{"gene":"RUFY3","run_date":"2026-06-10T07:46:28","timeline":{"discoveries":[{"year":2022,"finding":"RUFY3 acts as an Arl8b effector that links Arl8b to the JIP4-dynein-dynactin complex to facilitate retrograde lysosomal transport; RUFY3 knockdown disrupts Arl8b-positive endosome positioning and reduces Arl8b colocalization with Rab7-marked late endosomes, and reduces lysosome size rescuable by PIKFYVE inhibition.","method":"Co-immunoprecipitation, knockdown (siRNA/shRNA), live-cell imaging, colocalization analysis, lysosome size quantification","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP establishing complex membership, multiple orthogonal methods (co-IP, KD, imaging, rescue), replicated across multiple assays in one rigorous study","pmids":["35314681"],"is_preprint":false},{"year":2023,"finding":"RUFY3 exists as two isoforms; the FYVE domain-bearing isoform (iRUFY3) preferentially expressed in immune cells binds phosphatidylinositol 3-phosphate on endosomal membranes and is required for perinuclear positioning of ARL8b+/LAMP1+ endo-lysosomes in LPS-activated macrophages, controlling macrophage migration, MHC II antigen presentation, interferon-γ responses, and intracellular Salmonella replication.","method":"Isoform characterization, PI3P-binding assay, shRNA knockdown, conditional phagocyte-specific rufy3 inactivation in mice, immunofluorescence, migration assays, antigen presentation assays, in vivo infection models","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including in vitro biochemistry, cellular knockdown, and in vivo genetic model with defined functional readouts","pmids":["37463962"],"is_preprint":false},{"year":2007,"finding":"Singar1/RUFY3, a RUN domain-containing protein, suppresses formation of surplus axons in hippocampal neurons in a PI3K-dependent manner; overexpression suppresses shootin1-induced surplus axons, while RNAi knockdown increases neurons with multiple axons.","method":"Overexpression, RNA interference (RNAi), cultured hippocampal neurons, immunostaining, PI3K inhibitor rescue","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD/OE with defined neuronal polarity phenotype, PI3K epistasis established, single lab","pmids":["17439943"],"is_preprint":false},{"year":2017,"finding":"Rufy3 acts as an adapter protein recruited via glycoprotein M6A to lipid raft/detergent-resistant membrane domains; it forms a ternary complex assembling Rap2 in axonal growth cones and activates the Rac-GEF Tiam2/STEF downstream of Rap2 to control neuronal polarity and axon growth. In Rufy3-KO neurons, Tiam2/STEF accumulation at growth cones is inhibited.","method":"Rufy3 knockout mice, detergent-resistant membrane fractionation, co-immunoprecipitation, immunofluorescence colocalization, neuronal polarity assays","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO mouse model combined with biochemical fractionation and Co-IP establishing ternary complex, multiple orthogonal methods, defined pathway placement","pmids":["29089386"],"is_preprint":false},{"year":2014,"finding":"Rufy3 physically interacts with actin-bundling protein Fascin and the actin-binding protein Drebrin in neuronal growth cones; knockdown of Rufy3 impairs Fascin and F-actin distribution, increases multipolar neurons, and decreases axon length, while overexpression extends axons and redistributes Drebrin throughout the growth cone.","method":"Co-immunoprecipitation, immunofluorescence colocalization, shRNA knockdown, overexpression in mouse hippocampal neurons","journal":"Journal of Neurochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction plus KD/OE with defined phenotype, single lab, two orthogonal methods","pmids":["24720729"],"is_preprint":false},{"year":2019,"finding":"RUFY3 is required for caspase-mediated axon degeneration in TRKA+ sensory axons; deletion of Rufy3 protects axons from degeneration even when CASP3 is activated and competent to cleave substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a mechanism for local caspase-driven degeneration control.","method":"Mass spectrometry substrate identification, Rufy3 knockout (genetic deletion), in vitro axon degeneration assay, in vivo sensory axon degeneration model, phosphorylation site analysis","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic deletion with defined mechanistic epistasis (CASP3-competent but degeneration blocked), phosphorylation site identified, in vitro and in vivo validation","pmids":["31221560"],"is_preprint":false},{"year":2015,"finding":"PAK1 interacts with RUFY3 and promotes RUFY3 expression; RUFY3 overexpression induces formation of F-actin-enriched protrusive structures at the cell periphery and promotes gastric cancer cell migration and invasion; PAK1 inhibition attenuates RUFY3-induced migration and invasion.","method":"Co-immunoprecipitation, PAK1 inhibitor treatment, overexpression, siRNA knockdown, migration/invasion assays, F-actin staining","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction plus functional KD/OE with defined actin phenotype, single lab, two orthogonal methods","pmids":["25766321"],"is_preprint":false},{"year":2017,"finding":"RUFY3 physically interacts with transcription factor FOXK1 in colorectal cancer cells; siRNA-mediated repression of FOXK1 in RUFY3-overexpressing cells reverses EMT and metastatic phenotypes, placing FOXK1 downstream of RUFY3 in a pro-metastatic axis.","method":"Co-immunoprecipitation, siRNA knockdown, EMT marker analysis, in vivo orthotopic implantation","journal":"Scientific Reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction plus epistasis rescue experiment, single lab, in vitro and in vivo","pmids":["28623323"],"is_preprint":false},{"year":2017,"finding":"TGF-β1 induces RUFY3 expression in a dose-dependent manner in