{"gene":"TBC1D10B","run_date":"2026-04-28T21:42:58","timeline":{"discoveries":[{"year":2008,"finding":"TBC1D10B (FLJ13130) is a Rab-GAP with broad specificity that promotes GTPase activity of Rab3A, Rab22A, Rab27A, and Rab35 in vitro, and expression near the plasma membrane excludes endogenous Rab3A from dense-core vesicles; a catalytically inactive R134K mutant abolished these effects.","method":"In vitro GTPase assay, cell-based GTP-Rab3A depletion assay, catalytically inactive mutant (R134K)","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay with mutagenesis confirming catalytic requirement","pmids":["19077034"],"is_preprint":false},{"year":2012,"finding":"TBC1D10B (EPI64B) acts as a Rab35 GAP and functions as an effector of ARF6 to negatively regulate Rab35 activation at clathrin-coated pits, thereby controlling endocytic recycling and cytokinesis; activated ARF6 recruits EPI64B to reduce Rab35 loading into the endocytic pathway.","method":"Co-IP, epistasis with constitutively active/dominant negative ARF6 and Rab35 mutants, endocytic recycling assays, cytokinesis phenotype readout","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — reciprocal functional epistasis with multiple GTPase mutants replicated across assays, 128 citations","pmids":["22226746"],"is_preprint":false},{"year":2013,"finding":"TBC1D10B (EPI64B) acts as a GAP specifically for Rab27B (but not Rab3D) in pancreatic acinar cells, nearly completely abolishing GTP-Rab27B; overexpression enhances amylase release via Rab27B-dependent exocytosis, and GAP-inactive mutants abolish both effects.","method":"Adenovirus-mediated overexpression in isolated pancreatic acini, GTP-Rab27B pull-down assay, amylase secretion assay, knockout mouse acini, catalytically inactive mutant","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro/cell GAP activity assay with mutagenesis and genetic mouse model validation","pmids":["23671284"],"is_preprint":false},{"year":2015,"finding":"TBC1D10B (EPI64B) functions as a Rab35 GAP whose disappearance from clathrin-coated vesicles immediately after scission triggers Rab35 activation, enabling switch-like recruitment of the OCRL lipid phosphatase on newborn endosomes for PtdIns(4,5)P2 hydrolysis and subsequent cargo sorting.","method":"Live-cell imaging, siRNA knockdown, co-localization assays, endosomal cargo trafficking readout (CI-MPR retention)","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — live imaging with spatial/temporal resolution combined with siRNA and cargo tracking, 79 citations","pmids":["26725203"],"is_preprint":false},{"year":2019,"finding":"TBC1D10B lowers Erk1/2 and p38 signaling downstream of VEGFR2, reduces tube formation in vitro, and decreases surface expression of VEGFR2 and NRP1 on filopodia of activated endothelial cells, with opposite effects to paralog TBC1D10A.","method":"Overexpression in endothelial cells, western blotting for signaling, in vitro tube formation assay, immunofluorescence colocalization","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, overexpression phenotype with signaling readout but no direct Rab substrate identified","pmids":["31527750"],"is_preprint":false},{"year":2021,"finding":"BAG3 forms a complex with HSP70 and TBC1D10B that attenuates TBC1D10B's ability to inactivate RAB35, thereby supporting RAB35 activation and HRS recruitment to initiate ESCRT-mediated endosomal tau clearance.","method":"Mass spectrometry interactome, biochemical co-IP assays, live-cell imaging of endosomal dynamics, immunohistochemistry in human AD brain and P301S tau transgenic mice","journal":"Biological psychiatry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal biochemical interaction, multiple orthogonal assays, in vivo transgenic mouse validation","pmids":["35000752"],"is_preprint":false},{"year":2021,"finding":"TBC1D10B localizes to apical microvilli of epithelial cells via a localization domain spanning its RabGAP domains; CRISPR knockout of EPI64B reduces apical microvilli in Jeg-3 cells, likely through misregulation of Rab8 and Rab35, and disrupts apical junction morphology in Caco2 cells.","method":"CRISPR/Cas9 knockout, immunofluorescence localization, domain mapping, morphological phenotype analysis","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined apical morphology phenotype and domain localization mapping, but Rab8/35 involvement inferred rather than directly demonstrated","pmids":["34757852"],"is_preprint":false},{"year":2011,"finding":"siRNA-mediated depletion of TBC1D10B in human epithelial cells results in increased Salmonella typhimurium replication, identifying TBC1D10B as a host factor that restricts