{"gene":"WDFY1","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2015,"finding":"WDFY1 acts as a crucial adaptor protein in TLR3/4 signaling by physically interacting with TLR3 and TLR4 and mediating the recruitment of TRIF to these receptors, thereby enabling downstream NF-κB and IRF3 activation and type I interferon production. Overexpression potentiates signaling; depletion has the opposite effect.","method":"Co-immunoprecipitation, overexpression/knockdown experiments with NF-κB/IRF3 reporter assays, cytokine measurements","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP demonstrating physical interaction, combined with gain- and loss-of-function experiments showing consistent bidirectional effects, replicated in fish ortholog study (PMID:40748571)","pmids":["25736436"],"is_preprint":false},{"year":2022,"finding":"WDFY1 is a component of a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex that is essential for initiating lysophagy; the complex ubiquitinates LAMP2 on damaged lysosomes to trigger their autophagic clearance.","method":"Proteomic studies using transfection reagent-coated beads, further functional evaluations, E3 ligase activity assays, identification of LAMP2 as substrate","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — proteomic identification plus functional validation of complex activity and substrate ubiquitination, multiple orthogonal methods in single study","pmids":["36103833"],"is_preprint":false},{"year":2016,"finding":"NRP2 negatively regulates WDFY1 expression at the transcriptional level by preventing nuclear localization of the transcription factor FAC1, thereby maintaining WDFY1 at levels compatible with sustained endocytic activity in metastatic cancer cells.","method":"Transcriptional reporter assays, nuclear localization analysis of FAC1, NRP2 knockdown/overexpression with WDFY1 expression readouts","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (reporter assay, localization, expression) in single lab","pmids":["27026195"],"is_preprint":false},{"year":2019,"finding":"The transcription factor Hes1 suppresses type I IFN production by inhibiting WDFY1 expression indirectly: Hes1 directly binds and upregulates VEGF-C expression, which in turn downregulates WDFY1, attenuating TLR upstream signaling.","method":"Genome-wide ChIP-seq for Hes1 occupancy, Hes1 knockout mice, WDFY1 expression analysis, IFN production assays","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus KO mouse model with functional readouts, single lab","pmids":["31015298"],"is_preprint":false},{"year":2022,"finding":"WDFY1 protein is expressed in mouse testes, localizing to the cytoplasm of late pachytene spermatocytes to elongated spermatids; however, global Wdfy1 knockout male mice show normal spermatogenesis and fertility, demonstrating WDFY1 is dispensable for these processes.","method":"Transgenic FLAG-Wdfy1-mCherry reporter mice for localization; global Wdfy1 knockout mice with histological analysis of testes and fertility testing","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by transgenic reporter and KO phenotyping with histology, single lab","pmids":["35121371"],"is_preprint":false},{"year":2026,"finding":"Aged brain neurons secrete WDFY1 protein via extracellular vesicles (EVs) that are transferred to bone, where WDFY1 binds to the retromer complex to promote endosome-to-Golgi recycling of cathepsin D and peroxiredoxin 2, thereby inhibiting osteogenesis and augmenting adipogenesis, causing bone-fat imbalance and osteoporosis.","method":"Neuronal Wdfy1 knockout mice, EV inhibition experiments, co-immunoprecipitation (WDFY1-retromer interaction), gain/loss-of-function in brain-specific models, bone phenotype analysis","journal":"Nature aging","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for retromer binding plus multiple genetic models (whole brain, hippocampal, neuronal KO) with functional bone phenotype readouts, single study","pmids":["41491871"],"is_preprint":false},{"year":2025,"finding":"WDFY1 expression in follicular dendritic cells (FDCs) is required for immune complex (IC) presentation; WDFY1 knockout impairs IC binding to FDCs, reduces germinal center formation, and diminishes autoantibody production in a cGVHD lupus mouse model. B cells and T cells lacking WDFY1 retain normal intrinsic function, indicating the defect is FDC-intrinsic.","method":"WDFY1 knockout mice in cGVHD model, immunofluorescence for IC-FDC