{"gene":"ZNRF2","run_date":"2026-06-11T09:02:07","timeline":{"discoveries":[{"year":2012,"finding":"ZNRF2 is N-myristoylated, which localizes it to intracellular membranes and enhances its ubiquitin ligase activity. Upon phosphorylation by insulin/growth factor signaling, ZNRF2 binds 14-3-3 proteins and is released into the cytosol (reverse myristoyl switch). On membranes, ZNRF2 interacts with the Na+/K+ATPase α1 subunit via its UBZ domain, while its RING domain interacts with E2 ubiquitin-conjugating enzymes, predominantly Ubc13/Uev1a, mediating Lys63-linked ubiquitination of the Na+/K+ATPase α1 subunit. Knockdown of ZNRF2 inhibits ouabain-induced decrease of cell surface and total Na+/K+ATPase α1 levels.","method":"N-myristoylation analysis, phosphorylation assays, Co-immunoprecipitation, UBZ/RING domain interaction studies, in vitro ubiquitination assay (Lys63 linkage), cell fractionation, knockdown with functional readout (Na+/K+ATPase levels)","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (Co-IP, in vitro ubiquitination, domain mutagenesis, fractionation, KD phenotype) in a single rigorous study","pmids":["22797923"],"is_preprint":false},{"year":2016,"finding":"ZNRF2 interacts with mTOR on membranes and promotes amino acid-stimulated translocation of mTORC1 to lysosomes and its activation. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. ZNRF2 knockdown decreases cell size and proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145; this phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and creates a negative feedback on mTORC1. Ser145 is dephosphorylated by protein phosphatase 6. ZNRF2 acts upstream of Rag-GTPases and the V-ATPase in the amino acid sensing machinery.","method":"Co-immunoprecipitation (ZNRF2–mTOR, ZNRF2–V-ATPase), phosphosite mapping (Ser145), phosphatase identification (PP6), cell fractionation (vesicle-to-cytosol translocation), knockdown with cell size/proliferation/mTORC1 activation readouts, lysosomal acidity assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, phosphosite mutagenesis, fractionation, KD phenotype, phosphatase identification) in a single rigorous study","pmids":["27244671"],"is_preprint":false},{"year":2023,"finding":"CREB1 directly binds to the promoter sequence of ZNRF2 and activates its transcription in breast cancer cells. ZNRF2 knockdown reverses the proliferation-promoting effect of CREB1 on breast cancer cells, placing ZNRF2 downstream of CREB1.","method":"Chromatin immunoprecipitation / promoter binding assay, ZNRF2 knockdown rescue experiments, functional proliferation assays","journal":"Human cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-based promoter binding plus epistasis (KD rescue), single lab","pmids":["37165255"],"is_preprint":false},{"year":2025,"finding":"ZNRF2 ubiquitinates SLC3A2 at K147 to regulate its translocation to the plasma membrane. This membrane localization of SLC3A2 inhibits ferroptosis in lung adenocarcinoma cells. A blocking peptide (Peptide K147) prevents SLC3A2 plasma membrane transport, attenuates ZNRF2's anti-ferroptotic effect, and reduces tumor cell proliferation in vitro and in vivo.","method":"Ubiquitination site mapping (K147), Co-IP/pulldown, plasma membrane fractionation, ferroptosis assays, peptide blocking experiments, in vitro and in vivo tumor growth assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — substrate ubiquitination site identified with functional validation in vitro and in vivo, single lab","pmids":["40999004"],"is_preprint":false},{"year":2025,"finding":"ZNRF2 knockdown in an MPTP mouse model of Parkinson's disease exacerbates motor dysfunction, accelerates dopamine neuron degeneration, and elevates pro-inflammatory cytokines (IL-1β, IL-6) while suppressing anti-inflammatory cytokines (IL-4, IL-10). ZNRF2 knockdown significantly elevates phosphorylated mTOR levels after MPTP treatment; pharmacological inhibition of mTOR subsequently ameliorates the exacerbated neuroinflammation, placing ZNRF2 upstream of mTOR as a negative regulator of mTOR-mediated neuroinflammation.","method":"ZNRF2 