colorectal cancer cells, and RUFY3 knockdown inhibits TGF-β1-induced EMT, placing RUFY3 downstream of TGF-β1 signaling in EMT regulation.","method":"TGF-β1 dose-response treatment, siRNA knockdown, EMT marker western blot, invasion assay","journal":"Cancer Letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method per claim, no direct binding or reconstitution of TGF-β1–RUFY3 pathway","pmids":["28089833"],"is_preprint":false},{"year":2019,"finding":"HOXD9 directly associates with the RUFY3 promoter to transcriptionally activate RUFY3 expression, as demonstrated by ChIP and luciferase reporter assays; inhibition of RUFY3 attenuates HOXD9-driven gastric cancer cell proliferation, migration and invasiveness.","method":"Chromatin immunoprecipitation (ChIP), luciferase reporter assay, RUFY3 siRNA knockdown, in vitro and in vivo tumor growth assays","journal":"Journal of Experimental & Clinical Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus luciferase reporter establishing direct promoter binding, functional epistasis via KD, single lab","pmids":["31547840"],"is_preprint":false},{"year":2021,"finding":"RUFY3 promotes hepatocellular carcinoma cell growth, invasion, and metastasis through activation of NF-κB-mediated EMT signaling.","method":"siRNA knockdown, NF-κB activity assay, EMT marker western blot, xenograft and lung metastasis mouse models","journal":"Aging","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, mechanism inferred from pathway inhibitors without direct RUFY3-NF-κB interaction demonstrated","pmids":["34510031"],"is_preprint":false},{"year":2023,"finding":"RUFY3 localizes to focal adhesions and endosomal compartments along with HPIP and Rab5; RUFY3 and HPIP act as noncanonical guanine nucleotide exchange factors (GEFs) for Rab5, activating Rab5-dependent focal adhesion disassembly, FAK activation, fibronectin-associated β1 integrin trafficking, and cell migration.","method":"Co-immunoprecipitation, co-localization (immunofluorescence), Rab5 GEF activity assay, coiled-coil domain deletion mutants, siRNA knockdown, focal adhesion turnover assay, integrin trafficking assay","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct GEF activity assay plus domain mutagenesis and Co-IP, multiple orthogonal methods, single lab","pmids":["37797694"],"is_preprint":false},{"year":2022,"finding":"In subarachnoid hemorrhage, Rufy3 interacts with Rap1 and increases Rap1-GTP levels; Rufy3 overexpression activates the Rap1/Arap3/Rho/Fascin signaling pathway to promote axon repair and the Rap1/MEK/ERK/synapsin I pathway to enhance synaptic plasticity.","method":"Co-immunoprecipitation (Rufy3-Rap1), Rap1-GTP pull-down assay, lentiviral overexpression/knockdown in SAH rat model, western blot for pathway components, Rap1 agonist rescue","journal":"Molecular Brain","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP interaction plus GTP loading assay, pathway activation by western blot, in vivo lentiviral model with defined phenotype, single lab","pmids":["35461284"],"is_preprint":false},{"year":2025,"finding":"RUFY3 physically binds MAP4, and MAP4 binds CDK1; RUFY3 knockdown induces PANoptosis and inhibits colorectal cancer tumor growth in vitro and in vivo.","method":"Co-immunoprecipitation (Co-IP), siRNA knockdown, western blot, xenograft mouse model","journal":"FASEB Journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP demonstrating RUFY3-MAP4-CDK1 binding, mechanism downstream of this complex not fully established, single lab","pmids":["40879686"],"is_preprint":false}],"current_model":"RUFY3 is a RUN/FYVE domain-containing adapter protein that coordinates lysosomal and endosomal trafficking by linking the small GTPase Arl8b to the JIP4-dynein-dynactin retrograde motor complex, controls neuronal polarity and axon growth by forming a ternary complex with Rap2 and the Rac-GEF Tiam2/STEF at lipid raft domains, acts as a noncanonical GEF for Rab5 to regulate focal adhesion turnover and integrin trafficking, and is required for caspase-3-mediated axon degeneration in a dephosphorylation-dependent manner (S34), while also promoting tumor cell migration and invasion through interactions with PAK1, Fascin, and FOXK1 and regulation of actin dynamics."},"narrative":{"mechanistic_narrative":"RUFY3 is a RUN/FYVE domain adapter protein that couples small-GTPase signaling to membrane trafficking and actin-based cytoskeletal remodeling, governing both endo-lysosomal positioning and neuronal morphogenesis [PMID:35314681, PMID:29089386]. As an Arl8b effector, it bridges Arl8b to the JIP4-dynein-dynactin retrograde motor to control late endosome/lysosome positioning and size [PMID:35314681]; a FYVE-domain-bearing isoform binds endosomal PI3P and drives perinuclear endo-lysosome clustering in activated macrophages, where it shapes migration, MHC II antigen presentation, interferon-γ responses, and restriction of intracellular Salmonella [PMID:37463962]. RUFY3 also acts as a noncanonical guanine-nucleotide exchange factor for Rab5, partnering with HPIP at focal adhesions and endosomes to promote focal adhesion disassembly, FAK activation, β1-integrin trafficking, and cell migration [PMID:37797694]. In neurons, RUFY3 restricts surplus axon formation in a PI3K-dependent manner [PMID:17439943] and, recruited via M6A glycoprotein to lipid raft domains, assembles a ternary complex with Rap2 that activates the Rac-GEF Tiam2/STEF to direct axon growth and polarity [PMID:29089386]; it further organizes growth-cone actin through interactions with Fascin and Drebrin [PMID:24720729]. RUFY3 is required for caspase-3-mediated axon degeneration, with dephosphorylation at S34 permitting degeneration even when caspase-3 is active [PMID:31221560]. In cancer, RUFY3 promotes migration, invasion, and