intracellular bacterial replication.","method":"SILAC-based quantitative proteomics of Golgi-enriched fractions, siRNA knockdown, intracellular Salmonella replication assay","journal":"Proteomics","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative proteomics plus functional siRNA validation with defined phenotypic readout, but no direct molecular mechanism identified","pmids":["21919203"],"is_preprint":false},{"year":2024,"finding":"TBC1D10B colocalizes with Rit1 GTPase at phagocytic cup membranes in RAW264 macrophages; TBC1D10B decreases FcγR-mediated phagosome formation in both Rab-GAP activity-dependent and -independent manners. Rit1 (GTP-locked) promotes dissociation of TBC1D10B from phagocytic cups and rescues phagosome formation in TBC1D10B-expressing cells, placing Rit1 upstream of TBC1D10B in this pathway.","method":"Live-cell imaging, Rit1 knockout, GDP-locked and GTP-locked Rit1 mutant expression, TBC1D10B overexpression/knockout, phagosome formation quantification","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 — epistatic relationship established with GTPase mutants and KO, live imaging, but Rab substrate in this context not fully resolved","pmids":["39084876"],"is_preprint":false},{"year":2025,"finding":"TBC1D10B is required for tubular endosome formation in HeLa cells in a GAP-activity-dependent manner; knockdown or overexpression both reduce tubular endosome structures. TBC1D10B reduces active Rab22A levels and Rab22A-positive early endosome size, identifying Rab22A as its most probable substrate in this context.","method":"Comprehensive TBC/RabGAP siRNA screen, overexpression, active Rab22A pull-down, fluorescence microscopy of tubular endosomes","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 — systematic screen plus GAP-activity-dependent phenotype and active Rab22A measurement, but direct in vitro reconstitution not shown","pmids":["40241313"],"is_preprint":false}],"current_model":"TBC1D10B (EPI64B) is a TBC-domain RabGAP with broad substrate specificity (Rab3A, Rab22A, Rab27A, Rab27B, Rab35) that localizes to clathrin-coated pits/vesicles and apical microvilli, where it acts downstream of ARF6 and Rit1 to regulate endocytic recycling, ESCRT-mediated cargo sorting, exocytosis, tubular endosome formation, and FcγR-mediated phagocytosis; its activity is suppressed by a BAG3–HSP70 complex to support RAB35-dependent tau clearance."},"narrative":{"teleology":[{"year":2008,"claim":"Establishing TBC1D10B as a bona fide RabGAP: it was unknown whether TBC1D10B possessed catalytic activity, and in vitro assays demonstrated broad specificity toward Rab3A, Rab22A, Rab27A, and Rab35, with the R134K mutation ablating activity, proving the catalytic TBC domain is functional.","evidence":"In vitro GTPase assays with purified Rab proteins and catalytically dead R134K mutant in mammalian cells","pmids":["19077034"],"confidence":"High","gaps":["In vivo substrate selectivity undetermined","No structural basis for broad specificity","Regulation of TBC1D10B activity itself unknown"]},{"year":2011,"claim":"An early hint that TBC1D10B functions in membrane defense: siRNA depletion increased intracellular Salmonella replication, revealing a host-restrictive role, though the Rab substrate and mechanism were not identified.","evidence":"SILAC proteomics of Golgi-enriched fractions combined with siRNA knockdown and bacterial replication assay in human epithelial cells","pmids":["21919203"],"confidence":"Medium","gaps":["No direct Rab substrate identified in this context","Mechanism of bacterial restriction unknown","Single study without independent replication"]},{"year":2012,"claim":"Placing TBC1D10B in the ARF6–Rab35 signaling axis: it was unclear how Rab35 is inactivated at endocytic membranes, and this work showed ARF6 recruits TBC1D10B to clathrin-coated pits to suppress Rab35, linking it to endocytic recycling and cytokinesis.","evidence":"Co-IP, epistasis with constitutively active/dominant-negative ARF6 and Rab35 mutants, endocytic recycling and cytokinesis assays in mammalian cells","pmids":["22226746"],"confidence":"High","gaps":["Direct structural interaction between ARF6 and TBC1D10B not mapped","Relative contribution of TBC1D10B versus paralogs (EPI64/EPI64C) to Rab35 inactivation unclear"]},{"year":2013,"claim":"Demonstrating tissue-specific substrate usage: in pancreatic acinar cells TBC1D10B acts as a Rab27B-selective GAP to regulate exocytic amylase secretion, establishing that its broad in vitro specificity translates to context-dependent selectivity in vivo.","evidence":"Adenoviral overexpression and catalytic mutants in isolated mouse pancreatic