{"gene":"TBC1D10B","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2008,"finding":"TBC1D10B (FLJ13130) possesses broad Rab-GAP activity in vitro and in cells, promoting GTPase activity of Rab3A, Rab22A, Rab27A, and Rab35, but not Rab2A or Rab6A. A catalytically inactive R134K mutant abolished this activity, confirming the TBC domain is the catalytic unit. Expression of TBC1D10B (but not R134K) reduced GTP-Rab3A levels in PC12 cells and excluded endogenous Rab3A from dense-core vesicles.","method":"In vitro GTPase assay, cell-based Rab3A exclusion assay, catalytic mutant (R134K) comparison in PC12 cells","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro GTPase assay plus active-site mutagenesis plus cell-based assay, single lab but multiple orthogonal methods","pmids":["19077034"],"is_preprint":false},{"year":2012,"finding":"TBC1D10B (EPI64B) acts as a Rab35 GAP and functions as an effector of ARF6, placing it downstream of ARF6 in a signaling cascade. Activated ARF6 recruits EPI64B to negatively regulate Rab35 activation at clathrin-coated pits, thereby inhibiting Rab35-dependent endocytic recycling and causing cytokinesis defects.","method":"Epistasis (double-mutant analysis), live-cell imaging, co-immunoprecipitation, expression of constitutively active/dominant-negative GTPase mutants in human cells","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal epistasis with dominant mutants plus Co-IP plus imaging, single lab with multiple orthogonal methods","pmids":["22226746"],"is_preprint":false},{"year":2013,"finding":"TBC1D10B (EPI64B) functions as a GAP for Rab27B in pancreatic acinar cells: overexpression almost completely abolished GTP-Rab27B without affecting GTP-Rab3D, enhanced amylase release in a Rab27B-dependent manner, and GAP-dead mutations abolished both effects. Co-overexpression with Rab27B reversed Rab27B's inhibitory effect on amylase release.","method":"Adenovirus-mediated overexpression, GTP-Rab pulldown assay, amylase secretion assay, GAP-inactive mutant rescue, genetically modified mice (Rab27A knockout)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro/cell-based GTPase assay plus active-site mutagenesis plus secretion assay plus genetic mouse model, single lab with multiple orthogonal approaches","pmids":["23671284"],"is_preprint":false},{"year":2015,"finding":"TBC1D10B (EPI64B) localizes to clathrin-coated pits (CCPs) and its disappearance from newly formed clathrin-coated vesicles (CCVs) immediately after scission is required for Rab35 activation and subsequent OCRL lipid phosphatase recruitment. TBC1D10B acts as the Rab35 GAP/inhibitor whose spatial removal enables a switch-like activation of Rab35 on newborn endosomes.","method":"Live-cell imaging, fluorescence microscopy of CCV dynamics, depletion/overexpression of EPI64B combined with Rab35/OCRL localization assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct live-cell imaging of subcellular dynamics with functional consequence, combined with epistasis; replicated in independent lab (Echard group)","pmids":["26725203"],"is_preprint":false},{"year":2019,"finding":"TBC1D10B reduces ERK1/2 and p38 signaling downstream of VEGFR2 in endothelial cells and lowers VEGFR2 and NRP1 expression on filopodia of activated cells, leading to reduced tube formation in vitro. This is opposite to the effect of the paralog TBC1D10A.","method":"Overexpression in endothelial cells, Western blotting for ERK1/2 and p38 phosphorylation, immunofluorescence colocalization, tube formation assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, overexpression with signaling readout and localization, no direct substrate identification or mutagenesis","pmids":["31527750"],"is_preprint":false},{"year":2021,"finding":"TBC1D10B (EPI64B) localizes to apical microvilli of epithelial cells via a localization domain spanning the RabGAP domains. CRISPR/Cas9 knockout of EPI64B in Jeg-3 cells reduced apical microvilli density; double knockout with EPI64A caused more severe apical disruption, likely through misregulation of Rab8 and Rab35. EPI64B does not bind EBP50/NHERF1 (unlike EPI64A).","method":"CRISPR/Cas9 knockout, immunofluorescence microscopy, domain mapping, comparison of single/double knockouts","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO with specific apical morphology phenotype and domain mapping, single lab","pmids":["34757852"],"is_preprint":false},{"year":2021,"finding":"BAG3 forms a complex with HSP70 and TBC1D10B (EPI64B) that