EMT, interacting with PAK1 to build F-actin-rich protrusions [PMID:25766321] and acting through FOXK1 [PMID:28623323], while its expression is transcriptionally driven by HOXD9 [PMID:31547840].","teleology":[{"year":2007,"claim":"Established the first cellular role for RUFY3 by showing it constrains axon number, defining it as a regulator of neuronal polarity rather than an uncharacterized RUN-domain protein.","evidence":"Overexpression and RNAi in cultured hippocampal neurons with PI3K-inhibitor epistasis","pmids":["17439943"],"confidence":"Medium","gaps":["No molecular partner identified at this stage","Mechanistic link to PI3K not biochemically defined"]},{"year":2014,"claim":"Connected RUFY3's polarity function to actin organization by identifying physical interactions with Fascin and Drebrin in growth cones.","evidence":"Co-IP and colocalization plus shRNA/overexpression in mouse hippocampal neurons","pmids":["24720729"],"confidence":"Medium","gaps":["Direct vs indirect binding not resolved","Single lab, two orthogonal methods"]},{"year":2015,"claim":"Extended RUFY3's actin-remodeling role to tumor invasion by linking it to PAK1-driven protrusion formation.","evidence":"Co-IP, PAK1 inhibitor, KD/OE and invasion assays in gastric cancer cells","pmids":["25766321"],"confidence":"Medium","gaps":["Whether PAK1 acts upstream or downstream not fully separated","No structural basis for interaction"]},{"year":2017,"claim":"Placed RUFY3 in a defined neuronal signaling pathway, showing it scaffolds Rap2 and activates Tiam2/STEF at lipid rafts to drive axon growth.","evidence":"Rufy3-KO mice, detergent-resistant membrane fractionation, Co-IP and polarity assays","pmids":["29089386"],"confidence":"High","gaps":["Direct GEF activation mechanism of Tiam2/STEF not reconstituted","Role of M6A recruitment in other cell types untested"]},{"year":2017,"claim":"Defined a pro-metastatic axis in which RUFY3 acts through transcription factor FOXK1 to drive EMT.","evidence":"Co-IP, FOXK1 siRNA epistasis, EMT markers and orthotopic implantation in colorectal cancer","pmids":["28623323"],"confidence":"Medium","gaps":["How a trafficking adapter regulates a transcription factor unclear","Direct vs indirect interaction not resolved"]},{"year":2017,"claim":"Positioned RUFY3 as a downstream effector of TGF-β1 in EMT regulation.","evidence":"TGF-β1 dose-response and siRNA with EMT/invasion readouts in colorectal cancer cells","pmids":["28089833"],"confidence":"Low","gaps":["No direct binding or pathway reconstitution","Single method per claim, single lab"]},{"year":2019,"claim":"Identified an upstream transcriptional driver of RUFY3 by showing HOXD9 binds the RUFY3 promoter to activate its expression in gastric cancer.","evidence":"ChIP, luciferase reporter and siRNA epistasis with tumor growth assays","pmids":["31547840"],"confidence":"Medium","gaps":["Other transcriptional inputs not mapped","Single lab"]},{"year":2019,"claim":"Revealed a degeneration-specific function: RUFY3 is required for caspase-3-mediated axon degeneration, with S34 dephosphorylation as the permissive switch.","evidence":"Mass spec, Rufy3 genetic deletion, phosphosite analysis, in vitro and in vivo sensory axon degeneration","pmids":["31221560"],"confidence":"High","gaps":["Kinase/phosphatase controlling S34 not identified","Downstream effectors of dephosphorylated RUFY3 unknown"]},{"year":2021,"claim":"Linked RUFY3 to NF-κB-mediated EMT in hepatocellular carcinoma growth and metastasis.","evidence":"siRNA, NF-κB activity assay, EMT markers and xenograft/metastasis models","pmids":["34510031"],"confidence":"Low","gaps":["No direct RUFY3-NF-κB interaction demonstrated","Mechanism inferred from pathway inhibitors"]},{"year":2022,"claim":"Defined RUFY3's core trafficking mechanism by establishing it as an Arl8b effector that recruits the JIP4-dynein-dynactin retrograde motor to position lysosomes.","evidence":"Reciprocal Co-IP, knockdown, live-cell imaging, colocalization and PIKFYVE-rescue of lysosome size","pmids":["35314681"],"confidence":"High","gaps":["Stoichiometry of the Arl8b-RUFY3-JIP4 complex unresolved","How motor handoff is regulated unknown"]},{"year":2022,"claim":"Extended RUFY3-Rap signaling to injury repair, showing Rufy3 raises Rap1-GTP and activates downstream Rho/Fascin and MEK/ERK pathways for axon repair and synaptic plasticity.","evidence":"Co-IP, Rap1-GTP pull-down, lentiviral OE/KD in a subarachnoid hemorrhage rat model with pathway western blots","pmids":["35461284"],"confidence":"Medium","gaps":["Whether RUFY3 directly catalyzes Rap1 loading not tested","In vivo pathway dependencies inferred from western blot"]},{"year":2023,"claim":"Resolved isoform-specific specialization, showing a FYVE-bearing immune isoform binds PI3P to position endo-lysosomes and control macrophage immune functions and Salmonella restriction.","evidence":"Isoform characterization, PI3P-binding, phagocyte-specific conditional KO, and in vivo infection models","pmids":["37463962"],"confidence":"High","gaps":["Functional division between isoforms in non-immune cells unclear","How PI3P binding integrates with Arl8b effector role not defined"]},{"year":2023,"claim":"Assigned a catalytic activity to RUFY3, identifying it (with HPIP) as a noncanonical Rab5 GEF driving focal adhesion turnover and integrin trafficking.","evidence":"Rab5 GEF activity assay, coiled-coil deletion mutants, Co-IP, focal adhesion and integrin trafficking assays","pmids":["37797694"],"confidence":"Medium","gaps":["Structural basis of noncanonical GEF activity not solved","Single lab"]},{"year":2025,"claim":"Tied RUFY3 to cell-death control in cancer by linking a RUFY3-MAP4-CDK1 interaction to suppression of PANoptosis.","evidence":"Co-IP and siRNA with xenograft tumor models in