binding, mixed leukocyte reaction, B-cell reconstitution in Rag1-KO mice, autoantibody titers","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse model with multiple orthogonal functional assays (IC binding, GC formation, cell reconstitution), single lab","pmids":["40169152"],"is_preprint":false},{"year":2025,"finding":"In murine mesangial cells, a 500 bp distal fragment upstream of the wdfy1 5'-UTR drives approximately four-fold enhanced promoter activity; transcription factors Sp1, Ap-1, Hes1, and TCF7 bind sites in the promoter/5'-UTR to regulate WDFY1 expression; the 3'-UTR suppresses expression (~3.5-fold); IL-6 upregulates WDFY1 through Sp1.","method":"Luciferase reporter assays with promoter deletion mutants, siRNA knockdown of transcription factors, IL-6 stimulation of primary renal mesangial cells","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complementary luciferase and siRNA assays in primary cells, single lab","pmids":["40497974"],"is_preprint":false},{"year":2025,"finding":"Grass carp WDFY1 (CiWDFY1) physically interacts with TLR3, and TLR3 recruits both TRIF and CiWDFY1 to form an activated complex leading to IRF3 phosphorylation and nuclear translocation and IFN1 induction; CiWDFY1 knockdown disrupts the TLR3-TRIF physical interaction. Upon poly(I:C) stimulation, CiWDFY1 redistributes from diffuse cytoplasmic to perimembranous localization.","method":"Co-immunoprecipitation, co-localization assays, overexpression and knockdown in CIK cells, subcellular localization imaging, IRF3 phosphorylation/translocation assays","journal":"Fish physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and co-localization plus gain/loss-of-function in fish ortholog, consistent with mammalian findings; single lab","pmids":["40748571"],"is_preprint":false},{"year":2018,"finding":"WDFY1 siRNA knockdown decreases differentiation ability of primary neural stem cells; introduction of WDFY1 plasmid rescues reduced TLR4 expression and neurogenesis in neural stem cells from PRDX6-overexpressing transgenic mice, placing WDFY1 downstream of PRDX6 and upstream of TLR4 in a neurogenesis pathway.","method":"siRNA knockdown, plasmid rescue experiment in primary neural stem cells, neurogenesis assays, microarray identification of WDFY1 downregulation in PRDX6-Tg mice","journal":"Molecular neurobiology","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — rescue experiment and siRNA in primary cells support pathway placement, but mechanistic detail is limited and single lab","pmids":["30097850"],"is_preprint":false}],"current_model":"WDFY1 is a WD-repeat and FYVE-domain scaffolding/adaptor protein that bridges TLR3/TLR4 with the signalling adaptor TRIF to activate NF-κB, IRF3, and type I interferon production; participates as a subunit of a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex that ubiquitinates LAMP2 on damaged lysosomes to initiate lysophagy; interacts with the retromer complex in neurons to regulate endosome-to-Golgi recycling of cathepsin D and peroxiredoxin 2, and is transferred via extracellular vesicles from aged neurons to bone to drive bone-fat imbalance; and in follicular dendritic cells supports immune complex presentation and germinal center responses."},"narrative":{"mechanistic_narrative":"WDFY1 is a WD-repeat and FYVE-domain adaptor protein that functions at the interface of innate immune signalling and endolysosomal membrane traffic [PMID:25736436, PMID:36103833]. In Toll-like receptor signalling, WDFY1 physically associates with TLR3 and TLR4 and recruits the adaptor TRIF to these receptors, driving NF-κB and IRF3 activation and type I interferon production; gain of function potentiates and loss of function attenuates this output, and the role is conserved in a fish TLR3 ortholog where WDFY1 redistributes to a perimembranous compartment upon poly(I:C) stimulation [PMID:25736436, PMID:40748571]. Separately, WDFY1 is a substrate-determining subunit of a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase that ubiquitinates LAMP2 on damaged lysosomes to initiate lysophagy [PMID:36103833]. WDFY1 also binds the retromer complex to promote endosome-to-Golgi recycling of cathepsin D and peroxiredoxin 2; aged neurons package WDFY1 into extracellular vesicles that transfer this activity to bone, where it shifts the osteogenic–adipogenic balance toward osteoporosis [PMID:41491871]. At the organismal level WDFY1 supports immune complex presentation by