acini, GTP-Rab27B pull-down, amylase secretion assay","pmids":["23671284"],"confidence":"High","gaps":["How TBC1D10B discriminates Rab27B from Rab3D in acinar cells not resolved","Upstream signals directing TBC1D10B to exocytic granules unknown"]},{"year":2015,"claim":"Revealing the spatial logic of Rab35 activation: the disappearance of TBC1D10B from clathrin-coated vesicles immediately after scission was shown to be the trigger for switch-like Rab35 activation and OCRL recruitment, providing a timer mechanism for endosomal PtdIns(4,5)P₂ hydrolysis and cargo sorting.","evidence":"Live-cell imaging with temporal resolution of vesicle scission, siRNA knockdown, CI-MPR cargo sorting readout","pmids":["26725203"],"confidence":"High","gaps":["Mechanism of TBC1D10B dissociation from vesicles at scission not identified","Whether this timing mechanism operates in all cell types unknown"]},{"year":2019,"claim":"Extending TBC1D10B function to receptor signaling: overexpression in endothelial cells attenuated VEGFR2/NRP1 surface expression and Erk/p38 signaling, suggesting a role in receptor trafficking that influences angiogenic responses.","evidence":"Overexpression in endothelial cells, western blotting, tube formation assay, immunofluorescence","pmids":["31527750"],"confidence":"Medium","gaps":["Rab substrate responsible for VEGFR2 trafficking not identified","Only overexpression; loss-of-function not tested","Single lab observation"]},{"year":2021,"claim":"Identifying a chaperone-based suppression mechanism: BAG3–HSP70 complex binds TBC1D10B and attenuates its GAP activity toward RAB35, maintaining RAB35 in its active state to drive HRS recruitment and ESCRT-mediated endosomal tau clearance — connecting TBC1D10B to neurodegeneration-relevant proteostasis.","evidence":"Mass spectrometry interactome, co-IP, live-cell endosomal imaging, validation in P301S tau transgenic mice and human AD brain","pmids":["35000752"],"confidence":"High","gaps":["Direct biochemical reconstitution of BAG3–HSP70 inhibition of TBC1D10B GAP activity not shown","Whether this mechanism operates in neurons in vivo not directly tested"]},{"year":2021,"claim":"Establishing an apical morphogenesis role: TBC1D10B localizes to epithelial microvilli via its RabGAP domains, and CRISPR knockout reduces microvilli and disrupts apical junctions, linking its GAP activity to apical membrane organization.","evidence":"CRISPR/Cas9 knockout in Jeg-3 and Caco2 cells, domain mapping, immunofluorescence","pmids":["34757852"],"confidence":"Medium","gaps":["Rab8 and Rab35 involvement inferred but not directly measured","Mechanism linking GAP activity to microvillar assembly unknown","In vivo relevance in intestinal or placental epithelium not tested"]},{"year":2024,"claim":"Placing TBC1D10B downstream of Rit1 in phagocytosis: Rit1 GTPase promotes TBC1D10B dissociation from phagocytic cups, relieving its inhibitory effect on Fcγ receptor-mediated phagosome formation — revealing both GAP-dependent and GAP-independent inhibitory mechanisms.","evidence":"Live-cell imaging, Rit1 knockout and GTPase-locked mutants, TBC1D10B overexpression/knockout, phagosome quantification in RAW264 macrophages","pmids":["39084876"],"confidence":"Medium","gaps":["Rab substrate at the phagocytic cup not identified","GAP-independent inhibitory mechanism molecularly undefined","Single macrophage cell line used"]},{"year":2025,"claim":"Identifying Rab22A as the functional substrate for tubular endosome biogenesis: a systematic screen showed TBC1D10B is required for tubular endosome formation in a GAP-dependent manner, with active Rab22A as its principal target in this context.","evidence":"Comprehensive TBC/RabGAP siRNA screen, overexpression, active-Rab22A pull-down, fluorescence microscopy in HeLa cells","pmids":["40241313"],"confidence":"Medium","gaps":["Direct in vitro reconstitution of Rab22A GAP activity in this system not shown","How tubular endosome formation serves downstream trafficking unknown","Relationship between Rab22A and Rab35 regulation by TBC1D10B at endosomes unresolved"]},{"year":null,"claim":"Key unresolved questions include: the structural basis for TBC1D10B's broad yet context-dependent Rab selectivity, the molecular mechanism of its GAP-independent inhibition of phagocytosis, whether its loss contributes to human disease, and how upstream signals (ARF6, Rit1, BAG3–HSP70) are integrated to coordinate its activity across different membrane compartments.