{"gene":"ZNRF1","run_date":"2026-04-28T23:00:24","timeline":{"discoveries":[{"year":2011,"finding":"ZNRF1 acts as an E3 ubiquitin ligase that targets AKT for proteasomal degradation, thereby relieving AKT-mediated inhibitory phosphorylation of GSK3B, which then phosphorylates CRMP2 to drive microtubule reorganization and Wallerian degeneration in axons.","method":"Overexpression and knockdown in neurons, ubiquitin-proteasome assays, epistasis analysis with active/inactive GSK3B and CRMP2 mutants, axonal degeneration assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, genetic epistasis, pathway placement; replicated across subsequent studies","pmids":["22057101"],"is_preprint":false},{"year":2015,"finding":"Oxidative stress activates ZNRF1 E3 ligase activity via EGFR-mediated phosphorylation at tyrosine 103, requiring NADPH oxidase activity; this phosphorylation-activated ZNRF1 then degrades AKT to promote neuronal apoptosis and Wallerian degeneration.","method":"Phosphorylation-site mutagenesis (Y103), EGFR inhibition, NADPH oxidase inhibition, ubiquitin ligase activity assays, neuronal degeneration assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis of active-site residue combined with pharmacological epistasis and functional readout","pmids":["26572622"],"is_preprint":false},{"year":2017,"finding":"ZNRF1 physically interacts with caveolin-1 (CAV1) in response to LPS and mediates K48-linked ubiquitination and proteasomal degradation of CAV1, thereby modulating AKT-GSK3β signaling downstream of TLR4 to enhance pro-inflammatory cytokine production and suppress IL-10.","method":"Co-immunoprecipitation, ubiquitination assay, ZNRF1 KO mice with hematopoietic deletion, cytokine measurements, endotoxic shock model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, in vivo KO with defined phenotype, multiple orthogonal methods","pmids":["28593998"],"is_preprint":false},{"year":2012,"finding":"ZNRF1 localizes to intracellular membranes via N-myristoylation, interacts with the Na+/K+ATPase α1 subunit through its UBZ domain, and together with its RING domain engaging E2 Ubc13/Uev1a mediates K63-linked ubiquitination of the cytoplasmic loop of the Na+/K+ATPase α1 subunit.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, subcellular fractionation, ouabain treatment, ZNRF2 knockdown","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro ubiquitination reconstitution, domain mapping, reciprocal Co-IP","pmids":["22797923"],"is_preprint":false},{"year":2018,"finding":"The RING domain of ZNRF1 binds E2 ubiquitin-conjugating enzyme Ube2N with nanomolar affinity (~50 nM Kd), far higher than its affinity for Ube2D2 (~1 µM); crystal structure of ZNRF1 C-terminal domain in complex with Ube2N reveals the molecular basis, and excess ZNRF1 inhibits Ube2N-mediated ubiquitination, indicating a novel activity-regulation mechanism dependent on E3:E2 stoichiometry.","method":"Crystal structure determination, ITC/binding assays, in vitro ubiquitination assay, mutagenesis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and in vitro ubiquitination reconstitution","pmids":["29626159"],"is_preprint":false},{"year":2021,"finding":"ZNRF1 mediates ligand-induced ubiquitination of EGFR at lysine residues distinct from those targeted by CBL, promoting endosome-to-lysosome sorting and degradation of EGFR; ZNRF1 deletion delays receptor degradation, prolongs downstream signaling, and increases susceptibility to HSV-1 entry.","method":"ZNRF1 knockout cells, EGFR ubiquitination assay, endosomal trafficking assay, lysosomal degradation assay, site-specific ubiquitin mapping","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 2 — KO with defined trafficking and signaling phenotype, ubiquitin site mapping, epistasis with CBL","pmids":["33996800"],"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, promoting TLR3 sorting into multivesicular bodies/lysosomes and terminating type I interferon signaling.","method":"ZNRF1-deficient mice and cells, site-specific mutagenesis (Y103, K813), ubiquitination assays, endolysosomal trafficking assays, viral infection models (EMCV, SARS-CoV-2)","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis of phosphorylation and ubiquitination sites, in vivo KO, defined signaling and trafficking phenotype","pmids":["37158982"],"is_preprint":false},{"year":2009,"finding":"ZNRF1 interacts with beta-tubulin type 2 (Tubb2) via its RING finger and zinc finger domains; overexpression of ZNRF1 induces neurite-like morphological changes, and both RING and zinc finger domains are required for this effect.","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 — single Co-IP and yeast two-hybrid, single lab","pmids":["19737534"],"is_preprint":false},{"year":2016,"finding":"NADPH oxidases are required upstream of EGFR-dependent phosphorylation to activate ZNRF1, which then promotes AKT degradation and neuronal apoptosis; this pathway operates even when oxidative stress is applied exogenously.","method":"NADPH oxidase inhibition, EGFR inhibition, neuronal apoptosis assays, AKT degradation assays","journal":"Communicative & integrative biology","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological epistasis, extends prior mechanistic findings; single lab","pmids":["27195063"],"is_preprint":false},{"year":2023,"finding":"LZTFL1 tumor suppressor promotes AKT degradation through