attenuates TBC1D10B's ability to inactivate RAB35. This BAG3-HSP70-TBC1D10B axis supports RAB35 activation, HRS recruitment, and ESCRT-dependent endosomal tau clearance. TBC1D10B colocalization with BAG3 is significantly reduced in Alzheimer's disease brains.","method":"Mass spectrometry (BAG3 interactors), co-immunoprecipitation, biochemical assays for GTP-RAB35, live-cell imaging, immunohistochemistry in human AD brain and P301S mouse model, BAG3 overexpression","journal":"Biological psychiatry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus MS interactomics plus in vivo mouse model plus human tissue validation, single lab with multiple orthogonal methods","pmids":["35000752"],"is_preprint":false},{"year":2024,"finding":"TBC1D10B colocalizes with Rit1 GTPase at F-actin-rich phagocytic cup membranes in RAW264 macrophages. TBC1D10B overexpression and knockout both decrease FcγR-mediated phagosome formation in both GAP-activity-dependent and -independent manners. GTP-locked Rit1 promotes dissociation of TBC1D10B from phagocytic cups and restores phagosome formation, while GDP-locked Rit1 or Rit1 knockout inhibits TBC1D10B dissociation, defining a Rit1→TBC1D10B signaling axis in phagocytosis.","method":"Live-cell imaging, knockout studies (Rit1 and TBC1D10B), expression of GTP/GDP-locked mutants, fluorescence colocalization assay, phagosome formation quantification","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging plus KO plus constitutively active/inactive mutants, single lab with multiple orthogonal methods","pmids":["39084876"],"is_preprint":false},{"year":2025,"finding":"TBC1D10B is required for tubular endosome formation in HeLa cells in a GAP-activity-dependent manner: both knockdown and overexpression reduce tubular endosome structures. TBC1D10B reduces the amount of active (GTP-bound) Rab22A and reduces the size of Rab22A-positive early endosomes, identifying Rab22A as its most probable physiological substrate in this context.","method":"Comprehensive TBC/Rab-GAP siRNA screen, overexpression, GTP-Rab22A pulldown assay, fluorescence microscopy of tubular endosome structures","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic knockdown/overexpression screen plus active Rab pulldown, single lab; mechanistic link to Rab22A supported by GTPase assay","pmids":["40241313"],"is_preprint":false},{"year":2011,"finding":"siRNA-mediated depletion of TBC1D10B in human epithelial cells resulted in increased Salmonella typhimurium replication, identifying TBC1D10B as a host factor that restricts intracellular Salmonella infection.","method":"SILAC-based quantitative proteomics of Golgi fractions plus siRNA knockdown with Salmonella replication readout","journal":"Proteomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single siRNA knockdown with phenotypic readout, no mechanistic pathway placement, single lab","pmids":["21919203"],"is_preprint":false}],"current_model":"TBC1D10B (EPI64B) is a broad-specificity Rab-GTPase-activating protein (RabGAP) that inactivates multiple Rab isoforms—including Rab3A, Rab22A, Rab27A/B, and Rab35—via its catalytic TBC domain; it localizes to clathrin-coated pits and apical microvilli, where it operates downstream of ARF6 and upstream of Rab35 to control endocytic recycling, cytokinesis bridge stability, post-scission phosphoinositide remodeling (via Rab35-OCRL), tubular endosome formation, and epithelial apical morphology; additionally, a BAG3-HSP70 complex sequesters TBC1D10B to sustain RAB35 activity and ESCRT-dependent tau clearance, and TBC1D10B is released from phagocytic cups in a Rit1 GTPase-dependent manner to regulate FcγR-mediated phagocytosis."},"narrative":{"mechanistic_narrative":"TBC1D10B (EPI64B) is a broad-specificity Rab-GTPase-activating protein that uses its catalytic TBC domain to accelerate GTP hydrolysis on multiple Rab isoforms—including Rab3A, Rab22A, Rab27A, and Rab35—thereby switching these traffic regulators off; an active-site R134K substitution abolishes this activity, defining the TBC domain as the catalytic unit [PMID:19077034]. Its best-defined cellular role is in endocytic membrane trafficking, where it functions as an effector of ARF6 that is recruited to clathrin-coated pits to restrain Rab35; the spatially controlled disappearance of TBC1D10B from newly formed clathrin-coated vesicles after scission permits switch-like Rab35 activation and downstream OCRL recruitment, and loss of this regulation produces endocytic recycling and cytokinesis defects [PMID:22226746, PMID:26725203]. Through its GAP activity it also shapes endosomal architecture, being required for tubular endosome formation via inactivation of Rab22A [PMID:40241313], and it controls regulated secretion as a Rab27B GAP that enhances amylase release in pancreatic acinar cells [PMID:23671284]. At epithelial apical surfaces TBC1D10B localizes to microvilli through a domain spanning its RabGAP region and supports apical microvillar density, acting redundantly with the paralog EPI64A [PMID:34757852]. Beyond core trafficking, a BAG3–HSP70 complex sequesters TBC1D10B to attenuate its inactivation of RAB35, sustaining ESCRT-dependent endosomal tau clearance—a function whose colocalization is reduced in Alzheimer's disease brains [PMID:35000752], and TBC1D10B is released from phagocytic cups in a Rit1 GTPase-dependent manner to regulate FcγR-mediated phagocytosis [PMID:39084876].","teleology":[{"year":2008,"claim":"Established that TBC1D10B is a catalytically active, broad-specificity RabGAP rather than an orphan TBC-domain protein, defining its molecular activity and substrate range.","evidence":"In vitro GTPase assays across multiple Rabs plus R134K active-site mutant and Rab3A exclusion assay in PC12 cells","pmids":["19077034"],"confidence":"High","gaps":["Did not establish which Rab is the physiological substrate in any given cell type","No structural basis for substrate selectivity"]},{"year":2012,"claim":"Placed TBC1D10B in a signaling cascade as an ARF6 effector that negatively regulates Rab35 at clathrin-coated pits, linking its GAP activity to endocytic recycling and cytokinesis.","evidence":"Epistasis with dominant GTPase mutants, Co-IP, and live-cell imaging in human cells","pmids":["22226746"],"confidence":"High","gaps":["Mechanism of ARF6-dependent recruitment not resolved at structural level","Did not address other Rab substrates in this context"]},{"year":2013,"claim":"Showed TBC1D10B acts as a Rab27B GAP controlling regulated secretion, demonstrating a context-specific physiological substrate distinct from Rab35.","evidence":"Adenoviral overexpression, GTP-Rab pulldown, amylase secretion assays, GAP-dead rescue, Rab27A-knockout mice in pancreatic acinar cells","pmids":["23671284"],"confidence":"High","gaps":["Whether endogenous TBC1D10B levels limit secretion not shown","Selectivity for Rab27B over Rab3D mechanism unexplained"]},{"year":2015,"claim":"Resolved how a constitutive Rab35 inhibitor permits Rab35 activation, showing spatial removal of TBC1D10B from newborn vesicles generates a switch-like Rab35-OCRL response.","evidence":"Live-cell imaging of CCV dynamics with depletion/overexpression and Rab35/OCRL localization (independent lab)","pmids":["26725203"],"confidence":"High","gaps":["Trigger for TBC1D10B disappearance from vesicles not defined","Direct OCRL link is downstream of Rab35, not of TBC1D10B itself"]},{"year":2019,"claim":"Linked TBC1D10B to VEGFR2 signaling and angiogenesis, contrasting its action with paralog TBC1D10A.","evidence":"Overexpression in endothelial cells with ERK1/2 and p38 phospho-Western, immunofluorescence, and tube formation assay","pmids":["31527750"],"confidence":"Medium","gaps":["No substrate identification or GAP-dead control","Effect shown only by overexpression, not loss-of-function"]},{"year":2021,"claim":"Defined a role at epithelial apical surfaces, showing TBC1D10B supports microvillar density redundantly with EPI64A and localizes via its RabGAP-spanning domain without binding EBP50.","evidence":"CRISPR/Cas9 single and double knockouts, immunofluorescence, and domain mapping in Jeg-3 cells","pmids":["34757852"],"confidence":"Medium","gaps":["Causal Rab substrate (Rab8/Rab35) at microvilli inferred, not directly demonstrated","Functional consequence of microvillar loss not assessed"]},{"year":2021,"claim":"Identified a regulatory mechanism in which BAG3-HSP70 sequesters TBC1D10B to sustain RAB35 activity and ESCRT-dependent tau clearance, connecting its GAP regulation to neurodegeneration.","evidence":"Mass spectrometry, reciprocal Co-IP, GTP-RAB35 assays, live imaging, P301S mouse and human AD brain immunohistochemistry","pmids":["35000752"],"confidence":"High","gaps":["Whether reduced BAG3-TBC1D10B colocalization is cause or consequence of AD pathology unresolved","Stoichiometry of the BAG3-HSP70-TBC1D10B complex not defined"]},{"year":2024,"claim":"Placed TBC1D10B downstream of Rit1 in macrophage phagocytosis, showing GTP-Rit1-driven dissociation from