follicular dendritic cells and germinal center responses [PMID:40169152], and its expression is tightly controlled by transcriptional inputs including an NRP2/FAC1 axis, Hes1-VEGF-C, and IL-6/Sp1 signalling [PMID:27026195, PMID:31015298, PMID:40497974].","teleology":[{"year":2015,"claim":"Established WDFY1 as a positive adaptor in TLR3/TLR4 signalling, answering how these receptors couple to the TRIF branch driving interferon responses.","evidence":"Reciprocal Co-IP plus overexpression/knockdown with NF-κB/IRF3 reporters and cytokine readouts in mammalian cells","pmids":["25736436"],"confidence":"High","gaps":["Does not define which domain (WD vs FYVE) mediates TLR or TRIF binding","No structural model of the TLR–WDFY1–TRIF assembly"]},{"year":2016,"claim":"Identified an upstream transcriptional brake, showing NRP2 limits WDFY1 levels via FAC1 nuclear exclusion to tune endocytic activity in cancer cells.","evidence":"Transcriptional reporter assays, FAC1 localization analysis, and NRP2 perturbation with WDFY1 readouts","pmids":["27026195"],"confidence":"Medium","gaps":["Mechanism linking endocytic activity to WDFY1 abundance not biochemically resolved","Single-lab finding without in vivo validation"]},{"year":2018,"claim":"Placed WDFY1 within a PRDX6→WDFY1→TLR4 neurogenesis axis, connecting it to neural stem cell differentiation.","evidence":"siRNA knockdown and plasmid rescue of TLR4 expression and neurogenesis in primary neural stem cells from PRDX6-Tg mice","pmids":["30097850"],"confidence":"Low","gaps":["Limited mechanistic detail and single lab","Direct molecular link between WDFY1 and TLR4 expression unresolved"]},{"year":2019,"claim":"Defined a Hes1-VEGF-C cascade that suppresses WDFY1 to dampen type I IFN, integrating WDFY1 regulation into a transcriptional network.","evidence":"Genome-wide Hes1 ChIP-seq, Hes1 knockout mice, and WDFY1/IFN production analyses","pmids":["31015298"],"confidence":"Medium","gaps":["Indirect (VEGF-C-mediated) link to WDFY1 not mechanistically dissected","How VEGF-C downregulates WDFY1 unknown"]},{"year":2022,"claim":"Revealed a distinct E3-ligase role: WDFY1 forms a CUL4A-DDB1-WDFY1 complex that ubiquitinates LAMP2 to trigger lysophagy of damaged lysosomes.","evidence":"Proteomic capture on transfection-reagent beads, E3 ligase activity assays, and LAMP2 substrate identification","pmids":["36103833"],"confidence":"High","gaps":["How WDFY1 selects damaged lysosomes for the ligase complex unclear","Relationship between this role and TLR adaptor function not addressed"]},{"year":2022,"claim":"Tested physiological requirement in the germline, showing WDFY1 is expressed in spermatocytes/spermatids but dispensable for spermatogenesis and fertility.","evidence":"Transgenic FLAG-Wdfy1-mCherry reporter for localization and global Wdfy1 knockout with testis histology and fertility testing","pmids":["35121371"],"confidence":"Medium","gaps":["Cytoplasmic role in germ cells undefined","Possible functional redundancy not tested"]},{"year":2025,"claim":"Demonstrated an FDC-intrinsic requirement for WDFY1 in immune complex presentation and germinal center responses relevant to autoimmunity.","evidence":"WDFY1 knockout in a cGVHD lupus model with IC-FDC binding imaging, GC analysis, and B-cell reconstitution in Rag1-KO mice","pmids":["40169152"],"confidence":"Medium","gaps":["Molecular mechanism of WDFY1 in IC trafficking within FDCs unresolved","Whether this depends on its TLR or endosomal functions unknown"]},{"year":2025,"claim":"Mapped the cis- and trans-regulatory architecture of the WDFY1 locus, identifying Sp1, Ap-1, Hes1, TCF7 inputs and IL-6/Sp1-driven induction.","evidence":"Luciferase reporter assays with promoter/UTR deletions and TF siRNA knockdown plus IL-6 stimulation in primary mesangial cells","pmids":["40497974"],"confidence":"Medium","gaps":["TF binding inferred from reporter/knockdown rather than direct occupancy in some cases","Physiological context of mesangial WDFY1 regulation unclear"]},{"year":2026,"claim":"Uncovered an inter-organ role: neuron-derived WDFY1 in extracellular vesicles binds retromer in bone to recycle cathepsin D and peroxiredoxin 2, driving bone-fat imbalance in aging.","evidence":"Neuronal Wdfy1 knockout mice, EV inhibition, WDFY1-retromer Co-IP, and bone phenotype readouts","pmids":["41491871"],"confidence":"Medium","gaps":["Which retromer subunit WDFY1 contacts not defined","How EV-delivered WDFY1 alters osteo/adipogenic transcription unresolved"]},{"year":null,"claim":"It remains unknown how WDFY1's distinct molecular roles—TLR adaptor, CUL4A-DDB1 ligase subunit, and retromer-associated endosomal recycler—are coordinated or domain-segregated within a single protein.