ZNRF1-mediated ubiquitin-proteasome pathway to inhibit kidney tumor cell proliferation.","method":"Gain- and loss-of-function studies in kidney tumor cell lines, PDX model, AKT degradation assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — KO/OE with defined substrate degradation phenotype, PDX model; single lab","pmids":["36966254"],"is_preprint":false},{"year":2024,"finding":"Drosophila ZNRF1/2 homologue detour (CG14435) interacts with HOPS complex subunits (VPS18, VPS16A, VPS41) and promotes their ubiquitination to regulate autophagosome-lysosome fusion; mammalian ZNRF1 and ZNRF2 ablation increases basal autophagy in mammalian cells.","method":"Drosophila genetic depletion and overexpression, mass spectrometry interactome, ubiquitination assay, autophagy flux assays in mammalian cells","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 — MS interactome, ubiquitination assay; Drosophila ortholog with mammalian validation","pmids":["38360932"],"is_preprint":false},{"year":2025,"finding":"ZNRF1-dependent AKT degradation in neurons controls axon initial segment (AIS) position and promotes increased cell surface localization of voltage-gated sodium channel Nav1.2; ZNRF1 KO mice show AIS shift and enhanced fear memory.","method":"ZNRF1 KO mice, AIS morphology analysis, Nav1.2 surface biotinylation, fear conditioning behavior","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — KO with defined cellular and behavioral phenotype; single lab","pmids":["40331626"],"is_preprint":false},{"year":2025,"finding":"ZNRF1 in peripheral myeloid macrophages regulates surface MHC class II expression; ZNRF1-deficient macrophages display elevated surface MHC-II, leading to enhanced antigen-specific T cell proliferation and exacerbated autoimmune demyelination (EAE).","method":"Myeloid-specific ZNRF1 KO mice, flow cytometry for MHC-II surface expression, EAE model, T cell proliferation assays","journal":"Journal of neuroinflammation","confidence":"Medium","confidence_rationale":"Tier 2 — cell-type-specific KO with defined surface protein and immune phenotype; single lab","pmids":["41126242"],"is_preprint":false},{"year":2024,"finding":"ZNRF1 interacts with iron transport protein lipocalin-2 (LCN2) and this interaction is regulated by TFAM; ZNRF1 overexpression maintains mitochondrial integrity and inhibits ferroptosis in renal tubular epithelial cells.","method":"Co-immunoprecipitation, ZNRF1 overexpression in ferroptosis model, TFAM-deficient mice, transcriptional sequencing","journal":"European journal of pharmacology","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP, limited mechanistic follow-up, single lab","pmids":["39349116"],"is_preprint":false},{"year":2026,"finding":"ZNRF1 E3 ligase activity (requiring catalytic C184 residue) is required for terminal trafficking and surface exposure of Fas ligand (FasL) in macrophages by supporting the Munc18-2 (Stxbp2)-Syntaxin-3 (Stx3) interaction; ZNRF1 deficiency weakens this SNARE complex interaction and blocks FasL docking/fusion at the plasma membrane without affecting lysosome-related organelle polarization.","method":"Myeloid-specific ZNRF1 KO, catalytically inactive ZNRF1 C184A reconstitution, co-immunoprecipitation of Stxbp2-Stx3, confocal imaging of LAMP1 and FasL, surface FasL flow cytometry, FasL-dependent killing assays","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1-2 — catalytic mutant reconstitution, Co-IP of SNARE complex, multiple orthogonal functional assays","pmids":["41896526"],"is_preprint":false}],"current_model":"ZNRF1 is a membrane-associated RING/UBZ-domain E3 ubiquitin ligase that, upon activation by stress signals (oxidative stress via EGFR/NADPH oxidase-mediated phosphorylation at Y103 or Src kinase phosphorylation), ubiquitinates and degrades multiple substrates—including AKT (driving GSK3B-CRMP2-dependent axonal degeneration), CAV1 (modulating TLR4/AKT/GSK3β inflammatory signaling), EGFR and TLR3 (promoting lysosomal sorting to terminate signaling), and the Na+/K+ATPase α1 subunit (via K63-linked chains with Ube2N/Uvc13)—and also controls FasL surface trafficking through a Munc18-2-Syntaxin-3 SNARE axis, placing ZNRF1 as a central activity-regulated E3 ligase coordinating neuronal degeneration, immune receptor trafficking, and membrane protein homeostasis."},"narrative":{"teleology":[{"year":2009,"claim":"Identification of β-tubulin (Tubb2) as a ZNRF1 interactor established that ZNRF1 associates with cytoskeletal components and that its RING and zinc finger domains are required for morphological effects on neurites, providing the first functional hints beyond its predicted E3 ligase domain structure.","evidence":"Yeast two-hybrid screen and co-immunoprecipitation in neuronal-like cells with domain deletion analysis","pmids":["19737534"],"confidence":"Medium","gaps":["Single lab, single Y2H/Co-IP without reciprocal validation","Whether Tubb2 is a ubiquitination substrate was not tested","Morphological readout lacked mechanistic resolution"]},{"year":2011,"claim":"Demonstration that ZNRF1 ubiquitinates AKT for proteasomal degradation, relieving GSK3β inhibition and driving CRMP2 phosphorylation-dependent microtubule disassembly, established the first complete signaling pathway through which ZNRF1 promotes Wallerian degeneration.","evidence":"Neuronal overexpression/knockdown, ubiquitin-proteasome assays, epistasis