knockdown in MPTP mouse model, behavioral tests, immunohistochemistry, immunoblotting, ELISA, immunofluorescence, mTOR inhibitor rescue","journal":"Molecular neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (KD + mTOR inhibitor rescue) with multiple in vivo readouts, single lab","pmids":["40402410"],"is_preprint":false},{"year":2026,"finding":"ZNRF2 mediates proteasome-dependent degradation of CD-M6PR (cation-dependent mannose-6-phosphate receptor) in senescent cells. Elevated ZNRF2 expression in senescent cells (driven by stress-induced mTORC1 activation) reduces CD-M6PR protein levels, impairs lysosomal enzyme trafficking, and compromises autolysosomal function, thereby exacerbating cellular senescence. This defines an mTORC1–ZNRF2–CD-M6PR axis.","method":"Structural prediction and experimental validation of ZNRF2–CD-M6PR interaction, ubiquitination/proteasome inhibitor experiments, siRNA knockdown, cell senescence assays, lysosomal function assays, in vivo aged mouse/human tissue analysis","journal":"GeroScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — substrate identification with structural and experimental validation, proteasome degradation mechanistic link, pathway epistasis, single lab","pmids":["42065825"],"is_preprint":false}],"current_model":"ZNRF2 is an N-myristoylated RING-type E3 ubiquitin ligase that localizes to intracellular membranes, where it ubiquitinates substrates including the Na+/K+ATPase α1 subunit (via Ubc13/Uev1a, Lys63-linked chains) and SLC3A2 (at K147), and promotes mTORC1 lysosomal translocation and activation by interacting with mTOR and the V-ATPase; phosphorylation of ZNRF2 on Ser145 by mTORC1 (reversed by PP6) triggers 14-3-3 binding and cytosolic release, forming a negative feedback loop, while upstream CREB1 drives ZNRF2 transcription and stress-induced mTORC1 elevates ZNRF2 to degrade CD-M6PR and impair lysosomal function."},"narrative":{"mechanistic_narrative":"ZNRF2 is an N-myristoylated RING-type E3 ubiquitin ligase that operates at intracellular membranes to control substrate ubiquitination, lysosomal function, and mTORC1 signaling [PMID:22797923, PMID:27244671]. Membrane targeting via N-myristoylation enhances its ligase activity, and it engages substrates through its UBZ domain while recruiting E2 enzymes—predominantly Ubc13/Uev1a—through its RING domain to catalyze Lys63-linked ubiquitination of the Na+/K+ATPase α1 subunit [PMID:22797923]. ZNRF2 is governed by a reverse myristoyl switch: growth factor/insulin signaling drives its phosphorylation, 14-3-3 binding, and release from membranes into the cytosol [PMID:22797923]. It interacts with mTOR and the V-ATPase on lysosomal membranes, acting upstream of the Rag GTPases to promote amino acid-stimulated mTORC1 lysosomal translocation and activation while preserving lysosomal acidity; in turn, active mTORC1 phosphorylates ZNRF2 on Ser145 (reversed by protein phosphatase 6) to trigger its cytosolic release, forming a negative feedback loop, with ZNRF2 loss elevating mTOR activity [PMID:27244671, PMID:40402410]. Beyond Na+/K+ATPase, ZNRF2 ubiquitinates SLC3A2 at K147 to drive its plasma membrane localization and suppress ferroptosis, and mediates proteasome-dependent degradation of CD-M6PR to impair lysosomal enzyme trafficking in senescent cells [PMID:40999004, PMID:42065825]. ZNRF2 transcription is activated by CREB1, and ZNRF2 mediates CREB1's pro-proliferative effect in breast cancer [PMID:37165255].","teleology":[{"year":2012,"claim":"Established ZNRF2 as a membrane-targeted E3 ligase whose activity and localization are switch-controlled, answering how a RING ligase couples growth-factor signaling to substrate ubiquitination.","evidence":"N-myristoylation analysis, Co-IP, domain mutagenesis, in vitro Lys63 ubiquitination assay, fractionation, and knockdown of Na+/K+ATPase α1 in cells","pmids":["22797923"],"confidence":"High","gaps":["The kinase phosphorylating ZNRF2 to trigger the reverse myristoyl switch is not identified here","Whether Na+/K+ATPase α1 is the principal physiological substrate is not