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural assignment of WD vs FYVE domain to each function","No study integrating the immune, lysophagy, and endosomal roles in one system"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,6,8]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[1]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[5]}],"complexes":["CUL4A-DDB1-WDFY1 E3 ubiquitin ligase","retromer complex"],"partners":["TLR3","TLR4","TRIF","CUL4A","DDB1","LAMP2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IWB7","full_name":"WD repeat and FYVE domain-containing protein 1","aliases":["FYVE domain-containing protein localized to endosomes 1","FENS-1","Phosphoinositide-binding protein 1","WD40- and FYVE domain-containing protein 1","Zinc finger FYVE domain-containing protein 17"],"length_aa":410,"mass_kda":46.3,"function":"Positively regulates TLR3- and TLR4-mediated signaling pathways by bridging the interaction between TLR3 or TLR4 and TICAM1. Promotes TLR3/4 ligand-induced activation of transcription factors IRF3 and NF-kappa-B, as well as the production of IFN-beta and inflammatory cytokines (PubMed:25736436)","subcellular_location":"Early endosome","url":"https://www.uniprot.org/uniprotkb/Q8IWB7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/WDFY1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SCAMP2","stoichiometry":10.0},{"gene":"RAB11A","stoichiometry":0.2},{"gene":"RAB7A","stoichiometry":0.2},{"gene":"SCAMP1","stoichiometry":0.2},{"gene":"SEPT11","stoichiometry":0.2},{"gene":"SEPT2","stoichiometry":0.2},{"gene":"STX7","stoichiometry":0.2},{"gene":"VAMP2","stoichiometry":0.2},{"gene":"VAMP3","stoichiometry":0.2},{"gene":"VAMP8","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/WDFY1","total_profiled":1310},"omim":[{"mim_id":"618080","title":"WD REPEAT-AND FYVE DOMAIN-CONTAINING PROTEIN 1; WDFY1","url":"https://www.omim.org/entry/618080"},{"mim_id":"603030","title":"TOLL-LIKE RECEPTOR 4; TLR4","url":"https://www.omim.org/entry/603030"},{"mim_id":"603029","title":"TOLL-LIKE RECEPTOR 3; TLR3","url":"https://www.omim.org/entry/603029"},{"mim_id":"602070","title":"NEUROPILIN 2; NRP2","url":"https://www.omim.org/entry/602070"},{"mim_id":"601819","title":"BROMODOMAIN PHD FINGER TRANSCRIPTION FACTOR; BPTF","url":"https://www.omim.org/entry/601819"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Vesicles","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/WDFY1"},"hgnc":{"alias_symbol":["KIAA1435","FENS-1","WDF1","ZFYVE17"],"prev_symbol":[]},"alphafold":{"accession":"Q8IWB7","domains":[{"cath_id":"2.130.10.10","chopping":"14-278_357-398","consensus_level":"medium","plddt":95.7119,"start":14,"end":398},{"cath_id":"3.30.40.10","chopping":"297-354","consensus_level":"high","plddt":94.1771,"start":297,"end":354}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IWB7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IWB7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IWB7-F1-predicted_aligned_error_v6.png","plddt_mean":92.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=WDFY1","jax_strain_url":"https://www.jax.org/strain/search?query=WDFY1"},"sequence":{"accession":"Q8IWB7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IWB7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IWB7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IWB7"}},"corpus_meta":[{"pmid":"25736436","id":"PMC_25736436","title":"WDFY1 mediates TLR3/4 signaling by recruiting TRIF.","date":"2015","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/25736436","citation_count":79,"is_preprint":false},{"pmid":"36103833","id":"PMC_36103833","title":"Identification of CUL4A-DDB1-WDFY1 as an E3 ubiquitin ligase complex involved in initiation of lysophagy.","date":"2022","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/36103833","citation_count":40,"is_preprint":false},{"pmid":"30097850","id":"PMC_30097850","title":"PRDX6 Inhibits Neurogenesis through Downregulation of WDFY1-Mediated TLR4 Signal.","date":"2018","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/30097850","citation_count":35,"is_preprint":false},{"pmid":"27026195","id":"PMC_27026195","title":"NRP2 transcriptionally regulates its downstream effector WDFY1.