with constitutively active GSK3β and CRMP2 phosphomutants, axonal degeneration assays","pmids":["22057101"],"confidence":"High","gaps":["Upstream activation mechanism of ZNRF1 was unknown","Whether AKT degradation by ZNRF1 operates outside neurons was untested"]},{"year":2012,"claim":"Reconstitution of ZNRF1-mediated K63-linked ubiquitination of the Na⁺/K⁺-ATPase α1 subunit via Ubc13/Uev1a, and mapping of its N-myristoylation-dependent membrane association and UBZ-domain-mediated substrate recognition, defined the biochemical architecture of ZNRF1 as a membrane-associated E3 with chain-type specificity.","evidence":"In vitro ubiquitination reconstitution, domain mapping, subcellular fractionation, Co-IP","pmids":["22797923"],"confidence":"High","gaps":["Functional consequence of Na⁺/K⁺-ATPase K63-ubiquitination (degradation vs. trafficking) was not resolved","Structural basis of UBZ-substrate recognition unknown"]},{"year":2015,"claim":"Discovery that oxidative stress activates ZNRF1 through EGFR-dependent phosphorylation at Y103, downstream of NADPH oxidase, answered how ZNRF1 ligase activity is switched on and linked receptor tyrosine kinase signaling to axonal degeneration.","evidence":"Y103 mutagenesis, EGFR and NADPH oxidase pharmacological inhibition, ligase activity and neuronal degeneration assays","pmids":["26572622"],"confidence":"High","gaps":["Whether other kinases phosphorylate Y103 was not explored","Whether Y103 phosphorylation regulates all ZNRF1 substrates or only AKT was unknown"]},{"year":2017,"claim":"Identification of caveolin-1 as a ZNRF1 substrate in macrophages, degraded via K48-linked ubiquitination upon LPS stimulation, expanded ZNRF1 function from neurons to innate immunity, showing it amplifies TLR4-driven pro-inflammatory cytokine production by modulating AKT-GSK3β signaling.","evidence":"Reciprocal Co-IP, ubiquitination assays, hematopoietic ZNRF1 KO mice, endotoxic shock model with cytokine measurements","pmids":["28593998"],"confidence":"High","gaps":["Whether ZNRF1 is transcriptionally induced by LPS or post-translationally activated was not fully resolved","Direct ubiquitination site on CAV1 not mapped"]},{"year":2018,"claim":"Crystal structure of the ZNRF1 RING domain bound to Ube2N at ~50 nM affinity, compared with ~1 µM for Ube2D2, revealed the structural basis for E2 selectivity and an unusual stoichiometry-dependent inhibition mechanism whereby excess ZNRF1 suppresses ubiquitin chain assembly.","evidence":"X-ray crystallography, isothermal titration calorimetry, in vitro ubiquitination with mutagenesis","pmids":["29626159"],"confidence":"High","gaps":["Whether stoichiometric inhibition operates in vivo was not demonstrated","Full-length ZNRF1 structure including UBZ domain remains unsolved"]},{"year":2021,"claim":"Showing that ZNRF1 ubiquitinates EGFR at sites distinct from CBL to promote endosome-to-lysosome sorting established ZNRF1 as a non-redundant regulator of receptor tyrosine kinase trafficking, with loss of ZNRF1 prolonging downstream signaling and increasing HSV-1 susceptibility.","evidence":"ZNRF1 KO cells, ubiquitin site mapping on EGFR, endosomal/lysosomal trafficking and degradation assays","pmids":["33996800"],"confidence":"High","gaps":["Whether ZNRF1 targets other receptor tyrosine kinases beyond EGFR was not tested","Mechanism of ZNRF1 recruitment to endosomes not defined"]},{"year":2023,"claim":"Demonstration that c-Src phosphorylates ZNRF1 Y103 downstream of TLR3, enabling K63-linked ubiquitination of TLR3 at K813 for MVB/lysosomal sorting, unified the Y103 activation mechanism across RTK and innate immune signaling and identified a negative feedback loop terminating type I interferon responses.","evidence":"ZNRF1-deficient mice and cells, Y103 and K813 site-directed mutagenesis, ubiquitination and endolysosomal trafficking assays, EMCV and SARS-CoV-2 infection models","pmids":["37158982"],"confidence":"High","gaps":["Whether ZNRF1 regulates other TLRs via similar K63-ubiquitination was not tested","In vivo kinetics of TLR3 turnover in ZNRF1 KO during infection not fully characterized"]},{"year":2023,"claim":"LZTFL1-dependent recruitment of ZNRF1 to AKT for proteasomal degradation in kidney tumor cells established that the ZNRF1-AKT axis operates in cancer contexts and is regulated by upstream tumor suppressors.","evidence":"Gain- and loss-of-function in kidney tumor cell lines, patient-derived xenograft model","pmids":["36966254"],"confidence":"Medium","gaps":["How LZTFL1 recruits or activates ZNRF1 mechanistically is unclear","Whether Y103 phosphorylation is involved in tumor cell context not addressed"]},{"year":2024,"claim":"Discovery that the Drosophila ZNRF1/2 ortholog Detour ubiquitinates HOPS complex subunits (VPS18, VPS16A, VPS41) to regulate autophagosome-lysosome fusion, with mammalian ZNRF1/2 loss increasing basal autophagy, extended the substrate repertoire to vesicle tethering machinery and autophagy regulation.","evidence":"Drosophila genetic screen, mass spectrometry interactome, ubiquitination assay, autophagy flux assays in mammalian cells","pmids":["38360932"],"confidence":"Medium","gaps":["Drosophila ortholog may have divergent specificity from mammalian ZNRF1","Whether ZNRF1 specifically (vs. ZNRF2) ubiquitinates mammalian HOPS subunits not resolved","Ubiquitin chain type on HOPS subunits not