colorectal cancer","pmids":["40879686"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation","Mechanism downstream of the complex not established"]},{"year":null,"claim":"How RUFY3's distinct activities — Arl8b/PI3P-dependent lysosome positioning, noncanonical Rab5/Rap GEF function, and S34-regulated degeneration control — are coordinated within a single protein, and which are isoform- or context-specific, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model connecting RUN, FYVE and coiled-coil domain functions","Phosphoregulation linking trafficking and degeneration roles unmapped","Direct GEF mechanism for Rab5/Rap not structurally resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[4,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11,12]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1,11]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,11]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[4,6]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1,11]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,12]}],"complexes":["Arl8b-RUFY3-JIP4-dynein-dynactin complex","RUFY3-Rap2-Tiam2/STEF ternary complex"],"partners":["ARL8B","JIP4","RAP2","TIAM2","FSCN1","PAK1","FOXK1","RAB5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7L099","full_name":"Protein RUFY3","aliases":["RUN and FYVE domain-containing protein 3","Rap2-interacting protein x","RIPx","Single axon-regulated protein","Singar"],"length_aa":469,"mass_kda":53.0,"function":"ARL8 effector that promotes the coupling of endolysosomes to dynein-dynactin for retrograde transport along microtubules. Acts by binding both GTP-bound ARL8 and dynein-dynactin. In nonneuronal cells, promotes concentration of endolysosomes in the juxtanuclear area. In hippocampal neurons, drives retrograde transport of endolysosomes from the axon to the soma (PubMed:35314674). Plays a role in the generation of neuronal polarity formation and axon growth (By similarity). Implicated in the formation of a single axon by developing neurons (By similarity). May inhibit the formation of additional axons by inhibition of PI3K in minor neuronal processes (By similarity). Plays a role in the formation of F-actin-enriched protrusive structures at the cell periphery (PubMed:25766321). Plays a role in cytoskeletal organization by regulating the subcellular localization of FSCN1 and DBN1 at axonal growth cones (By similarity)","subcellular_location":"Cytoplasm; Endomembrane system; Cell projection, invadopodium; Perikaryon; Cell projection; Cell projection, growth cone; Cell projection, filopodium; Cell projection, lamellipodium; Lysosome","url":"https://www.uniprot.org/uniprotkb/Q7L099/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RUFY3","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/RUFY3","total_profiled":1310},"omim":[{"mim_id":"620994","title":"RUN AND FYVE DOMAINS-CONTAINING PROTEIN 4; RUFY4","url":"https://www.omim.org/entry/620994"},{"mim_id":"611194","title":"RUN AND FYVE DOMAINS-CONTAINING PROTEIN 3; RUFY3","url":"https://www.omim.org/entry/611194"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"retina","ntpm":111.4}],"url":"https://www.proteinatlas.org/search/RUFY3"},"hgnc":{"alias_symbol":["RIPx","KIAA0871","Singar1","ZFYVE30"],"prev_symbol":[]},"alphafold":{"accession":"Q7L099","domains":[{"cath_id":"1.20.58.900","chopping":"23-47_61-119_126-242","consensus_level":"high","plddt":86.41,"start":23,"end":242},{"cath_id":"-","chopping":"280-324_366-454","consensus_level":"medium","plddt":93.5493,"start":280,"end":454}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7L099","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q7L099-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q7L099-F1-predicted_aligned_error_v6.png","plddt_mean":81.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RUFY3","jax_strain_url":"https://www.jax.org/strain/search?query=RUFY3"},"sequence":{"accession":"Q7L099","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7L099.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7L099/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7L099"}},"corpus_meta":[{"pmid":"35314681","id":"PMC_35314681","title":"RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/35314681","citation_count":68,"is_preprint":false},{"pmid":"31547840","id":"PMC_31547840","title":"HOXD9 promotes the growth, invasion and metastasis of gastric cancer cells by transcriptional activation of RUFY3.","date":"2019","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/31547840","citation_count":56,"is_preprint":false},{"pmid":"17439943","id":"PMC_17439943","title":"Singar1, a novel RUN domain-containing protein, suppresses formation of surplus axons for neuronal polarity.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17439943","citation_count":53,"is_preprint":false},{"pmid":"25766321","id":"PMC_25766321","title":"PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion.","date":"2015","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/25766321","citation_count":46,"is_preprint":false},{"pmid":"10498718","id":"PMC_10498718","title":"The ripX locus of Bacillus subtilis encodes a site-specific recombinase involved in proper chromosome partitioning.","date":"1999","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/10498718","citation_count":41,"is_preprint":false},{"pmid":"24720729","id":"PMC_24720729","title":"Rufy3, a protein specifically expressed in neurons, interacts with actin-bundling protein Fascin to control the growth of axons.","date":"2014","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24720729","citation_count":34,"is_preprint":false},{"pmid":"28623323","id":"PMC_28623323","title":"RUFY3 interaction with FOXK1 promotes invasion and metastasis in colorectal cancer.