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure available","No Mendelian or somatic disease association established by direct evidence","Integrated signaling logic across ARF6/Rit1/BAG3 inputs not modeled"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[0,1,2,3,5,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,3,5,9]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,6,8]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3,5,9]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,3,5,9]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,4,5]}],"complexes":[],"partners":["ARF6","RAB35","RAB27B","RAB22A","BAG3","HSP70","RIT1","RAB3A"],"other_free_text":[]},"mechanistic_narrative":"TBC1D10B (EPI64B) is a TBC-domain Rab GTPase-activating protein (RabGAP) with broad substrate specificity that orchestrates membrane identity transitions during endocytic recycling, exocytosis, phagocytosis, and cargo sorting. It catalyzes GTP hydrolysis on Rab3A, Rab22A, Rab27A, Rab27B, and Rab35 in vitro and in cells, with catalytic arginine residue R134 essential for activity [PMID:19077034, PMID:23671284, PMID:22226746]. At clathrin-coated pits, ARF6-dependent recruitment of TBC1D10B suppresses Rab35 until vesicle scission, whereupon its departure triggers switch-like Rab35 activation and OCRL-mediated PtdIns(4,5)P₂ hydrolysis on newborn endosomes; a BAG3–HSP70 complex attenuates TBC1D10B activity to sustain RAB35-dependent ESCRT-mediated tau clearance [PMID:26725203, PMID:22226746, PMID:35000752]. TBC1D10B also localizes to apical microvilli where its loss disrupts microvillar and junctional architecture, regulates tubular endosome formation through Rab22A inactivation, and modulates Fcγ receptor–mediated phagocytosis downstream of the Rit1 GTPase [PMID:34757852, PMID:40241313, PMID:39084876]."},"prefetch_data":{"uniprot":{"accession":"Q4KMP7","full_name":"TBC1 domain family member 10B","aliases":["Rab27A-GAP-beta"],"length_aa":808,"mass_kda":87.2,"function":"Acts as a GTPase-activating protein for RAB3A, RAB22A, RAB27A, and RAB35. Does not act on RAB2A and RAB6A","subcellular_location":"Cytoplasm; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q4KMP7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TBC1D10B","classification":"Not Classified","n_dependent_lines":59,"n_total_lines":1208,"dependency_fraction":0.048841059602649006},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"RIOK1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TBC1D10B","total_profiled":1310},"omim":[{"mim_id":"613620","title":"TBC1 DOMAIN FAMILY, MEMBER 10B; TBC1D10B","url":"https://www.omim.org/entry/613620"},{"mim_id":"610020","title":"TBC1 DOMAIN FAMILY, MEMBER 10A; TBC1D10A","url":"https://www.omim.org/entry/610020"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TBC1D10B"},"hgnc":{"alias_symbol":["DKFZP434P1750","Rab27A-GAPbeta","FLJ13130","EPI64B"],"prev_symbol":[]},"alphafold":{"accession":"Q4KMP7","domains":[{"cath_id":"1.10.8.270","chopping":"364-476","consensus_level":"medium","plddt":96.5976,"start":364,"end":476},{"cath_id":"1.10.472.80","chopping":"481-633","consensus_level":"medium","plddt":94.2456,"start":481,"end":633},{"cath_id":"1.10.287","chopping":"326-360","consensus_level":"medium","plddt":95.9903,"start":326,"end":360}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q4KMP7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q4KMP7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q4KMP7-F1-predicted_aligned_error_v6.png","plddt_mean":67.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TBC1D10B","jax_strain_url":"https://www.jax.org/strain/search?query=TBC1D10B"},"sequence":{"accession":"Q4KMP7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q4KMP7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q4KMP7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q4KMP7"}},"corpus_meta":[{"pmid":"22226746","id":"PMC_22226746","title":"An ARF6/Rab35 GTPase cascade for endocytic recycling and successful cytokinesis.","date":"2012","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/22226746","citation_count":128,"is_preprint":false},{"pmid":"26725203","id":"PMC_26725203","title":"Rab35 GTPase Triggers Switch-like Recruitment of the Lowe Syndrome Lipid Phosphatase OCRL on Newborn Endosomes.","date":"2015","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/26725203","citation_count":79,"is_preprint":false},{"pmid":"19077034","id":"PMC_19077034","title":"Identification and characterization of a novel Tre-2/Bub2/Cdc16 (TBC) protein that possesses Rab3A-GAP activity.","date":"2008","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/19077034","citation_count":46,"is_preprint":false},{"pmid":"31527750","id":"PMC_31527750","title":"Regulation of VEGFR2 trafficking and signaling by Rab GTPase-activating proteins.