determined"]},{"year":2025,"claim":"ZNRF1 KO mice revealed that ZNRF1-dependent AKT degradation positions the axon initial segment and controls Nav1.2 surface expression, linking the E3 ligase to neuronal excitability and fear memory, extending the AKT substrate axis to a systems-level behavioral output.","evidence":"ZNRF1 KO mice, AIS morphometry, Nav1.2 surface biotinylation, fear conditioning behavioral assay","pmids":["40331626"],"confidence":"Medium","gaps":["Whether Nav1.2 is a direct ZNRF1 substrate or an indirect consequence of AKT-GSK3β signaling not resolved","Single lab finding"]},{"year":2025,"claim":"Myeloid-specific ZNRF1 KO showed elevated surface MHC class II on macrophages, enhanced T cell priming, and exacerbated EAE, establishing ZNRF1 as a negative regulator of antigen presentation and autoimmune neuroinflammation.","evidence":"Myeloid-specific ZNRF1 KO mice, flow cytometry, EAE model, T cell proliferation assays","pmids":["41126242"],"confidence":"Medium","gaps":["Whether ZNRF1 directly ubiquitinates MHC-II or acts indirectly (e.g., via CAV1 or trafficking) not determined","Single lab, single disease model"]},{"year":2026,"claim":"Reconstitution experiments with catalytically dead ZNRF1 (C184A) demonstrated that ZNRF1 E3 ligase activity supports the Munc18-2–Syntaxin-3 SNARE complex required for FasL surface trafficking, revealing a non-degradative role in vesicle docking/fusion at the plasma membrane.","evidence":"Myeloid-specific ZNRF1 KO, C184A catalytic mutant reconstitution, Stxbp2-Stx3 Co-IP, confocal imaging, surface FasL flow cytometry, FasL-dependent killing assays","pmids":["41896526"],"confidence":"High","gaps":["Direct ubiquitination substrate enabling Munc18-2–Stx3 interaction not identified","Whether this SNARE-regulatory function extends to other secretory cargoes unknown"]},{"year":null,"claim":"Key unresolved questions include: the identity of the direct ubiquitination target through which ZNRF1 controls the Munc18-2–Stx3 SNARE complex; whether ZNRF1 directly ubiquitinates MHC-II; the full-length ZNRF1 structure including UBZ domain; and how stoichiometry-dependent E2 inhibition operates in physiological settings.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length structural model","Direct MHC-II ubiquitination by ZNRF1 not tested","SNARE-regulatory substrate unidentified","In vivo relevance of stoichiometric E2 inhibition not demonstrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,5,6,14]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,3,4,5,6,14]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,14]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5,6]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[3,5]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,3,4,5,6,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,5,6,9]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,6,12]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[5,6,14]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[10]}],"complexes":[],"partners":["AKT1","CAV1","EGFR","TLR3","UBE2N","ATP1A1","STXBP2","STX3"],"other_free_text":[]},"mechanistic_narrative":"ZNRF1 is a membrane-associated RING-type E3 ubiquitin ligase that functions as a stress- and signal-activated hub coordinating protein degradation, receptor trafficking, and membrane protein homeostasis across neuronal and immune cell contexts. Its catalytic activity is controlled by tyrosine 103 phosphorylation—mediated by EGFR (downstream of NADPH oxidase-generated ROS) or c-Src kinase (downstream of TLR3)—enabling ZNRF1 to ubiquitinate substrates including AKT (K48-linked, proteasomal degradation driving GSK3β-CRMP2–dependent axonal degeneration and axon initial segment positioning), caveolin-1 (K48-linked, amplifying TLR4-driven inflammation), EGFR (endosome-to-lysosome sorting distinct from CBL), and TLR3 (K63-linked, multivesicular body sorting to terminate type I interferon signaling) [PMID:22057101, PMID:26572622, PMID:28593998, PMID:33996800, PMID:37158982]. Structurally, its RING domain engages the E2 Ube2N with nanomolar affinity, and an unusual stoichiometry-dependent inhibition mechanism modulates ubiquitin chain assembly; its UBZ domain mediates substrate recognition, as shown for the Na⁺/K⁺-ATPase α1 subunit [PMID:29626159, PMID:22797923]. Beyond degradative ubiquitination, ZNRF1 E3 ligase activity supports the Munc18-2–Syntaxin-3 SNARE interaction required for FasL surface trafficking in macrophages and regulates MHC class II surface expression on myeloid cells, linking it to cytotoxic effector function and antigen presentation [PMID:41896526, PMID:41126242]."},"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":21,"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":5,"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":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},{"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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8577,"output_tokens":3651,"usd":0.040248},"stage2":{"model":"claude-opus-4-6","input_tokens":7076,"output_tokens":3967,"usd":0.201832},"total_usd":0.24208,"stage1_batch_id":"msgbatch_01BparvgRZ9S2LMidvaoo86K","stage2_batch_id":"msgbatch_01NkBfv9SVZWthhS7j6mU6G3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"ZNRF1 acts as an E3 ubiquitin ligase that targets AKT for proteasomal degradation, thereby relieving AKT-mediated inhibitory phosphorylation of GSK3B, which then phosphorylates CRMP2 to drive microtubule reorganization and Wallerian degeneration in axons.