resolved"]},{"year":2016,"claim":"Placed ZNRF2 in the amino acid sensing machinery upstream of Rag GTPases and the V-ATPase, defining a phosphorylation-dependent negative feedback loop with mTORC1.","evidence":"Co-IP (mTOR, V-ATPase), Ser145 phosphosite mapping, PP6 identification, fractionation, and knockdown with cell size/proliferation/mTORC1 readouts","pmids":["27244671"],"confidence":"High","gaps":["The ubiquitination substrate by which ZNRF2 promotes mTORC1 translocation is not defined","Mechanism linking ZNRF2 to V-ATPase regulation of acidity is unresolved"]},{"year":2023,"claim":"Identified an upstream transcriptional input, showing CREB1 directly activates ZNRF2 to drive proliferation, connecting ZNRF2 to oncogenic signaling.","evidence":"ChIP/promoter binding assay and ZNRF2 knockdown rescue of CREB1-driven proliferation in breast cancer cells","pmids":["37165255"],"confidence":"Medium","gaps":["Single lab, single cancer context","Which ZNRF2 catalytic activity mediates the proliferative effect is not shown"]},{"year":2025,"claim":"Extended ZNRF2 substrate repertoire to SLC3A2, linking site-specific ubiquitination to membrane transport, ferroptosis suppression, and tumor growth.","evidence":"K147 ubiquitination site mapping, Co-IP, membrane fractionation, ferroptosis and peptide-blocking assays, in vitro and in vivo tumor growth","pmids":["40999004"],"confidence":"Medium","gaps":["Ubiquitin chain linkage type on SLC3A2 not specified","Single lab finding"]},{"year":2025,"claim":"Demonstrated an in vivo physiological role for ZNRF2 as a negative regulator of mTOR-mediated neuroinflammation, consistent with the mTORC1 feedback model.","evidence":"ZNRF2 knockdown in MPTP Parkinson's mouse model with behavioral, cytokine, and mTOR-inhibitor rescue readouts","pmids":["40402410"],"confidence":"Medium","gaps":["Direct molecular target of ZNRF2 in neurons not identified","Single lab, model-specific"]},{"year":2026,"claim":"Defined an mTORC1–ZNRF2–CD-M6PR axis in senescence, showing stress-elevated ZNRF2 degrades CD-M6PR to impair lysosomal function.","evidence":"Structural prediction plus experimental validation of ZNRF2–CD-M6PR interaction, proteasome-inhibitor ubiquitination experiments, siRNA, senescence and lysosomal assays, aged mouse/human tissue analysis","pmids":["42065825"],"confidence":"Medium","gaps":["Ubiquitin chain architecture directing proteasomal versus lysosomal fate not detailed","Single lab"]},{"year":null,"claim":"How ZNRF2 substrate selection and chain-type output are governed across its membrane substrates, and whether its dual role (mTORC1 promotion versus mTOR feedback inhibition) is context-dependent, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying structural model of substrate recognition","Reconciliation of pro- and anti-mTOR roles across tissues is unestablished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,3,5]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,5]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1,5]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,4]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,5]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[1,5]}],"complexes":[],"partners":["MTOR","ATP1A1","SLC3A2","YWHA (14-3-3)","V-ATPASE","PPP6C","CREB1","CD-M6PR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NHG8","full_name":"E3 ubiquitin-protein ligase ZNRF2","aliases":["Protein Ells2","RING finger protein 202","RING-type E3 ubiquitin transferase ZNRF2","Zinc/RING finger protein 2"],"length_aa":242,"mass_kda":24.1,"function":"E3 ubiquitin-protein ligase that plays a role in the establishment and maintenance of neuronal transmission and plasticity. Ubiquitinates the Na(+)/K(+) ATPase alpha-1 subunit/ATP1A1 and thereby influences its endocytosis and/or degradation (PubMed:22797923). Acts also as a positive regulator of mTORC1 activation by amino acids, which functions upstream of the V-ATPase and of Rag-GTPases (PubMed:27244671). In turn, phosphorylation by mTOR leads to its inhibition via targeting to the cytosol allowing a self-regulating