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27026195","citation_count":22,"is_preprint":false},{"pmid":"31015298","id":"PMC_31015298","title":"Hes1 attenuates type I IFN responses via VEGF-C and WDFY1.","date":"2019","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31015298","citation_count":20,"is_preprint":false},{"pmid":"25736437","id":"PMC_25736437","title":"Catching the adaptor-WDFY1, a new player in the TLR-TRIF pathway.","date":"2015","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/25736437","citation_count":10,"is_preprint":false},{"pmid":"32477064","id":"PMC_32477064","title":"Lack of Helios During Neural Development Induces Adult Schizophrenia-Like Behaviors Associated With Aberrant Levels of the TRIF-Recruiter Protein WDFY1.","date":"2020","source":"Frontiers in cellular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/32477064","citation_count":10,"is_preprint":false},{"pmid":"35121371","id":"PMC_35121371","title":"WDFY1, a WD40 repeat protein, is not essential for spermatogenesis and male fertility in mice.","date":"2022","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/35121371","citation_count":7,"is_preprint":false},{"pmid":"41491871","id":"PMC_41491871","title":"Brain neuron-derived WDFY1 induces bone loss.","date":"2026","source":"Nature aging","url":"https://pubmed.ncbi.nlm.nih.gov/41491871","citation_count":3,"is_preprint":false},{"pmid":"40169152","id":"PMC_40169152","title":"WDFY1-expressing follicular dendritic cells play a critical role in lupus development in cGVHD mouse model.","date":"2025","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/40169152","citation_count":1,"is_preprint":false},{"pmid":"40497974","id":"PMC_40497974","title":"Regulation of WDFY1 Expression by miRNAs, Transcription Factors, and IL-6 in Murine Mesangial Cells.","date":"2025","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/40497974","citation_count":0,"is_preprint":false},{"pmid":"40748571","id":"PMC_40748571","title":"Grass carp (Ctenopharyngodon idella) WDFY1 activates IFN1 response via TLR3-TRIF-IRF3 axis.","date":"2025","source":"Fish physiology and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40748571","citation_count":0,"is_preprint":false},{"pmid":"39839352","id":"PMC_39839352","title":"Corrigendum: Lack of Helios during neural development induces adult schizophrenia-like behaviors associated with aberrant levels of the TRIF-recruiter protein WDFY1.","date":"2025","source":"Frontiers in cellular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/39839352","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8013,"output_tokens":2696,"usd":0.032239,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9876,"output_tokens":3342,"usd":0.066465,"stage2_stop_reason":"end_turn"},"total_usd":0.098704,"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\": 2015,\n      \"finding\": \"WDFY1 acts as a crucial adaptor protein in TLR3/4 signaling by physically interacting with TLR3 and TLR4 and mediating the recruitment of TRIF to these receptors, thereby enabling downstream NF-κB and IRF3 activation and type I interferon production. Overexpression potentiates signaling; depletion has the opposite effect.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown experiments with NF-κB/IRF3 reporter assays, cytokine measurements\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP demonstrating physical interaction, combined with gain- and loss-of-function experiments showing consistent bidirectional effects, replicated in fish ortholog study (PMID:40748571)\",\n      \"pmids\": [\"25736436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"WDFY1 is a component of a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex that is essential for initiating lysophagy; the complex ubiquitinates LAMP2 on damaged lysosomes to trigger their autophagic clearance.\",\n      \"method\": \"Proteomic studies using transfection reagent-coated beads, further functional evaluations, E3 ligase activity assays, identification of LAMP2 as substrate\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic identification plus functional validation of complex activity and substrate ubiquitination, multiple orthogonal methods in single study\",\n      \"pmids\": [\"36103833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NRP2 negatively regulates WDFY1 expression at the transcriptional level by preventing nuclear localization of the transcription factor FAC1, thereby maintaining WDFY1 at levels compatible with sustained endocytic activity in metastatic cancer cells.