\",\n      \"method\": \"Overexpression and knockdown in neurons, ubiquitin-proteasome assays, epistasis analysis with active/inactive GSK3B and CRMP2 mutants, axonal degeneration assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, genetic epistasis, pathway placement; replicated across subsequent studies\",\n      \"pmids\": [\"22057101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Oxidative stress activates ZNRF1 E3 ligase activity via EGFR-mediated phosphorylation at tyrosine 103, requiring NADPH oxidase activity; this phosphorylation-activated ZNRF1 then degrades AKT to promote neuronal apoptosis and Wallerian degeneration.\",\n      \"method\": \"Phosphorylation-site mutagenesis (Y103), EGFR inhibition, NADPH oxidase inhibition, ubiquitin ligase activity assays, neuronal degeneration assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis of active-site residue combined with pharmacological epistasis and functional readout\",\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 K48-linked ubiquitination and proteasomal degradation of CAV1, thereby modulating AKT-GSK3β signaling downstream of TLR4 to enhance pro-inflammatory cytokine production and suppress IL-10.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ZNRF1 KO mice with hematopoietic deletion, cytokine measurements, endotoxic shock model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, in vivo KO with defined phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"28593998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ZNRF1 localizes to intracellular membranes via N-myristoylation, interacts with the Na+/K+ATPase α1 subunit through its UBZ domain, and together with its RING domain engaging E2 Ubc13/Uev1a mediates K63-linked ubiquitination of the cytoplasmic loop of the Na+/K+ATPase α1 subunit.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, subcellular fractionation, ouabain treatment, ZNRF2 knockdown\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitination reconstitution, domain mapping, reciprocal Co-IP\",\n      \"pmids\": [\"22797923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The RING domain of ZNRF1 binds E2 ubiquitin-conjugating enzyme Ube2N with nanomolar affinity (~50 nM Kd), far higher than its affinity for Ube2D2 (~1 µM); crystal structure of ZNRF1 C-terminal domain in complex with Ube2N reveals the molecular basis, and excess ZNRF1 inhibits Ube2N-mediated ubiquitination, indicating a novel activity-regulation mechanism dependent on E3:E2 stoichiometry.\",\n      \"method\": \"Crystal structure determination, ITC/binding assays, in vitro ubiquitination assay, mutagenesis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and in vitro ubiquitination reconstitution\",\n      \"pmids\": [\"29626159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ZNRF1 mediates ligand-induced ubiquitination of EGFR at lysine residues distinct from those targeted by CBL, promoting endosome-to-lysosome sorting and degradation of EGFR; ZNRF1 deletion delays receptor degradation, prolongs downstream signaling, and increases susceptibility to HSV-1 entry.\",\n      \"method\": \"ZNRF1 knockout cells, EGFR ubiquitination assay, endosomal trafficking assay, lysosomal degradation assay, site-specific ubiquitin mapping\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with defined trafficking and signaling phenotype, ubiquitin site mapping, epistasis with CBL\",\n      \"pmids\": [\"33996800\"],\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, promoting TLR3 sorting into multivesicular bodies/lysosomes and terminating type I interferon signaling.\",\n      \"method\": \"ZNRF1-deficient mice and cells, site-specific mutagenesis (Y103, K813), ubiquitination assays, endolysosomal trafficking assays, viral infection models (EMCV, SARS-CoV-2)\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis of phosphorylation and ubiquitination sites, in vivo KO, defined signaling and trafficking phenotype\",\n      \"pmids\": [\"37158982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ZNRF1 interacts with beta-tubulin type 2 (Tubb2) via its RING finger and zinc finger domains; overexpression of ZNRF1 induces neurite-like morphological changes, and both RING and zinc finger domains are required for this effect.\",\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 — single Co-IP and yeast two-hybrid, single lab\",\n      \"pmids\": [\"19737534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NADPH oxidases are required upstream of EGFR-dependent phosphorylation to activate ZNRF1, which then promotes AKT degradation and neuronal apoptosis; this pathway operates even when oxidative stress is applied exogenously.\",\n      \"method\": \"NADPH oxidase inhibition, EGFR inhibition, neuronal apoptosis assays, AKT degradation assays\",\n      \"journal\": \"Communicative & integrative biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological epistasis, extends prior mechanistic findings; single lab\",\n      \"pmids\": [\"27195063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LZTFL1 tumor suppressor promotes AKT degradation through ZNRF1-mediated ubiquitin-proteasome pathway to inhibit kidney tumor cell proliferation.