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31527750","citation_count":27,"is_preprint":false},{"pmid":"35000752","id":"PMC_35000752","title":"BAG3 Regulation of RAB35 Mediates the Endosomal Sorting Complexes Required for Transport/Endolysosome Pathway and Tau Clearance.","date":"2021","source":"Biological psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/35000752","citation_count":23,"is_preprint":false},{"pmid":"21919203","id":"PMC_21919203","title":"Quantitative proteomic identification of host factors involved in the Salmonella typhimurium infection cycle.","date":"2011","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/21919203","citation_count":21,"is_preprint":false},{"pmid":"28334711","id":"PMC_28334711","title":"Quantitative Proteomics Analysis of Ischemia/Reperfusion Injury-Modulated Proteins in Cardiac Microvascular Endothelial Cells and the Protective Role of Tongxinluo.","date":"2017","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/28334711","citation_count":19,"is_preprint":false},{"pmid":"23671284","id":"PMC_23671284","title":"EPI64B acts as a GTPase-activating protein for Rab27B in pancreatic acinar cells.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23671284","citation_count":15,"is_preprint":false},{"pmid":"32397798","id":"PMC_32397798","title":"STAT4-mediated down-regulation of miR-3619-5p facilitates stomach adenocarcinoma by modulating TBC1D10B.","date":"2020","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/32397798","citation_count":9,"is_preprint":false},{"pmid":"35589867","id":"PMC_35589867","title":"Evaluation of cfDNA as an early detection assay for dense tissue breast cancer.","date":"2022","source":"Scientific 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GTPase-Activating Proteins Required for Tubular Endosome Formation.","date":"2025","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/40241313","citation_count":0,"is_preprint":false},{"pmid":"41532798","id":"PMC_41532798","title":"The Proteome of Human Amyloid Beta Oligomers.","date":"2026","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41532798","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10592,"output_tokens":2545,"usd":0.034975},"stage2":{"model":"claude-opus-4-6","input_tokens":5865,"output_tokens":3040,"usd":0.157988},"total_usd":0.192963,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"TBC1D10B (FLJ13130) is a Rab-GAP with broad specificity that promotes GTPase activity of Rab3A, Rab22A, Rab27A, and Rab35 in vitro, and expression near the plasma membrane excludes endogenous Rab3A from dense-core vesicles; a catalytically inactive R134K mutant abolished these effects.\",\n      \"method\": \"In vitro GTPase assay, cell-based GTP-Rab3A depletion assay, catalytically inactive mutant (R134K)\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with mutagenesis confirming catalytic requirement\",\n      \"pmids\": [\"19077034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TBC1D10B (EPI64B) acts as a Rab35 GAP and functions as an effector of ARF6 to negatively regulate Rab35 activation at clathrin-coated pits, thereby controlling endocytic recycling and cytokinesis; activated ARF6 recruits EPI64B to reduce Rab35 loading into the endocytic pathway.\",\n      \"method\": \"Co-IP, epistasis with constitutively active/dominant negative ARF6 and Rab35 mutants, endocytic recycling assays, cytokinesis phenotype readout\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal functional epistasis with multiple GTPase mutants replicated across assays, 128 citations\",\n      \"pmids\": [\"22226746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TBC1D10B (EPI64B) acts as a GAP specifically for Rab27B (but not Rab3D) in pancreatic acinar cells, nearly completely abolishing GTP-Rab27B; overexpression enhances amylase release via Rab27B-dependent exocytosis, and GAP-inactive mutants abolish both effects.\",\n      \"method\": \"Adenovirus-mediated overexpression in isolated pancreatic acini, GTP-Rab27B pull-down assay, amylase secretion assay, knockout mouse acini, catalytically inactive mutant\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro/cell GAP activity assay with mutagenesis and genetic mouse model validation\",\n      \"pmids\": [\"23671284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TBC1D10B (EPI64B) functions as a Rab35 GAP whose disappearance from clathrin-coated vesicles immediately after scission triggers Rab35 activation, enabling switch-like recruitment of the OCRL lipid phosphatase on newborn endosomes for PtdIns(4,5)P2 hydrolysis and subsequent cargo sorting.