feedback mechanism (PubMed:27244671)","subcellular_location":"Endosome membrane; Lysosome membrane; Presynaptic cell membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q8NHG8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ZNRF2","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SLC37A4","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/search/ZNRF2","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":"Uncertain","locations":[{"location":"Nucleoplasm","reliability":"Uncertain"},{"location":"Vesicles","reliability":"Uncertain"},{"location":"Nucleoli fibrillar center","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ZNRF2"},"hgnc":{"alias_symbol":["RNF202"],"prev_symbol":[]},"alphafold":{"accession":"Q8NHG8","domains":[{"cath_id":"3.30.160","chopping":"156-184","consensus_level":"medium","plddt":88.0238,"start":156,"end":184},{"cath_id":"3.30.40.10","chopping":"186-237","consensus_level":"medium","plddt":93.7729,"start":186,"end":237}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NHG8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NHG8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NHG8-F1-predicted_aligned_error_v6.png","plddt_mean":65.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ZNRF2","jax_strain_url":"https://www.jax.org/strain/search?query=ZNRF2"},"sequence":{"accession":"Q8NHG8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NHG8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NHG8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NHG8"}},"corpus_meta":[{"pmid":"30811764","id":"PMC_30811764","title":"Long non-coding RNA TTN-AS1 facilitates tumorigenesis of papillary thyroid cancer through modulating the miR-153-3p/ZNRF2 axis.","date":"2019","source":"The journal of gene medicine","url":"https://pubmed.ncbi.nlm.nih.gov/30811764","citation_count":50,"is_preprint":false},{"pmid":"27244671","id":"PMC_27244671","title":"The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids.","date":"2016","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/27244671","citation_count":31,"is_preprint":false},{"pmid":"28416774","id":"PMC_28416774","title":"MicroRNA-100 suppresses human osteosarcoma cell proliferation and chemo-resistance via ZNRF2.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/28416774","citation_count":29,"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":"27775798","id":"PMC_27775798","title":"The role of ZNRF2 in the growth of non-small cell lung cancer.","date":"2016","source":"European review for medical and pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/27775798","citation_count":8,"is_preprint":false},{"pmid":"37551845","id":"PMC_37551845","title":"A pancancer analysis of the oncogenic role of ZNRF2 in human tumours.","date":"2023","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37551845","citation_count":4,"is_preprint":false},{"pmid":"39953597","id":"PMC_39953597","title":"ZNRF2 is essential for gliomagenesis through orchestrating glycolysis and acts as a promising therapeutic target in glioma.","date":"2025","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39953597","citation_count":3,"is_preprint":false},{"pmid":"37165255","id":"PMC_37165255","title":"ZNRF2 as an oncogene is transcriptionally regulated by CREB1 in breast cancer models.","date":"2023","source":"Human cell","url":"https://pubmed.ncbi.nlm.nih.gov/37165255","citation_count":3,"is_preprint":false},{"pmid":"40987203","id":"PMC_40987203","title":"ZNRF2 integrates ubiquitination-driven ferroptosis and mitochondrial quality control in renal ischemia-reperfusion injury.","date":"2025","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/40987203","citation_count":1,"is_preprint":false},{"pmid":"40999004","id":"PMC_40999004","title":"Targeting ZNRF2-mediated SLC3A2 plasma membrane translocation enhances ferroptosis in lung adenocarcinoma.","date":"2025","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/40999004","citation_count":1,"is_preprint":false},{"pmid":"40402410","id":"PMC_40402410","title":"Deletion of ZNRF2 Exacerbates MPTP-Induced Parkinson's