\",\n      \"method\": \"Gain- and loss-of-function studies in kidney tumor cell lines, PDX model, AKT degradation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO/OE with defined substrate degradation phenotype, PDX model; single lab\",\n      \"pmids\": [\"36966254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Drosophila ZNRF1/2 homologue detour (CG14435) interacts with HOPS complex subunits (VPS18, VPS16A, VPS41) and promotes their ubiquitination to regulate autophagosome-lysosome fusion; mammalian ZNRF1 and ZNRF2 ablation increases basal autophagy in mammalian cells.\",\n      \"method\": \"Drosophila genetic depletion and overexpression, mass spectrometry interactome, ubiquitination assay, autophagy flux assays in mammalian cells\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS interactome, ubiquitination assay; Drosophila ortholog with mammalian validation\",\n      \"pmids\": [\"38360932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZNRF1-dependent AKT degradation in neurons controls axon initial segment (AIS) position and promotes increased cell surface localization of voltage-gated sodium channel Nav1.2; ZNRF1 KO mice show AIS shift and enhanced fear memory.\",\n      \"method\": \"ZNRF1 KO mice, AIS morphology analysis, Nav1.2 surface biotinylation, fear conditioning behavior\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with defined cellular and behavioral phenotype; single lab\",\n      \"pmids\": [\"40331626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZNRF1 in peripheral myeloid macrophages regulates surface MHC class II expression; ZNRF1-deficient macrophages display elevated surface MHC-II, leading to enhanced antigen-specific T cell proliferation and exacerbated autoimmune demyelination (EAE).\",\n      \"method\": \"Myeloid-specific ZNRF1 KO mice, flow cytometry for MHC-II surface expression, EAE model, T cell proliferation assays\",\n      \"journal\": \"Journal of neuroinflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with defined surface protein and immune phenotype; single lab\",\n      \"pmids\": [\"41126242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ZNRF1 interacts with iron transport protein lipocalin-2 (LCN2) and this interaction is regulated by TFAM; ZNRF1 overexpression maintains mitochondrial integrity and inhibits ferroptosis in renal tubular epithelial cells.\",\n      \"method\": \"Co-immunoprecipitation, ZNRF1 overexpression in ferroptosis model, TFAM-deficient mice, transcriptional sequencing\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP, limited mechanistic follow-up, single lab\",\n      \"pmids\": [\"39349116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ZNRF1 E3 ligase activity (requiring catalytic C184 residue) is required for terminal trafficking and surface exposure of Fas ligand (FasL) in macrophages by supporting the Munc18-2 (Stxbp2)-Syntaxin-3 (Stx3) interaction; ZNRF1 deficiency weakens this SNARE complex interaction and blocks FasL docking/fusion at the plasma membrane without affecting lysosome-related organelle polarization.\",\n      \"method\": \"Myeloid-specific ZNRF1 KO, catalytically inactive ZNRF1 C184A reconstitution, co-immunoprecipitation of Stxbp2-Stx3, confocal imaging of LAMP1 and FasL, surface FasL flow cytometry, FasL-dependent killing assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — catalytic mutant reconstitution, Co-IP of SNARE complex, multiple orthogonal functional assays\",\n      \"pmids\": [\"41896526\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ZNRF1 is a membrane-associated RING/UBZ-domain E3 ubiquitin ligase that, upon activation by stress signals (oxidative stress via EGFR/NADPH oxidase-mediated phosphorylation at Y103 or Src kinase phosphorylation), ubiquitinates and degrades multiple substrates—including AKT (driving GSK3B-CRMP2-dependent axonal degeneration), CAV1 (modulating TLR4/AKT/GSK3β inflammatory signaling), EGFR and TLR3 (promoting lysosomal sorting to terminate signaling), and the Na+/K+ATPase α1 subunit (via K63-linked chains with Ube2N/Uvc13)—and also controls FasL surface trafficking through a Munc18-2-Syntaxin-3 SNARE axis, placing ZNRF1 as a central activity-regulated E3 ligase coordinating neuronal degeneration, immune receptor trafficking, and membrane protein homeostasis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ZNRF1 is a membrane-associated RING-type E3 ubiquitin ligase that functions as a stress- and signal-activated hub coordinating protein degradation, receptor trafficking, and membrane protein homeostasis across neuronal and immune cell contexts. Its catalytic activity is controlled by tyrosine 103 phosphorylation—mediated by EGFR (downstream of NADPH oxidase-generated ROS) or c-Src kinase (downstream of TLR3)—enabling ZNRF1 to ubiquitinate substrates including AKT (K48-linked, proteasomal degradation driving GSK3β-CRMP2–dependent axonal degeneration and axon initial segment positioning), caveolin-1 (K48-linked, amplifying TLR4-driven inflammation), EGFR (endosome-to-lysosome sorting distinct from CBL), and TLR3 (K63-linked, multivesicular body sorting to terminate type I interferon signaling) [PMID:22057101, PMID:26572622, PMID:28593998, PMID:33996800, PMID:37158982]. Structurally, its RING domain engages the E2 Ube2N with nanomolar affinity, and an unusual stoichiometry-dependent inhibition mechanism modulates ubiquitin chain assembly; its UBZ domain mediates substrate recognition, as shown for the Na⁺/K⁺-ATPase α1 subunit [PMID:29626159, PMID:22797923]. Beyond degradative ubiquitination, ZNRF1 E3 ligase activity supports the Munc18-2–Syntaxin-3 SNARE interaction required for FasL surface trafficking in macrophages and regulates MHC class II surface expression on myeloid cells, linking it to cytotoxic effector function and antigen presentation [PMID:41896526, PMID:41126242].