\",\n      \"method\": \"Live-cell imaging, siRNA knockdown, co-localization assays, endosomal cargo trafficking readout (CI-MPR retention)\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live imaging with spatial/temporal resolution combined with siRNA and cargo tracking, 79 citations\",\n      \"pmids\": [\"26725203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TBC1D10B lowers Erk1/2 and p38 signaling downstream of VEGFR2, reduces tube formation in vitro, and decreases surface expression of VEGFR2 and NRP1 on filopodia of activated endothelial cells, with opposite effects to paralog TBC1D10A.\",\n      \"method\": \"Overexpression in endothelial cells, western blotting for signaling, in vitro tube formation assay, immunofluorescence colocalization\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, overexpression phenotype with signaling readout but no direct Rab substrate identified\",\n      \"pmids\": [\"31527750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BAG3 forms a complex with HSP70 and TBC1D10B that attenuates TBC1D10B's ability to inactivate RAB35, thereby supporting RAB35 activation and HRS recruitment to initiate ESCRT-mediated endosomal tau clearance.\",\n      \"method\": \"Mass spectrometry interactome, biochemical co-IP assays, live-cell imaging of endosomal dynamics, immunohistochemistry in human AD brain and P301S tau transgenic mice\",\n      \"journal\": \"Biological psychiatry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal biochemical interaction, multiple orthogonal assays, in vivo transgenic mouse validation\",\n      \"pmids\": [\"35000752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TBC1D10B localizes to apical microvilli of epithelial cells via a localization domain spanning its RabGAP domains; CRISPR knockout of EPI64B reduces apical microvilli in Jeg-3 cells, likely through misregulation of Rab8 and Rab35, and disrupts apical junction morphology in Caco2 cells.\",\n      \"method\": \"CRISPR/Cas9 knockout, immunofluorescence localization, domain mapping, morphological phenotype analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined apical morphology phenotype and domain localization mapping, but Rab8/35 involvement inferred rather than directly demonstrated\",\n      \"pmids\": [\"34757852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"siRNA-mediated depletion of TBC1D10B in human epithelial cells results in increased Salmonella typhimurium replication, identifying TBC1D10B as a host factor that restricts intracellular bacterial replication.\",\n      \"method\": \"SILAC-based quantitative proteomics of Golgi-enriched fractions, siRNA knockdown, intracellular Salmonella replication assay\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative proteomics plus functional siRNA validation with defined phenotypic readout, but no direct molecular mechanism identified\",\n      \"pmids\": [\"21919203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TBC1D10B colocalizes with Rit1 GTPase at phagocytic cup membranes in RAW264 macrophages; TBC1D10B decreases FcγR-mediated phagosome formation in both Rab-GAP activity-dependent and -independent manners. Rit1 (GTP-locked) promotes dissociation of TBC1D10B from phagocytic cups and rescues phagosome formation in TBC1D10B-expressing cells, placing Rit1 upstream of TBC1D10B in this pathway.\",\n      \"method\": \"Live-cell imaging, Rit1 knockout, GDP-locked and GTP-locked Rit1 mutant expression, TBC1D10B overexpression/knockout, phagosome formation quantification\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistatic relationship established with GTPase mutants and KO, live imaging, but Rab substrate in this context not fully resolved\",\n      \"pmids\": [\"39084876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TBC1D10B is required for tubular endosome formation in HeLa cells in a GAP-activity-dependent manner; knockdown or overexpression both reduce tubular endosome structures. TBC1D10B reduces active Rab22A levels and Rab22A-positive early endosome size, identifying Rab22A as its most probable substrate in this context.\",\n      \"method\": \"Comprehensive TBC/RabGAP siRNA screen, overexpression, active Rab22A pull-down, fluorescence microscopy of tubular endosomes\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic screen plus GAP-activity-dependent phenotype and active Rab22A measurement, but direct in vitro reconstitution not shown\",\n      \"pmids\": [\"40241313\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TBC1D10B (EPI64B) is a TBC-domain RabGAP with broad substrate specificity (Rab3A, Rab22A, Rab27A, Rab27B, Rab35) that localizes to clathrin-coated pits/vesicles and apical microvilli, where it acts downstream of ARF6 and Rit1 to regulate endocytic recycling, ESCRT-mediated cargo sorting, exocytosis, tubular endosome formation, and FcγR-mediated phagocytosis; its activity is suppressed by a BAG3–HSP70 complex to support RAB35-dependent tau clearance.