phagocytic cups regulates FcγR-mediated phagosome formation in both GAP-dependent and -independent ways.","evidence":"Live-cell imaging, Rit1 and TBC1D10B knockouts, GTP/GDP-locked Rit1 mutants in RAW264 macrophages","pmids":["39084876"],"confidence":"Medium","gaps":["GAP-independent mechanism of phagocytosis regulation undefined","Direct Rit1-TBC1D10B physical interaction not biochemically established"]},{"year":2025,"claim":"Identified Rab22A as a probable physiological substrate by showing TBC1D10B is required for tubular endosome formation through Rab22A inactivation.","evidence":"TBC/Rab-GAP siRNA screen, overexpression, GTP-Rab22A pulldown, and tubular endosome imaging in HeLa cells","pmids":["40241313"],"confidence":"Medium","gaps":["Why both knockdown and overexpression reduce tubular endosomes mechanistically unclear","Relationship to Rab35-based functions not integrated"]},{"year":null,"claim":"How TBC1D10B's broad in vitro substrate range is restricted to specific Rabs in specific compartments, and what governs its recruitment versus removal, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate selectivity","Determinants of compartment-specific recruitment unknown","Integration of secretory, endosomal, phagocytic, and neurodegenerative roles into one regulatory logic missing"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,3,8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,7]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,3,8]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7]}],"complexes":["BAG3-HSP70-TBC1D10B complex"],"partners":["ARF6","RAB35","RAB27B","RAB22A","BAG3","HSP70","RIT1"],"other_free_text":[]}},"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":129,"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":47,"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":28,"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":24,"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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Beta Oligomers.","date":"2026","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41532798","citation_count":0,"is_preprint":false},{"pmid":"41114811","id":"PMC_41114811","title":"Frequent TCR rearrangements in pediatric B-cell lymphoblastic acute leukemia: genomic and phenotypic features.","date":"2025","source":"Annals of hematology","url":"https://pubmed.ncbi.nlm.nih.gov/41114811","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11382,"output_tokens":2972,"usd":0.039363,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10207,"output_tokens":3395,"usd":0.067955,"stage2_stop_reason":"end_turn"},"total_usd":0.107318,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"TBC1D10B (FLJ13130) possesses broad Rab-GAP activity in vitro and in cells, promoting GTPase activity of Rab3A, Rab22A, Rab27A, and Rab35, but not Rab2A or Rab6A. A catalytically inactive R134K mutant abolished this activity, confirming the TBC domain is the catalytic unit. Expression of TBC1D10B (but not R134K) reduced GTP-Rab3A levels in PC12 cells and excluded endogenous Rab3A from dense-core vesicles.\",\n      \"method\": \"In vitro GTPase assay, cell-based Rab3A exclusion assay, catalytic mutant (R134K) comparison in PC12 cells\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro GTPase assay plus active-site mutagenesis plus cell-based assay, single lab but multiple orthogonal methods\",\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, placing it downstream of ARF6 in a signaling cascade. Activated ARF6 recruits EPI64B to negatively regulate Rab35 activation at clathrin-coated pits, thereby inhibiting Rab35-dependent endocytic recycling and causing cytokinesis defects.\",\n      \"method\": \"Epistasis (double-mutant analysis), live-cell imaging, co-immunoprecipitation, expression of constitutively active/dominant-negative GTPase mutants in human cells\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal epistasis with dominant mutants plus Co-IP plus imaging, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22226746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TBC1D10B (EPI64B) functions as a GAP for Rab27B in pancreatic acinar cells: overexpression almost completely abolished GTP-Rab27B without affecting GTP-Rab3D, enhanced amylase release in a Rab27B-dependent manner, and GAP-dead mutations abolished both effects. Co-overexpression with Rab27B reversed Rab27B's inhibitory effect on amylase release.