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of β-tubulin (Tubb2) as a ZNRF1 interactor established that ZNRF1 associates with cytoskeletal components and that its RING and zinc finger domains are required for morphological effects on neurites, providing the first functional hints beyond its predicted E3 ligase domain structure.\",\n      \"evidence\": \"Yeast two-hybrid screen and co-immunoprecipitation in neuronal-like cells with domain deletion analysis\",\n      \"pmids\": [\"19737534\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single Y2H/Co-IP without reciprocal validation\", \"Whether Tubb2 is a ubiquitination substrate was not tested\", \"Morphological readout lacked mechanistic resolution\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstration that ZNRF1 ubiquitinates AKT for proteasomal degradation, relieving GSK3β inhibition and driving CRMP2 phosphorylation-dependent microtubule disassembly, established the first complete signaling pathway through which ZNRF1 promotes Wallerian degeneration.\",\n      \"evidence\": \"Neuronal overexpression/knockdown, ubiquitin-proteasome assays, epistasis with constitutively active GSK3β and CRMP2 phosphomutants, axonal degeneration assays\",\n      \"pmids\": [\"22057101\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream activation mechanism of ZNRF1 was unknown\", \"Whether AKT degradation by ZNRF1 operates outside neurons was untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Reconstitution of ZNRF1-mediated K63-linked ubiquitination of the Na⁺/K⁺-ATPase α1 subunit via Ubc13/Uev1a, and mapping of its N-myristoylation-dependent membrane association and UBZ-domain-mediated substrate recognition, defined the biochemical architecture of ZNRF1 as a membrane-associated E3 with chain-type specificity.\",\n      \"evidence\": \"In vitro ubiquitination reconstitution, domain mapping, subcellular fractionation, Co-IP\",\n      \"pmids\": [\"22797923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Na⁺/K⁺-ATPase K63-ubiquitination (degradation vs. trafficking) was not resolved\", \"Structural basis of UBZ-substrate recognition unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovery that oxidative stress activates ZNRF1 through EGFR-dependent phosphorylation at Y103, downstream of NADPH oxidase, answered how ZNRF1 ligase activity is switched on and linked receptor tyrosine kinase signaling to axonal degeneration.\",\n      \"evidence\": \"Y103 mutagenesis, EGFR and NADPH oxidase pharmacological inhibition, ligase activity and neuronal degeneration assays\",\n      \"pmids\": [\"26572622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other kinases phosphorylate Y103 was not explored\", \"Whether Y103 phosphorylation regulates all ZNRF1 substrates or only AKT was unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of caveolin-1 as a ZNRF1 substrate in macrophages, degraded via K48-linked ubiquitination upon LPS stimulation, expanded ZNRF1 function from neurons to innate immunity, showing it amplifies TLR4-driven pro-inflammatory cytokine production by modulating AKT-GSK3β signaling.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assays, hematopoietic ZNRF1 KO mice, endotoxic shock model with cytokine measurements\",\n      \"pmids\": [\"28593998\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZNRF1 is transcriptionally induced by LPS or post-translationally activated was not fully resolved\", \"Direct ubiquitination site on CAV1 not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Crystal structure of the ZNRF1 RING domain bound to Ube2N at ~50 nM affinity, compared with ~1 µM for Ube2D2, revealed the structural basis for E2 selectivity and an unusual stoichiometry-dependent inhibition mechanism whereby excess ZNRF1 suppresses ubiquitin chain assembly.\",\n      \"evidence\": \"X-ray crystallography, isothermal titration calorimetry, in vitro ubiquitination with mutagenesis\",\n      \"pmids\": [\"29626159\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether stoichiometric inhibition operates in vivo was not demonstrated\", \"Full-length ZNRF1 structure including UBZ domain remains unsolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showing that ZNRF1 ubiquitinates EGFR at sites distinct from CBL to promote endosome-to-lysosome sorting established ZNRF1 as a non-redundant regulator of receptor tyrosine kinase trafficking, with loss of ZNRF1 prolonging downstream signaling and increasing HSV-1 susceptibility.\",\n      \"evidence\": \"ZNRF1 KO cells, ubiquitin site mapping on EGFR, endosomal/lysosomal trafficking and degradation assays\",\n      \"pmids\": [\"33996800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZNRF1 targets other receptor tyrosine kinases beyond EGFR was not tested\", \"Mechanism of ZNRF1 recruitment to endosomes not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstration that c-Src phosphorylates ZNRF1 Y103 downstream of TLR3, enabling K63-linked ubiquitination of TLR3 at K813 for MVB/lysosomal sorting, unified the Y103 activation mechanism across RTK and innate immune signaling and identified a negative feedback loop terminating type I interferon responses.