\",\n      \"method\": \"Transcriptional reporter assays, nuclear localization analysis of FAC1, NRP2 knockdown/overexpression with WDFY1 expression readouts\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (reporter assay, localization, expression) in single lab\",\n      \"pmids\": [\"27026195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The transcription factor Hes1 suppresses type I IFN production by inhibiting WDFY1 expression indirectly: Hes1 directly binds and upregulates VEGF-C expression, which in turn downregulates WDFY1, attenuating TLR upstream signaling.\",\n      \"method\": \"Genome-wide ChIP-seq for Hes1 occupancy, Hes1 knockout mice, WDFY1 expression analysis, IFN production assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus KO mouse model with functional readouts, single lab\",\n      \"pmids\": [\"31015298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"WDFY1 protein is expressed in mouse testes, localizing to the cytoplasm of late pachytene spermatocytes to elongated spermatids; however, global Wdfy1 knockout male mice show normal spermatogenesis and fertility, demonstrating WDFY1 is dispensable for these processes.\",\n      \"method\": \"Transgenic FLAG-Wdfy1-mCherry reporter mice for localization; global Wdfy1 knockout mice with histological analysis of testes and fertility testing\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by transgenic reporter and KO phenotyping with histology, single lab\",\n      \"pmids\": [\"35121371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Aged brain neurons secrete WDFY1 protein via extracellular vesicles (EVs) that are transferred to bone, where WDFY1 binds to the retromer complex to promote endosome-to-Golgi recycling of cathepsin D and peroxiredoxin 2, thereby inhibiting osteogenesis and augmenting adipogenesis, causing bone-fat imbalance and osteoporosis.\",\n      \"method\": \"Neuronal Wdfy1 knockout mice, EV inhibition experiments, co-immunoprecipitation (WDFY1-retromer interaction), gain/loss-of-function in brain-specific models, bone phenotype analysis\",\n      \"journal\": \"Nature aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for retromer binding plus multiple genetic models (whole brain, hippocampal, neuronal KO) with functional bone phenotype readouts, single study\",\n      \"pmids\": [\"41491871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"WDFY1 expression in follicular dendritic cells (FDCs) is required for immune complex (IC) presentation; WDFY1 knockout impairs IC binding to FDCs, reduces germinal center formation, and diminishes autoantibody production in a cGVHD lupus mouse model. B cells and T cells lacking WDFY1 retain normal intrinsic function, indicating the defect is FDC-intrinsic.\",\n      \"method\": \"WDFY1 knockout mice in cGVHD model, immunofluorescence for IC-FDC binding, mixed leukocyte reaction, B-cell reconstitution in Rag1-KO mice, autoantibody titers\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse model with multiple orthogonal functional assays (IC binding, GC formation, cell reconstitution), single lab\",\n      \"pmids\": [\"40169152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In murine mesangial cells, a 500 bp distal fragment upstream of the wdfy1 5'-UTR drives approximately four-fold enhanced promoter activity; transcription factors Sp1, Ap-1, Hes1, and TCF7 bind sites in the promoter/5'-UTR to regulate WDFY1 expression; the 3'-UTR suppresses expression (~3.5-fold); IL-6 upregulates WDFY1 through Sp1.\",\n      \"method\": \"Luciferase reporter assays with promoter deletion mutants, siRNA knockdown of transcription factors, IL-6 stimulation of primary renal mesangial cells\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complementary luciferase and siRNA assays in primary cells, single lab\",\n      \"pmids\": [\"40497974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Grass carp WDFY1 (CiWDFY1) physically interacts with TLR3, and TLR3 recruits both TRIF and CiWDFY1 to form an activated complex leading to IRF3 phosphorylation and nuclear translocation and IFN1 induction; CiWDFY1 knockdown disrupts the TLR3-TRIF physical interaction. Upon poly(I:C) stimulation, CiWDFY1 redistributes from diffuse cytoplasmic to perimembranous localization.