Disease by Activating mTOR-Mediated Neuroinflammatory Pathways.","date":"2025","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/40402410","citation_count":1,"is_preprint":false},{"pmid":"42065825","id":"PMC_42065825","title":"ZNRF2-mediated CD-M6PR degradation and lysosomal dysfunction aggravate cellular senescence and aging.","date":"2026","source":"GeroScience","url":"https://pubmed.ncbi.nlm.nih.gov/42065825","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8086,"output_tokens":2120,"usd":0.028029,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9134,"output_tokens":2769,"usd":0.057447,"stage2_stop_reason":"end_turn"},"total_usd":0.085476,"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\": 2012,\n      \"finding\": \"ZNRF2 is N-myristoylated, which localizes it to intracellular membranes and enhances its ubiquitin ligase activity. Upon phosphorylation by insulin/growth factor signaling, ZNRF2 binds 14-3-3 proteins and is released into the cytosol (reverse myristoyl switch). On membranes, ZNRF2 interacts with the Na+/K+ATPase α1 subunit via its UBZ domain, while its RING domain interacts with E2 ubiquitin-conjugating enzymes, predominantly Ubc13/Uev1a, mediating Lys63-linked ubiquitination of the Na+/K+ATPase α1 subunit. Knockdown of ZNRF2 inhibits ouabain-induced decrease of cell surface and total Na+/K+ATPase α1 levels.\",\n      \"method\": \"N-myristoylation analysis, phosphorylation assays, Co-immunoprecipitation, UBZ/RING domain interaction studies, in vitro ubiquitination assay (Lys63 linkage), cell fractionation, knockdown with functional readout (Na+/K+ATPase levels)\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (Co-IP, in vitro ubiquitination, domain mutagenesis, fractionation, KD phenotype) in a single rigorous study\",\n      \"pmids\": [\"22797923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ZNRF2 interacts with mTOR on membranes and promotes amino acid-stimulated translocation of mTORC1 to lysosomes and its activation. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. ZNRF2 knockdown decreases cell size and proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145; this phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and creates a negative feedback on mTORC1. Ser145 is dephosphorylated by protein phosphatase 6. ZNRF2 acts upstream of Rag-GTPases and the V-ATPase in the amino acid sensing machinery.\",\n      \"method\": \"Co-immunoprecipitation (ZNRF2–mTOR, ZNRF2–V-ATPase), phosphosite mapping (Ser145), phosphatase identification (PP6), cell fractionation (vesicle-to-cytosol translocation), knockdown with cell size/proliferation/mTORC1 activation readouts, lysosomal acidity assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, phosphosite mutagenesis, fractionation, KD phenotype, phosphatase identification) in a single rigorous study\",\n      \"pmids\": [\"27244671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CREB1 directly binds to the promoter sequence of ZNRF2 and activates its transcription in breast cancer cells. ZNRF2 knockdown reverses the proliferation-promoting effect of CREB1 on breast cancer cells, placing ZNRF2 downstream of CREB1.\",\n      \"method\": \"Chromatin immunoprecipitation / promoter binding assay, ZNRF2 knockdown rescue experiments, functional proliferation assays\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-based promoter binding plus epistasis (KD rescue), single lab\",\n      \"pmids\": [\"37165255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZNRF2 ubiquitinates SLC3A2 at K147 to regulate its translocation to the plasma membrane. This membrane localization of SLC3A2 inhibits ferroptosis in lung adenocarcinoma cells. A blocking peptide (Peptide K147) prevents SLC3A2 plasma membrane transport, attenuates ZNRF2's anti-ferroptotic effect, and reduces tumor cell proliferation in vitro and in vivo.