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TBC1D10B (EPI64B) is a TBC-domain Rab GTPase-activating protein (RabGAP) with broad substrate specificity that orchestrates membrane identity transitions during endocytic recycling, exocytosis, phagocytosis, and cargo sorting. It catalyzes GTP hydrolysis on Rab3A, Rab22A, Rab27A, Rab27B, and Rab35 in vitro and in cells, with catalytic arginine residue R134 essential for activity [PMID:19077034, PMID:23671284, PMID:22226746]. At clathrin-coated pits, ARF6-dependent recruitment of TBC1D10B suppresses Rab35 until vesicle scission, whereupon its departure triggers switch-like Rab35 activation and OCRL-mediated PtdIns(4,5)P₂ hydrolysis on newborn endosomes; a BAG3–HSP70 complex attenuates TBC1D10B activity to sustain RAB35-dependent ESCRT-mediated tau clearance [PMID:26725203, PMID:22226746, PMID:35000752]. TBC1D10B also localizes to apical microvilli where its loss disrupts microvillar and junctional architecture, regulates tubular endosome formation through Rab22A inactivation, and modulates Fcγ receptor–mediated phagocytosis downstream of the Rit1 GTPase [PMID:34757852, PMID:40241313, PMID:39084876].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Establishing TBC1D10B as a bona fide RabGAP: it was unknown whether TBC1D10B possessed catalytic activity, and in vitro assays demonstrated broad specificity toward Rab3A, Rab22A, Rab27A, and Rab35, with the R134K mutation ablating activity, proving the catalytic TBC domain is functional.\",\n      \"evidence\": \"In vitro GTPase assays with purified Rab proteins and catalytically dead R134K mutant in mammalian cells\",\n      \"pmids\": [\"19077034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo substrate selectivity undetermined\", \"No structural basis for broad specificity\", \"Regulation of TBC1D10B activity itself unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"An early hint that TBC1D10B functions in membrane defense: siRNA depletion increased intracellular Salmonella replication, revealing a host-restrictive role, though the Rab substrate and mechanism were not identified.\",\n      \"evidence\": \"SILAC proteomics of Golgi-enriched fractions combined with siRNA knockdown and bacterial replication assay in human epithelial cells\",\n      \"pmids\": [\"21919203\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct Rab substrate identified in this context\", \"Mechanism of bacterial restriction unknown\", \"Single study without independent replication\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placing TBC1D10B in the ARF6–Rab35 signaling axis: it was unclear how Rab35 is inactivated at endocytic membranes, and this work showed ARF6 recruits TBC1D10B to clathrin-coated pits to suppress Rab35, linking it to endocytic recycling and cytokinesis.\",\n      \"evidence\": \"Co-IP, epistasis with constitutively active/dominant-negative ARF6 and Rab35 mutants, endocytic recycling and cytokinesis assays in mammalian cells\",\n      \"pmids\": [\"22226746\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural interaction between ARF6 and TBC1D10B not mapped\", \"Relative contribution of TBC1D10B versus paralogs (EPI64/EPI64C) to Rab35 inactivation unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrating tissue-specific substrate usage: in pancreatic acinar cells TBC1D10B acts as a Rab27B-selective GAP to regulate exocytic amylase secretion, establishing that its broad in vitro specificity translates to context-dependent selectivity in vivo.\",\n      \"evidence\": \"Adenoviral overexpression and catalytic mutants in isolated mouse pancreatic acini, GTP-Rab27B pull-down, amylase secretion assay\",\n      \"pmids\": [\"23671284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TBC1D10B discriminates Rab27B from Rab3D in acinar cells not resolved\", \"Upstream signals directing TBC1D10B to exocytic granules unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealing the spatial logic of Rab35 activation: the disappearance of TBC1D10B from clathrin-coated vesicles immediately after scission was shown to be the trigger for switch-like Rab35 activation and OCRL recruitment, providing a timer mechanism for endosomal PtdIns(4,5)P₂ hydrolysis and cargo sorting.