\",\n      \"method\": \"Co-immunoprecipitation, co-localization assays, overexpression and knockdown in CIK cells, subcellular localization imaging, IRF3 phosphorylation/translocation assays\",\n      \"journal\": \"Fish physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and co-localization plus gain/loss-of-function in fish ortholog, consistent with mammalian findings; single lab\",\n      \"pmids\": [\"40748571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"WDFY1 siRNA knockdown decreases differentiation ability of primary neural stem cells; introduction of WDFY1 plasmid rescues reduced TLR4 expression and neurogenesis in neural stem cells from PRDX6-overexpressing transgenic mice, placing WDFY1 downstream of PRDX6 and upstream of TLR4 in a neurogenesis pathway.\",\n      \"method\": \"siRNA knockdown, plasmid rescue experiment in primary neural stem cells, neurogenesis assays, microarray identification of WDFY1 downregulation in PRDX6-Tg mice\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — rescue experiment and siRNA in primary cells support pathway placement, but mechanistic detail is limited and single lab\",\n      \"pmids\": [\"30097850\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"WDFY1 is a WD-repeat and FYVE-domain scaffolding/adaptor protein that bridges TLR3/TLR4 with the signalling adaptor TRIF to activate NF-κB, IRF3, and type I interferon production; participates as a subunit of a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex that ubiquitinates LAMP2 on damaged lysosomes to initiate lysophagy; interacts with the retromer complex in neurons to regulate endosome-to-Golgi recycling of cathepsin D and peroxiredoxin 2, and is transferred via extracellular vesicles from aged neurons to bone to drive bone-fat imbalance; and in follicular dendritic cells supports immune complex presentation and germinal center responses.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"WDFY1 is a WD-repeat and FYVE-domain adaptor protein that functions at the interface of innate immune signalling and endolysosomal membrane traffic [#0, #1]. In Toll-like receptor signalling, WDFY1 physically associates with TLR3 and TLR4 and recruits the adaptor TRIF to these receptors, driving NF-\\u03baB and IRF3 activation and type I interferon production; gain of function potentiates and loss of function attenuates this output, and the role is conserved in a fish TLR3 ortholog where WDFY1 redistributes to a perimembranous compartment upon poly(I:C) stimulation [#0, #8]. Separately, WDFY1 is a substrate-determining subunit of a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase that ubiquitinates LAMP2 on damaged lysosomes to initiate lysophagy [#1]. WDFY1 also binds the retromer complex to promote endosome-to-Golgi recycling of cathepsin D and peroxiredoxin 2; aged neurons package WDFY1 into extracellular vesicles that transfer this activity to bone, where it shifts the osteogenic\\u2013adipogenic balance toward osteoporosis [#5]. At the organismal level WDFY1 supports immune complex presentation by follicular dendritic cells and germinal center responses [#6], and its expression is tightly controlled by transcriptional inputs including an NRP2/FAC1 axis, Hes1-VEGF-C, and IL-6/Sp1 signalling [#2, #3, #7].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Established WDFY1 as a positive adaptor in TLR3/TLR4 signalling, answering how these receptors couple to the TRIF branch driving interferon responses.\",\n      \"evidence\": \"Reciprocal Co-IP plus overexpression/knockdown with NF-\\u03baB/IRF3 reporters and cytokine readouts in mammalian cells\",\n      \"pmids\": [\"25736436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define which domain (WD vs FYVE) mediates TLR or TRIF binding\", \"No structural model of the TLR\\u2013WDFY1\\u2013TRIF assembly\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified an upstream transcriptional brake, showing NRP2 limits WDFY1 levels via FAC1 nuclear exclusion to tune endocytic activity in cancer cells.\",\n      \"evidence\": \"Transcriptional reporter assays, FAC1 localization analysis, and NRP2 perturbation with WDFY1 readouts\",\n      \"pmids\": [\"27026195\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking endocytic activity to WDFY1 abundance not biochemically resolved\", \"Single-lab finding without in vivo validation\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placed WDFY1 within a PRDX6\\u2192WDFY1\\u2192TLR4 neurogenesis axis, connecting it to neural stem cell differentiation.