\",\n      \"evidence\": \"ZNRF1-deficient mice and cells, Y103 and K813 site-directed mutagenesis, ubiquitination and endolysosomal trafficking assays, EMCV and SARS-CoV-2 infection models\",\n      \"pmids\": [\"37158982\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ZNRF1 regulates other TLRs via similar K63-ubiquitination was not tested\", \"In vivo kinetics of TLR3 turnover in ZNRF1 KO during infection not fully characterized\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"LZTFL1-dependent recruitment of ZNRF1 to AKT for proteasomal degradation in kidney tumor cells established that the ZNRF1-AKT axis operates in cancer contexts and is regulated by upstream tumor suppressors.\",\n      \"evidence\": \"Gain- and loss-of-function in kidney tumor cell lines, patient-derived xenograft model\",\n      \"pmids\": [\"36966254\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How LZTFL1 recruits or activates ZNRF1 mechanistically is unclear\", \"Whether Y103 phosphorylation is involved in tumor cell context not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that the Drosophila ZNRF1/2 ortholog Detour ubiquitinates HOPS complex subunits (VPS18, VPS16A, VPS41) to regulate autophagosome-lysosome fusion, with mammalian ZNRF1/2 loss increasing basal autophagy, extended the substrate repertoire to vesicle tethering machinery and autophagy regulation.\",\n      \"evidence\": \"Drosophila genetic screen, mass spectrometry interactome, ubiquitination assay, autophagy flux assays in mammalian cells\",\n      \"pmids\": [\"38360932\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Drosophila ortholog may have divergent specificity from mammalian ZNRF1\", \"Whether ZNRF1 specifically (vs. ZNRF2) ubiquitinates mammalian HOPS subunits not resolved\", \"Ubiquitin chain type on HOPS subunits not determined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"ZNRF1 KO mice revealed that ZNRF1-dependent AKT degradation positions the axon initial segment and controls Nav1.2 surface expression, linking the E3 ligase to neuronal excitability and fear memory, extending the AKT substrate axis to a systems-level behavioral output.\",\n      \"evidence\": \"ZNRF1 KO mice, AIS morphometry, Nav1.2 surface biotinylation, fear conditioning behavioral assay\",\n      \"pmids\": [\"40331626\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Nav1.2 is a direct ZNRF1 substrate or an indirect consequence of AKT-GSK3β signaling not resolved\", \"Single lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Myeloid-specific ZNRF1 KO showed elevated surface MHC class II on macrophages, enhanced T cell priming, and exacerbated EAE, establishing ZNRF1 as a negative regulator of antigen presentation and autoimmune neuroinflammation.\",\n      \"evidence\": \"Myeloid-specific ZNRF1 KO mice, flow cytometry, EAE model, T cell proliferation assays\",\n      \"pmids\": [\"41126242\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ZNRF1 directly ubiquitinates MHC-II or acts indirectly (e.g., via CAV1 or trafficking) not determined\", \"Single lab, single disease model\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Reconstitution experiments with catalytically dead ZNRF1 (C184A) demonstrated that ZNRF1 E3 ligase activity supports the Munc18-2–Syntaxin-3 SNARE complex required for FasL surface trafficking, revealing a non-degradative role in vesicle docking/fusion at the plasma membrane.\",\n      \"evidence\": \"Myeloid-specific ZNRF1 KO, C184A catalytic mutant reconstitution, Stxbp2-Stx3 Co-IP, confocal imaging, surface FasL flow cytometry, FasL-dependent killing assays\",\n      \"pmids\": [\"41896526\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ubiquitination substrate enabling Munc18-2–Stx3 interaction not identified\", \"Whether this SNARE-regulatory function extends to other secretory cargoes unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the identity of the direct ubiquitination target through which ZNRF1 controls the Munc18-2–Stx3 SNARE complex; whether ZNRF1 directly ubiquitinates MHC-II; the full-length ZNRF1 structure including UBZ domain; and how stoichiometry-dependent E2 inhibition operates in physiological settings.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length structural model\", \"Direct MHC-II ubiquitination by ZNRF1 not tested\", \"SNARE-regulatory substrate unidentified\", \"In vivo relevance of stoichiometric E2 inhibition not demonstrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6, 14]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 3, 4, 5, 6, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 14]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [3, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 5, 6, 9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 6, 12]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [5, 6, 14]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"AKT1\",\n      \"CAV1\",\n      \"EGFR\",\n      \"TLR3\",\n      \"UBE2N\",\n      \"ATP1A1\",\n      \"STXBP2\",\n      \"STX3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}