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28623323","citation_count":26,"is_preprint":false},{"pmid":"28089833","id":"PMC_28089833","title":"Rufy3 promotes metastasis through epithelial-mesenchymal transition in colorectal cancer.","date":"2017","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/28089833","citation_count":25,"is_preprint":false},{"pmid":"32183439","id":"PMC_32183439","title":"Ralstonia solanacearum elicitor RipX Induces Defense Reaction by Suppressing the Mitochondrial atpA Gene in Host Plant.","date":"2020","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32183439","citation_count":18,"is_preprint":false},{"pmid":"29089386","id":"PMC_29089386","title":"Rufy3 is an adapter protein for small GTPases that activates a Rac guanine nucleotide exchange factor to control neuronal polarity.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29089386","citation_count":17,"is_preprint":false},{"pmid":"31221560","id":"PMC_31221560","title":"Neuronally Enriched RUFY3 Is Required for Caspase-Mediated Axon Degeneration.","date":"2019","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/31221560","citation_count":12,"is_preprint":false},{"pmid":"34510031","id":"PMC_34510031","title":"RUFY3 promotes the progression of hepatocellular carcinoma through activating NF-κB-mediated epithelial-mesenchymal transition.","date":"2021","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/34510031","citation_count":10,"is_preprint":false},{"pmid":"35461284","id":"PMC_35461284","title":"Roles of Rufy3 in experimental subarachnoid hemorrhage-induced early brain injury via accelerating neuronal axon repair and synaptic plasticity.","date":"2022","source":"Molecular brain","url":"https://pubmed.ncbi.nlm.nih.gov/35461284","citation_count":10,"is_preprint":false},{"pmid":"23305333","id":"PMC_23305333","title":"Interaction of the putative tyrosine recombinases RipX (UU145), XerC (UU222), and CodV (UU529) of Ureaplasma parvum serovar 3 with specific DNA.","date":"2013","source":"FEMS microbiology letters","url":"https://pubmed.ncbi.nlm.nih.gov/23305333","citation_count":6,"is_preprint":false},{"pmid":"37463962","id":"PMC_37463962","title":"RUFY3 regulates endolysosomes perinuclear positioning, antigen presentation and migration in activated phagocytes.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37463962","citation_count":5,"is_preprint":false},{"pmid":"38915053","id":"PMC_38915053","title":"circSORBS1 inhibits lung cancer progression by sponging miR-6779-5p and directly binding RUFY3 mRNA.","date":"2024","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38915053","citation_count":4,"is_preprint":false},{"pmid":"37797694","id":"PMC_37797694","title":"HPIP and RUFY3 are noncanonical guanine nucleotide exchange factors of Rab5 to regulate endocytosis-coupled focal adhesion turnover.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37797694","citation_count":1,"is_preprint":false},{"pmid":"40879686","id":"PMC_40879686","title":"Rufy3 Knockdown Induces PANoptosis Through MAP4/CDK1 Axis to Inhibit Colorectal Cancer Growth: Evidence From In Vitro and In Vivo Models.","date":"2025","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/40879686","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10942,"output_tokens":3553,"usd":0.043061,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11184,"output_tokens":4370,"usd":0.082585,"stage2_stop_reason":"end_turn"},"total_usd":0.125646,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"RUFY3 acts as an Arl8b effector that links Arl8b to the JIP4-dynein-dynactin complex to facilitate retrograde lysosomal transport; RUFY3 knockdown disrupts Arl8b-positive endosome positioning and reduces Arl8b colocalization with Rab7-marked late endosomes, and reduces lysosome size rescuable by PIKFYVE inhibition.\",\n      \"method\": \"Co-immunoprecipitation, knockdown (siRNA/shRNA), live-cell imaging, colocalization analysis, lysosome size quantification\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP establishing complex membership, multiple orthogonal methods (co-IP, KD, imaging, rescue), replicated across multiple assays in one rigorous study\",\n      \"pmids\": [\"35314681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RUFY3 exists as two isoforms; the FYVE domain-bearing isoform (iRUFY3) preferentially expressed in immune cells binds phosphatidylinositol 3-phosphate on endosomal membranes and is required for perinuclear positioning of ARL8b+/LAMP1+ endo-lysosomes in LPS-activated macrophages, controlling macrophage migration, MHC II antigen presentation, interferon-γ responses, and intracellular Salmonella replication.\",\n      \"method\": \"Isoform characterization, PI3P-binding assay, shRNA knockdown, conditional phagocyte-specific rufy3 inactivation in mice, immunofluorescence, migration assays, antigen presentation assays, in vivo infection models\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including in vitro biochemistry, cellular knockdown, and in vivo genetic model with defined functional readouts\",\n      \"pmids\": [\"37463962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Singar1/RUFY3, a RUN domain-containing protein, suppresses formation of surplus axons in hippocampal neurons in a PI3K-dependent manner; overexpression suppresses shootin1-induced surplus axons, while RNAi knockdown increases neurons with multiple axons.\",\n      \"method\": \"Overexpression, RNA interference (RNAi), cultured hippocampal neurons, immunostaining, PI3K inhibitor rescue\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD/OE with defined neuronal polarity phenotype, PI3K epistasis established, single lab\",\n      \"pmids\": [\"17439943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Rufy3 acts as an adapter protein recruited via glycoprotein M6A to lipid raft/detergent-resistant membrane domains; it forms a ternary complex assembling Rap2 in axonal growth cones and activates the Rac-GEF Tiam2/STEF downstream of Rap2 to control neuronal polarity and axon growth. In Rufy3-KO neurons, Tiam2/STEF accumulation at growth cones is inhibited.