\",\n      \"method\": \"Ubiquitination site mapping (K147), Co-IP/pulldown, plasma membrane fractionation, ferroptosis assays, peptide blocking experiments, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — substrate ubiquitination site identified with functional validation in vitro and in vivo, single lab\",\n      \"pmids\": [\"40999004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ZNRF2 knockdown in an MPTP mouse model of Parkinson's disease exacerbates motor dysfunction, accelerates dopamine neuron degeneration, and elevates pro-inflammatory cytokines (IL-1β, IL-6) while suppressing anti-inflammatory cytokines (IL-4, IL-10). ZNRF2 knockdown significantly elevates phosphorylated mTOR levels after MPTP treatment; pharmacological inhibition of mTOR subsequently ameliorates the exacerbated neuroinflammation, placing ZNRF2 upstream of mTOR as a negative regulator of mTOR-mediated neuroinflammation.\",\n      \"method\": \"ZNRF2 knockdown in MPTP mouse model, behavioral tests, immunohistochemistry, immunoblotting, ELISA, immunofluorescence, mTOR inhibitor rescue\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (KD + mTOR inhibitor rescue) with multiple in vivo readouts, single lab\",\n      \"pmids\": [\"40402410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ZNRF2 mediates proteasome-dependent degradation of CD-M6PR (cation-dependent mannose-6-phosphate receptor) in senescent cells. Elevated ZNRF2 expression in senescent cells (driven by stress-induced mTORC1 activation) reduces CD-M6PR protein levels, impairs lysosomal enzyme trafficking, and compromises autolysosomal function, thereby exacerbating cellular senescence. This defines an mTORC1–ZNRF2–CD-M6PR axis.\",\n      \"method\": \"Structural prediction and experimental validation of ZNRF2–CD-M6PR interaction, ubiquitination/proteasome inhibitor experiments, siRNA knockdown, cell senescence assays, lysosomal function assays, in vivo aged mouse/human tissue analysis\",\n      \"journal\": \"GeroScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — substrate identification with structural and experimental validation, proteasome degradation mechanistic link, pathway epistasis, single lab\",\n      \"pmids\": [\"42065825\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ZNRF2 is an N-myristoylated RING-type E3 ubiquitin ligase that localizes to intracellular membranes, where it ubiquitinates substrates including the Na+/K+ATPase α1 subunit (via Ubc13/Uev1a, Lys63-linked chains) and SLC3A2 (at K147), and promotes mTORC1 lysosomal translocation and activation by interacting with mTOR and the V-ATPase; phosphorylation of ZNRF2 on Ser145 by mTORC1 (reversed by PP6) triggers 14-3-3 binding and cytosolic release, forming a negative feedback loop, while upstream CREB1 drives ZNRF2 transcription and stress-induced mTORC1 elevates ZNRF2 to degrade CD-M6PR and impair lysosomal function.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ZNRF2 is an N-myristoylated RING-type E3 ubiquitin ligase that operates at intracellular membranes to control substrate ubiquitination, lysosomal function, and mTORC1 signaling [#0, #1]. Membrane targeting via N-myristoylation enhances its ligase activity, and it engages substrates through its UBZ domain while recruiting E2 enzymes—predominantly Ubc13/Uev1a—through its RING domain to catalyze Lys63-linked ubiquitination of the Na+/K+ATPase α1 subunit [#0]. ZNRF2 is governed by a reverse myristoyl switch: growth factor/insulin signaling drives its phosphorylation, 14-3-3 binding, and release from membranes into the cytosol [#0]. It interacts with mTOR and the V-ATPase on lysosomal membranes, acting upstream of the Rag GTPases to promote amino acid-stimulated mTORC1 lysosomal translocation and activation while preserving lysosomal acidity; in turn, active mTORC1 phosphorylates ZNRF2 on Ser145 (reversed by protein phosphatase 6) to trigger its cytosolic release, forming a negative feedback loop, with ZNRF2 loss elevating mTOR activity [#1, #4]. Beyond Na+/K+ATPase, ZNRF2 ubiquitinates SLC3A2 at K147 to drive its plasma membrane localization and suppress ferroptosis, and mediates proteasome-dependent degradation of CD-M6PR to impair lysosomal enzyme trafficking in senescent cells [#3, #5]. ZNRF2 transcription is activated by CREB1, and ZNRF2 mediates CREB1's pro-proliferative effect in breast cancer [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established ZNRF2 as a membrane-targeted E3 ligase whose activity and localization are switch-controlled, answering how a RING ligase couples growth-factor signaling to substrate ubiquitination.