\",\n      \"evidence\": \"Live-cell imaging with temporal resolution of vesicle scission, siRNA knockdown, CI-MPR cargo sorting readout\",\n      \"pmids\": [\"26725203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of TBC1D10B dissociation from vesicles at scission not identified\", \"Whether this timing mechanism operates in all cell types unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extending TBC1D10B function to receptor signaling: overexpression in endothelial cells attenuated VEGFR2/NRP1 surface expression and Erk/p38 signaling, suggesting a role in receptor trafficking that influences angiogenic responses.\",\n      \"evidence\": \"Overexpression in endothelial cells, western blotting, tube formation assay, immunofluorescence\",\n      \"pmids\": [\"31527750\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Rab substrate responsible for VEGFR2 trafficking not identified\", \"Only overexpression; loss-of-function not tested\", \"Single lab observation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying a chaperone-based suppression mechanism: BAG3–HSP70 complex binds TBC1D10B and attenuates its GAP activity toward RAB35, maintaining RAB35 in its active state to drive HRS recruitment and ESCRT-mediated endosomal tau clearance — connecting TBC1D10B to neurodegeneration-relevant proteostasis.\",\n      \"evidence\": \"Mass spectrometry interactome, co-IP, live-cell endosomal imaging, validation in P301S tau transgenic mice and human AD brain\",\n      \"pmids\": [\"35000752\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical reconstitution of BAG3–HSP70 inhibition of TBC1D10B GAP activity not shown\", \"Whether this mechanism operates in neurons in vivo not directly tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Establishing an apical morphogenesis role: TBC1D10B localizes to epithelial microvilli via its RabGAP domains, and CRISPR knockout reduces microvilli and disrupts apical junctions, linking its GAP activity to apical membrane organization.\",\n      \"evidence\": \"CRISPR/Cas9 knockout in Jeg-3 and Caco2 cells, domain mapping, immunofluorescence\",\n      \"pmids\": [\"34757852\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Rab8 and Rab35 involvement inferred but not directly measured\", \"Mechanism linking GAP activity to microvillar assembly unknown\", \"In vivo relevance in intestinal or placental epithelium not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placing TBC1D10B downstream of Rit1 in phagocytosis: Rit1 GTPase promotes TBC1D10B dissociation from phagocytic cups, relieving its inhibitory effect on Fcγ receptor-mediated phagosome formation — revealing both GAP-dependent and GAP-independent inhibitory mechanisms.\",\n      \"evidence\": \"Live-cell imaging, Rit1 knockout and GTPase-locked mutants, TBC1D10B overexpression/knockout, phagosome quantification in RAW264 macrophages\",\n      \"pmids\": [\"39084876\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Rab substrate at the phagocytic cup not identified\", \"GAP-independent inhibitory mechanism molecularly undefined\", \"Single macrophage cell line used\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying Rab22A as the functional substrate for tubular endosome biogenesis: a systematic screen showed TBC1D10B is required for tubular endosome formation in a GAP-dependent manner, with active Rab22A as its principal target in this context.\",\n      \"evidence\": \"Comprehensive TBC/RabGAP siRNA screen, overexpression, active-Rab22A pull-down, fluorescence microscopy in HeLa cells\",\n      \"pmids\": [\"40241313\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro reconstitution of Rab22A GAP activity in this system not shown\", \"How tubular endosome formation serves downstream trafficking unknown\", \"Relationship between Rab22A and Rab35 regulation by TBC1D10B at endosomes unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structural basis for TBC1D10B's broad yet context-dependent Rab selectivity, the molecular mechanism of its GAP-independent inhibition of phagocytosis, whether its loss contributes to human disease, and how upstream signals (ARF6, Rit1, BAG3–HSP70) are integrated to coordinate its activity across different membrane compartments.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure available\", \"No Mendelian or somatic disease association established by direct evidence\", \"Integrated signaling logic across ARF6/Rit1/BAG3 inputs not modeled\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 6, 8]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3, 5, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 3, 5, 9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ARF6\", \"RAB35\", \"RAB27B\", \"RAB22A\", \"BAG3\", \"HSP70\", \"RIT1\", \"RAB3A\"],\n    \"other_free_text\": []\n  }\n}\n```"}