\",\n      \"method\": \"Rufy3 knockout mice, detergent-resistant membrane fractionation, co-immunoprecipitation, immunofluorescence colocalization, neuronal polarity assays\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO mouse model combined with biochemical fractionation and Co-IP establishing ternary complex, multiple orthogonal methods, defined pathway placement\",\n      \"pmids\": [\"29089386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Rufy3 physically interacts with actin-bundling protein Fascin and the actin-binding protein Drebrin in neuronal growth cones; knockdown of Rufy3 impairs Fascin and F-actin distribution, increases multipolar neurons, and decreases axon length, while overexpression extends axons and redistributes Drebrin throughout the growth cone.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence colocalization, shRNA knockdown, overexpression in mouse hippocampal neurons\",\n      \"journal\": \"Journal of Neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction plus KD/OE with defined phenotype, single lab, two orthogonal methods\",\n      \"pmids\": [\"24720729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RUFY3 is required for caspase-mediated axon degeneration in TRKA+ sensory axons; deletion of Rufy3 protects axons from degeneration even when CASP3 is activated and competent to cleave substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a mechanism for local caspase-driven degeneration control.\",\n      \"method\": \"Mass spectrometry substrate identification, Rufy3 knockout (genetic deletion), in vitro axon degeneration assay, in vivo sensory axon degeneration model, phosphorylation site analysis\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic deletion with defined mechanistic epistasis (CASP3-competent but degeneration blocked), phosphorylation site identified, in vitro and in vivo validation\",\n      \"pmids\": [\"31221560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PAK1 interacts with RUFY3 and promotes RUFY3 expression; RUFY3 overexpression induces formation of F-actin-enriched protrusive structures at the cell periphery and promotes gastric cancer cell migration and invasion; PAK1 inhibition attenuates RUFY3-induced migration and invasion.\",\n      \"method\": \"Co-immunoprecipitation, PAK1 inhibitor treatment, overexpression, siRNA knockdown, migration/invasion assays, F-actin staining\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction plus functional KD/OE with defined actin phenotype, single lab, two orthogonal methods\",\n      \"pmids\": [\"25766321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RUFY3 physically interacts with transcription factor FOXK1 in colorectal cancer cells; siRNA-mediated repression of FOXK1 in RUFY3-overexpressing cells reverses EMT and metastatic phenotypes, placing FOXK1 downstream of RUFY3 in a pro-metastatic axis.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, EMT marker analysis, in vivo orthotopic implantation\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction plus epistasis rescue experiment, single lab, in vitro and in vivo\",\n      \"pmids\": [\"28623323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TGF-β1 induces RUFY3 expression in a dose-dependent manner in colorectal cancer cells, and RUFY3 knockdown inhibits TGF-β1-induced EMT, placing RUFY3 downstream of TGF-β1 signaling in EMT regulation.\",\n      \"method\": \"TGF-β1 dose-response treatment, siRNA knockdown, EMT marker western blot, invasion assay\",\n      \"journal\": \"Cancer Letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method per claim, no direct binding or reconstitution of TGF-β1–RUFY3 pathway\",\n      \"pmids\": [\"28089833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HOXD9 directly associates with the RUFY3 promoter to transcriptionally activate RUFY3 expression, as demonstrated by ChIP and luciferase reporter assays; inhibition of RUFY3 attenuates HOXD9-driven gastric cancer cell proliferation, migration and invasiveness.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), luciferase reporter assay, RUFY3 siRNA knockdown, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Journal of Experimental & Clinical Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus luciferase reporter establishing direct promoter binding, functional epistasis via KD, single lab\",\n      \"pmids\": [\"31547840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RUFY3 promotes hepatocellular carcinoma cell growth, invasion, and metastasis through activation of NF-κB-mediated EMT signaling.\",\n      \"method\": \"siRNA knockdown, NF-κB activity assay, EMT marker western blot, xenograft and lung metastasis mouse models\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, mechanism inferred from pathway inhibitors without direct RUFY3-NF-κB interaction demonstrated\",\n      \"pmids\": [\"34510031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RUFY3 localizes to focal adhesions and endosomal compartments along with HPIP and Rab5; RUFY3 and HPIP act as noncanonical guanine nucleotide exchange factors (GEFs) for Rab5, activating Rab5-dependent focal adhesion disassembly, FAK activation, fibronectin-associated β1 integrin trafficking, and cell migration.