\",\n      \"evidence\": \"siRNA knockdown and plasmid rescue of TLR4 expression and neurogenesis in primary neural stem cells from PRDX6-Tg mice\",\n      \"pmids\": [\"30097850\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Limited mechanistic detail and single lab\", \"Direct molecular link between WDFY1 and TLR4 expression unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined a Hes1-VEGF-C cascade that suppresses WDFY1 to dampen type I IFN, integrating WDFY1 regulation into a transcriptional network.\",\n      \"evidence\": \"Genome-wide Hes1 ChIP-seq, Hes1 knockout mice, and WDFY1/IFN production analyses\",\n      \"pmids\": [\"31015298\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Indirect (VEGF-C-mediated) link to WDFY1 not mechanistically dissected\", \"How VEGF-C downregulates WDFY1 unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a distinct E3-ligase role: WDFY1 forms a CUL4A-DDB1-WDFY1 complex that ubiquitinates LAMP2 to trigger lysophagy of damaged lysosomes.\",\n      \"evidence\": \"Proteomic capture on transfection-reagent beads, E3 ligase activity assays, and LAMP2 substrate identification\",\n      \"pmids\": [\"36103833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How WDFY1 selects damaged lysosomes for the ligase complex unclear\", \"Relationship between this role and TLR adaptor function not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Tested physiological requirement in the germline, showing WDFY1 is expressed in spermatocytes/spermatids but dispensable for spermatogenesis and fertility.\",\n      \"evidence\": \"Transgenic FLAG-Wdfy1-mCherry reporter for localization and global Wdfy1 knockout with testis histology and fertility testing\",\n      \"pmids\": [\"35121371\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytoplasmic role in germ cells undefined\", \"Possible functional redundancy not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated an FDC-intrinsic requirement for WDFY1 in immune complex presentation and germinal center responses relevant to autoimmunity.\",\n      \"evidence\": \"WDFY1 knockout in a cGVHD lupus model with IC-FDC binding imaging, GC analysis, and B-cell reconstitution in Rag1-KO mice\",\n      \"pmids\": [\"40169152\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of WDFY1 in IC trafficking within FDCs unresolved\", \"Whether this depends on its TLR or endosomal functions unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapped the cis- and trans-regulatory architecture of the WDFY1 locus, identifying Sp1, Ap-1, Hes1, TCF7 inputs and IL-6/Sp1-driven induction.\",\n      \"evidence\": \"Luciferase reporter assays with promoter/UTR deletions and TF siRNA knockdown plus IL-6 stimulation in primary mesangial cells\",\n      \"pmids\": [\"40497974\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TF binding inferred from reporter/knockdown rather than direct occupancy in some cases\", \"Physiological context of mesangial WDFY1 regulation unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Uncovered an inter-organ role: neuron-derived WDFY1 in extracellular vesicles binds retromer in bone to recycle cathepsin D and peroxiredoxin 2, driving bone-fat imbalance in aging.\",\n      \"evidence\": \"Neuronal Wdfy1 knockout mice, EV inhibition, WDFY1-retromer Co-IP, and bone phenotype readouts\",\n      \"pmids\": [\"41491871\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which retromer subunit WDFY1 contacts not defined\", \"How EV-delivered WDFY1 alters osteo/adipogenic transcription unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how WDFY1's distinct molecular roles\\u2014TLR adaptor, CUL4A-DDB1 ligase subunit, and retromer-associated endosomal recycler\\u2014are coordinated or domain-segregated within a single protein.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural assignment of WD vs FYVE domain to each function\", \"No study integrating the immune, lysophagy, and endosomal roles in one system\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 6, 8]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\n      \"CUL4A-DDB1-WDFY1 E3 ubiquitin ligase\",\n      \"retromer complex\"\n    ],\n    \"partners\": [\n      \"TLR3\",\n      \"TLR4\",\n      \"TRIF\",\n      \"CUL4A\",\n      \"DDB1\",\n      \"LAMP2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":5,"faith_total":5,"faith_pct":100.0}}