [\"19737534\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ubiquitination substrate identified\", \"Tubb2 not shown to be a ubiquitination target\", \"Functional consequence limited to morphology\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"This defined ZNRF1's first substrate-level mechanism: it degrades AKT via the proteasome to derepress GSK3B and drive CRMP2 phosphorylation, establishing a linear pathway for Wallerian axonal degeneration.\",\n      \"evidence\": \"Overexpression/knockdown in neurons, AKT rescue, GSK3B/CRMP2 epistasis, pharmacological GSK3B inhibition\",\n      \"pmids\": [\"22057101\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of substrate recognition of AKT not defined\", \"Activation trigger for ZNRF1 not yet identified\", \"Ubiquitin chain linkage not specified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Biochemical dissection established the catalytic and membrane-targeting machinery: N-myristoylation localizes ZNRF1 to membranes, the UBZ domain docks substrate (Na+/K+ATPase a1), and the RING engages Ubc13/Uev1a to build K63 chains.\",\n      \"evidence\": \"N-myristoylation assays, domain-mapping Co-IP, in vitro K63 ubiquitination, knockdown with surface biotinylation\",\n      \"pmids\": [\"22797923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Na+/K+ATPase ubiquitination on activity not fully resolved\", \"ZNRF1 vs ZNRF2 division of labor unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"This identified the activating signal for ZNRF1: oxidative stress drives EGFR-dependent phosphorylation at Y103, coupling NADPH oxidase signaling to AKT degradation and neuronal death.\",\n      \"evidence\": \"Y103 mutagenesis, EGFR and NADPH oxidase inhibition, ubiquitin-proteasome assays, degeneration readouts\",\n      \"pmids\": [\"26572622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Y103 phosphorylation alters ligase activity structurally not defined\", \"Whether EGFR directly phosphorylates ZNRF1 not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extending ZNRF1 into innate immunity, this showed ZNRF1 ubiquitinates and degrades caveolin-1 downstream of TLR4 to amplify pro-inflammatory cytokine output via AKT-GSK3B.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assays, hematopoietic ZNRF1 knockout mice, endotoxic shock model\",\n      \"pmids\": [\"28593998\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin linkage type on CAV1 stated only as K-linked\", \"Site of CAV1 ubiquitination not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Structural work explained ZNRF1's E2 selectivity, showing an unusually tight RING–Ube2N interface (Kd ~50 nM vs ~1 uM for Ube2D2) and a concentration-dependent autoinhibition by excess ZNRF1.\",\n      \"evidence\": \"Crystal structure, ITC, site-directed mutagenesis, in vitro ubiquitination\",\n      \"pmids\": [\"29626159\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of autoinhibition not tested in cells\", \"Full-length ZNRF1 structure not determined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"This established ZNRF1 as a trafficking regulator distinct from CBL, ubiquitinating EGFR at unique lysines to drive endosome-to-lysosome sorting; its loss prolongs signaling and increases HSV-1 entry.\",\n      \"evidence\": \"ZNRF1 knockout cells, lysine-site mapping vs CBL, endosomal trafficking and degradation kinetics, HSV-1 infection assay\",\n      \"pmids\": [\"33996800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific EGFR lysines targeted not fully enumerated\", \"Coordination with ESCRT machinery not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"This unified the activation logic with a new substrate: TLR3 engagement activates c-Src to phosphorylate ZNRF1 at Y103, enabling K63-ubiquitination of TLR3 at K813 and its MVB/lysosomal degradation to terminate interferon signaling.\",\n      \"evidence\": \"ZNRF1 knockout mice, dual site mapping (Y103, TLR3 K813), trafficking assays, SARS-CoV-2 and EMCV infection models\",\n      \"pmids\": [\"37158982\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same Y103-phospho mechanism applies to all substrates not tested\", \"Direct c-Src–ZNRF1 contact not structurally defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"This placed ZNRF1 in a tumor-suppressive axis, showing LZTFL1 destabilizes AKT through ZNRF1 to limit kidney tumor cell proliferation.\",\n      \"evidence\": \"Gain/loss of function in kidney tumor lines, PDX model, AKT stability assays\",\n      \"pmids\": [\"36966254\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"LZTFL1-ZNRF1 physical interaction mechanism not detailed\", \"Whether LZTFL1 acts as adaptor or activator unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Comparative genetics implicated the ZNRF1/2 module in autophagy, showing the Drosophila ortholog ubiquitinates HOPS subunits and regulates autophagosome–lysosome fusion, with mammalian knockdown altering autophagic flux.\",\n      \"evidence\": \"Drosophila detour genetics, MS interactome, HOPS subunit ubiquitination, mammalian knockdown autophagy flux\",\n      \"pmids\": [\"38360932\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian data are knockdown only\", \"Functional consequence of HOPS ubiquitination on fusion not directly shown in mammals\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A renal study reported ZNRF1 interacts with lipocalin-2 and preserves mitochondrial integrity and limits fibrosis, extending ZNRF1 to iron-handling and ferroptosis contexts.\",\n      \"evidence\": \"Co-IP (ZNRF1-LCN2), TFAM knockout mice, ferroptosis assays, ZNRF1 overexpression in CKD model\",\n      \"pmids\": [\"39349116\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"No link between ZNRF1 E3 activity and LCN2 fate established\", \"Mechanism of mitochondrial protection unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"This connected ZNRF1's AKT-degradation activity to neuronal excitability, showing it induces axon initial segment shift and Nav1.2 surface relocalization, with knockout mice showing enhanced fear memory.\",\n      \"evidence\": \"ZNRF1 knockout mice, AIS morphology, Nav1.2 surface localization, fear conditioning\",\n      \"pmids\": [\"40331626\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between AKT degradation and AIS remodeling not defined\", \"Whether Nav1.2 is a direct trafficking target unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"This showed myeloid ZNRF1 suppresses macrophage MHC class II surface expression after EAE, dampening T-cell-driven neuroinflammation.\",\n      \"evidence\": \"Myeloid-specific ZNRF1 knockout mice, EAE model, flow cytometry for MHC-II and T cell polarization\",\n      \"pmids\": [\"41126242\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination substrate mediating MHC-II regulation not identified\", \"Whether effect is trafficking- or stability-based unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"This defined a catalytic-activity-dependent role in immune effector delivery, showing ZNRF1 (requiring C184) is needed for FasL surface exposure in macrophages via a Munc18-2–Syntaxin-3 docking axis controlling lysosome-related organelle fusion.\",\n      \"evidence\": \"Myeloid ZNRF1 knockout mice, C184A catalytic rescue, Stxbp2-Stx3 Co-IP, LAMP1/FasL imaging, FasL flow cytometry, T cell killing assay\",\n      \"pmids\": [\"41896526\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitination substrate within the SNARE/SM machinery not identified\", \"Whether Munc18-2 or Stx3 is the ubiquitinated target unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single membrane-anchored E3 selects among its diverse substrates (AKT, CAV1, EGFR, TLR3, Na+/K+ATPase) and whether Y103 phosphorylation is the universal switch governing this selectivity remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified substrate-recognition model across the substrate repertoire\", \"Substrate-specific adaptor proteins largely uncharacterized\", \"Linkage between activation state and substrate choice not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 3, 4, 13]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 3, 5, 7]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [3, 4, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5, 7]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [5, 7, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 7, 12, 13]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 3, 5, 7]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [5, 7, 13]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 1, 11]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UBE2N\", \"CAV1\", \"EGFR\", \"TLR3\", \"ATP1A1\", \"TUBB2\", \"STXBP2\", \"STX3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}