\",\n      \"method\": \"Co-immunoprecipitation, co-localization (immunofluorescence), Rab5 GEF activity assay, coiled-coil domain deletion mutants, siRNA knockdown, focal adhesion turnover assay, integrin trafficking assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct GEF activity assay plus domain mutagenesis and Co-IP, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"37797694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In subarachnoid hemorrhage, Rufy3 interacts with Rap1 and increases Rap1-GTP levels; Rufy3 overexpression activates the Rap1/Arap3/Rho/Fascin signaling pathway to promote axon repair and the Rap1/MEK/ERK/synapsin I pathway to enhance synaptic plasticity.\",\n      \"method\": \"Co-immunoprecipitation (Rufy3-Rap1), Rap1-GTP pull-down assay, lentiviral overexpression/knockdown in SAH rat model, western blot for pathway components, Rap1 agonist rescue\",\n      \"journal\": \"Molecular Brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP interaction plus GTP loading assay, pathway activation by western blot, in vivo lentiviral model with defined phenotype, single lab\",\n      \"pmids\": [\"35461284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RUFY3 physically binds MAP4, and MAP4 binds CDK1; RUFY3 knockdown induces PANoptosis and inhibits colorectal cancer tumor growth in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation (Co-IP), siRNA knockdown, western blot, xenograft mouse model\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP demonstrating RUFY3-MAP4-CDK1 binding, mechanism downstream of this complex not fully established, single lab\",\n      \"pmids\": [\"40879686\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RUFY3 is a RUN/FYVE domain-containing adapter protein that coordinates lysosomal and endosomal trafficking by linking the small GTPase Arl8b to the JIP4-dynein-dynactin retrograde motor complex, controls neuronal polarity and axon growth by forming a ternary complex with Rap2 and the Rac-GEF Tiam2/STEF at lipid raft domains, acts as a noncanonical GEF for Rab5 to regulate focal adhesion turnover and integrin trafficking, and is required for caspase-3-mediated axon degeneration in a dephosphorylation-dependent manner (S34), while also promoting tumor cell migration and invasion through interactions with PAK1, Fascin, and FOXK1 and regulation of actin dynamics.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RUFY3 is a RUN/FYVE domain adapter protein that couples small-GTPase signaling to membrane trafficking and actin-based cytoskeletal remodeling, governing both endo-lysosomal positioning and neuronal morphogenesis [#0, #3]. As an Arl8b effector, it bridges Arl8b to the JIP4-dynein-dynactin retrograde motor to control late endosome/lysosome positioning and size [#0]; a FYVE-domain-bearing isoform binds endosomal PI3P and drives perinuclear endo-lysosome clustering in activated macrophages, where it shapes migration, MHC II antigen presentation, interferon-γ responses, and restriction of intracellular Salmonella [#1]. RUFY3 also acts as a noncanonical guanine-nucleotide exchange factor for Rab5, partnering with HPIP at focal adhesions and endosomes to promote focal adhesion disassembly, FAK activation, β1-integrin trafficking, and cell migration [#11]. In neurons, RUFY3 restricts surplus axon formation in a PI3K-dependent manner [#2] and, recruited via M6A glycoprotein to lipid raft domains, assembles a ternary complex with Rap2 that activates the Rac-GEF Tiam2/STEF to direct axon growth and polarity [#3]; it further organizes growth-cone actin through interactions with Fascin and Drebrin [#4]. RUFY3 is required for caspase-3-mediated axon degeneration, with dephosphorylation at S34 permitting degeneration even when caspase-3 is active [#5]. In cancer, RUFY3 promotes migration, invasion, and EMT, interacting with PAK1 to build F-actin-rich protrusions [#6] and acting through FOXK1 [#7], while its expression is transcriptionally driven by HOXD9 [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the first cellular role for RUFY3 by showing it constrains axon number, defining it as a regulator of neuronal polarity rather than an uncharacterized RUN-domain protein.\",\n      \"evidence\": \"Overexpression and RNAi in cultured hippocampal neurons with PI3K-inhibitor epistasis\",\n      \"pmids\": [\"17439943\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular partner identified at this stage\", \"Mechanistic link to PI3K not biochemically defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected RUFY3's polarity function to actin organization by identifying physical interactions with Fascin and Drebrin in growth cones.\",\n      \"evidence\": \"Co-IP and colocalization plus shRNA/overexpression in mouse hippocampal neurons\",\n      \"pmids\": [\"24720729\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect binding not resolved\", \"Single lab, two orthogonal methods\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended RUFY3's actin-remodeling role to tumor invasion by linking it to PAK1-driven protrusion formation.\",\n      \"evidence\": \"Co-IP, PAK1 inhibitor, KD/OE and invasion assays in gastric cancer cells\",\n      \"pmids\": [\"25766321\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PAK1 acts upstream or downstream not fully separated\", \"No structural basis for interaction\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed RUFY3 in a defined neuronal signaling pathway, showing it scaffolds Rap2 and activates Tiam2/STEF at lipid rafts to drive axon growth.\",\n      \"evidence\": \"Rufy3-KO mice, detergent-resistant membrane fractionation, Co-IP and polarity assays\",\n      \"pmids\": [\"29089386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct GEF activation mechanism of Tiam2/STEF not reconstituted\", \"Role of M6A recruitment in other cell types untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined a pro-metastatic axis in which RUFY3 acts through transcription factor FOXK1 to drive EMT.