\",\n      \"evidence\": \"N-myristoylation analysis, Co-IP, domain mutagenesis, in vitro Lys63 ubiquitination assay, fractionation, and knockdown of Na+/K+ATPase α1 in cells\",\n      \"pmids\": [\"22797923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The kinase phosphorylating ZNRF2 to trigger the reverse myristoyl switch is not identified here\", \"Whether Na+/K+ATPase α1 is the principal physiological substrate is not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed ZNRF2 in the amino acid sensing machinery upstream of Rag GTPases and the V-ATPase, defining a phosphorylation-dependent negative feedback loop with mTORC1.\",\n      \"evidence\": \"Co-IP (mTOR, V-ATPase), Ser145 phosphosite mapping, PP6 identification, fractionation, and knockdown with cell size/proliferation/mTORC1 readouts\",\n      \"pmids\": [\"27244671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The ubiquitination substrate by which ZNRF2 promotes mTORC1 translocation is not defined\", \"Mechanism linking ZNRF2 to V-ATPase regulation of acidity is unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified an upstream transcriptional input, showing CREB1 directly activates ZNRF2 to drive proliferation, connecting ZNRF2 to oncogenic signaling.\",\n      \"evidence\": \"ChIP/promoter binding assay and ZNRF2 knockdown rescue of CREB1-driven proliferation in breast cancer cells\",\n      \"pmids\": [\"37165255\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single cancer context\", \"Which ZNRF2 catalytic activity mediates the proliferative effect is not shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended ZNRF2 substrate repertoire to SLC3A2, linking site-specific ubiquitination to membrane transport, ferroptosis suppression, and tumor growth.\",\n      \"evidence\": \"K147 ubiquitination site mapping, Co-IP, membrane fractionation, ferroptosis and peptide-blocking assays, in vitro and in vivo tumor growth\",\n      \"pmids\": [\"40999004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin chain linkage type on SLC3A2 not specified\", \"Single lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated an in vivo physiological role for ZNRF2 as a negative regulator of mTOR-mediated neuroinflammation, consistent with the mTORC1 feedback model.\",\n      \"evidence\": \"ZNRF2 knockdown in MPTP Parkinson's mouse model with behavioral, cytokine, and mTOR-inhibitor rescue readouts\",\n      \"pmids\": [\"40402410\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular target of ZNRF2 in neurons not identified\", \"Single lab, model-specific\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined an mTORC1–ZNRF2–CD-M6PR axis in senescence, showing stress-elevated ZNRF2 degrades CD-M6PR to impair lysosomal function.\",\n      \"evidence\": \"Structural prediction plus experimental validation of ZNRF2–CD-M6PR interaction, proteasome-inhibitor ubiquitination experiments, siRNA, senescence and lysosomal assays, aged mouse/human tissue analysis\",\n      \"pmids\": [\"42065825\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin chain architecture directing proteasomal versus lysosomal fate not detailed\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ZNRF2 substrate selection and chain-type output are governed across its membrane substrates, and whether its dual role (mTORC1 promotion versus mTOR feedback inhibition) is context-dependent, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying structural model of substrate recognition\", \"Reconciliation of pro- and anti-mTOR roles across tissues is unestablished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"mTOR\", \"ATP1A1\", \"SLC3A2\", \"YWHA (14-3-3)\", \"V-ATPase\", \"PPP6C\", \"CREB1\", \"CD-M6PR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}