\",\n      \"evidence\": \"Co-IP, FOXK1 siRNA epistasis, EMT markers and orthotopic implantation in colorectal cancer\",\n      \"pmids\": [\"28623323\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How a trafficking adapter regulates a transcription factor unclear\", \"Direct vs indirect interaction not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Positioned RUFY3 as a downstream effector of TGF-β1 in EMT regulation.\",\n      \"evidence\": \"TGF-β1 dose-response and siRNA with EMT/invasion readouts in colorectal cancer cells\",\n      \"pmids\": [\"28089833\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct binding or pathway reconstitution\", \"Single method per claim, single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified an upstream transcriptional driver of RUFY3 by showing HOXD9 binds the RUFY3 promoter to activate its expression in gastric cancer.\",\n      \"evidence\": \"ChIP, luciferase reporter and siRNA epistasis with tumor growth assays\",\n      \"pmids\": [\"31547840\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Other transcriptional inputs not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a degeneration-specific function: RUFY3 is required for caspase-3-mediated axon degeneration, with S34 dephosphorylation as the permissive switch.\",\n      \"evidence\": \"Mass spec, Rufy3 genetic deletion, phosphosite analysis, in vitro and in vivo sensory axon degeneration\",\n      \"pmids\": [\"31221560\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase/phosphatase controlling S34 not identified\", \"Downstream effectors of dephosphorylated RUFY3 unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked RUFY3 to NF-κB-mediated EMT in hepatocellular carcinoma growth and metastasis.\",\n      \"evidence\": \"siRNA, NF-κB activity assay, EMT markers and xenograft/metastasis models\",\n      \"pmids\": [\"34510031\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct RUFY3-NF-κB interaction demonstrated\", \"Mechanism inferred from pathway inhibitors\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined RUFY3's core trafficking mechanism by establishing it as an Arl8b effector that recruits the JIP4-dynein-dynactin retrograde motor to position lysosomes.\",\n      \"evidence\": \"Reciprocal Co-IP, knockdown, live-cell imaging, colocalization and PIKFYVE-rescue of lysosome size\",\n      \"pmids\": [\"35314681\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the Arl8b-RUFY3-JIP4 complex unresolved\", \"How motor handoff is regulated unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended RUFY3-Rap signaling to injury repair, showing Rufy3 raises Rap1-GTP and activates downstream Rho/Fascin and MEK/ERK pathways for axon repair and synaptic plasticity.\",\n      \"evidence\": \"Co-IP, Rap1-GTP pull-down, lentiviral OE/KD in a subarachnoid hemorrhage rat model with pathway western blots\",\n      \"pmids\": [\"35461284\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RUFY3 directly catalyzes Rap1 loading not tested\", \"In vivo pathway dependencies inferred from western blot\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved isoform-specific specialization, showing a FYVE-bearing immune isoform binds PI3P to position endo-lysosomes and control macrophage immune functions and Salmonella restriction.\",\n      \"evidence\": \"Isoform characterization, PI3P-binding, phagocyte-specific conditional KO, and in vivo infection models\",\n      \"pmids\": [\"37463962\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional division between isoforms in non-immune cells unclear\", \"How PI3P binding integrates with Arl8b effector role not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Assigned a catalytic activity to RUFY3, identifying it (with HPIP) as a noncanonical Rab5 GEF driving focal adhesion turnover and integrin trafficking.\",\n      \"evidence\": \"Rab5 GEF activity assay, coiled-coil deletion mutants, Co-IP, focal adhesion and integrin trafficking assays\",\n      \"pmids\": [\"37797694\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of noncanonical GEF activity not solved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Tied RUFY3 to cell-death control in cancer by linking a RUFY3-MAP4-CDK1 interaction to suppression of PANoptosis.\",\n      \"evidence\": \"Co-IP and siRNA with xenograft tumor models in colorectal cancer\",\n      \"pmids\": [\"40879686\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"Mechanism downstream of the complex not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RUFY3's distinct activities — Arl8b/PI3P-dependent lysosome positioning, noncanonical Rab5/Rap GEF function, and S34-regulated degeneration control — are coordinated within a single protein, and which are isoform- or context-specific, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model connecting RUN, FYVE and coiled-coil domain functions\", \"Phosphoregulation linking trafficking and degeneration roles unmapped\", \"Direct GEF mechanism for Rab5/Rap not structurally resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11, 12]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1, 11]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 11]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [4, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 11]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 12]}\n    ],\n    \"complexes\": [\n      \"Arl8b-RUFY3-JIP4-dynein-dynactin complex\",\n      \"RUFY3-Rap2-Tiam2/STEF ternary complex\"\n    ],\n    \"partners\": [\n      \"ARL8B\",\n      \"JIP4\",\n      \"RAP2\",\n      \"TIAM2\",\n      \"FSCN1\",\n      \"PAK1\",\n      \"FOXK1\",\n      \"RAB5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}