\",\n      \"method\": \"Adenovirus-mediated overexpression, GTP-Rab pulldown assay, amylase secretion assay, GAP-inactive mutant rescue, genetically modified mice (Rab27A knockout)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro/cell-based GTPase assay plus active-site mutagenesis plus secretion assay plus genetic mouse model, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"23671284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TBC1D10B (EPI64B) localizes to clathrin-coated pits (CCPs) and its disappearance from newly formed clathrin-coated vesicles (CCVs) immediately after scission is required for Rab35 activation and subsequent OCRL lipid phosphatase recruitment. TBC1D10B acts as the Rab35 GAP/inhibitor whose spatial removal enables a switch-like activation of Rab35 on newborn endosomes.\",\n      \"method\": \"Live-cell imaging, fluorescence microscopy of CCV dynamics, depletion/overexpression of EPI64B combined with Rab35/OCRL localization assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct live-cell imaging of subcellular dynamics with functional consequence, combined with epistasis; replicated in independent lab (Echard group)\",\n      \"pmids\": [\"26725203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TBC1D10B reduces ERK1/2 and p38 signaling downstream of VEGFR2 in endothelial cells and lowers VEGFR2 and NRP1 expression on filopodia of activated cells, leading to reduced tube formation in vitro. This is opposite to the effect of the paralog TBC1D10A.\",\n      \"method\": \"Overexpression in endothelial cells, Western blotting for ERK1/2 and p38 phosphorylation, immunofluorescence colocalization, tube formation assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, overexpression with signaling readout and localization, no direct substrate identification or mutagenesis\",\n      \"pmids\": [\"31527750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TBC1D10B (EPI64B) localizes to apical microvilli of epithelial cells via a localization domain spanning the RabGAP domains. CRISPR/Cas9 knockout of EPI64B in Jeg-3 cells reduced apical microvilli density; double knockout with EPI64A caused more severe apical disruption, likely through misregulation of Rab8 and Rab35. EPI64B does not bind EBP50/NHERF1 (unlike EPI64A).\",\n      \"method\": \"CRISPR/Cas9 knockout, immunofluorescence microscopy, domain mapping, comparison of single/double knockouts\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO with specific apical morphology phenotype and domain mapping, single lab\",\n      \"pmids\": [\"34757852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BAG3 forms a complex with HSP70 and TBC1D10B (EPI64B) that attenuates TBC1D10B's ability to inactivate RAB35. This BAG3-HSP70-TBC1D10B axis supports RAB35 activation, HRS recruitment, and ESCRT-dependent endosomal tau clearance. TBC1D10B colocalization with BAG3 is significantly reduced in Alzheimer's disease brains.\",\n      \"method\": \"Mass spectrometry (BAG3 interactors), co-immunoprecipitation, biochemical assays for GTP-RAB35, live-cell imaging, immunohistochemistry in human AD brain and P301S mouse model, BAG3 overexpression\",\n      \"journal\": \"Biological psychiatry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus MS interactomics plus in vivo mouse model plus human tissue validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35000752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TBC1D10B colocalizes with Rit1 GTPase at F-actin-rich phagocytic cup membranes in RAW264 macrophages. TBC1D10B overexpression and knockout both decrease FcγR-mediated phagosome formation in both GAP-activity-dependent and -independent manners. GTP-locked Rit1 promotes dissociation of TBC1D10B from phagocytic cups and restores phagosome formation, while GDP-locked Rit1 or Rit1 knockout inhibits TBC1D10B dissociation, defining a Rit1→TBC1D10B signaling axis in phagocytosis.\",\n      \"method\": \"Live-cell imaging, knockout studies (Rit1 and TBC1D10B), expression of GTP/GDP-locked mutants, fluorescence colocalization assay, phagosome formation quantification\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging plus KO plus constitutively active/inactive mutants, single lab with multiple orthogonal methods\",\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: both knockdown and overexpression reduce tubular endosome structures. TBC1D10B reduces the amount of active (GTP-bound) Rab22A and reduces the size of Rab22A-positive early endosomes, identifying Rab22A as its most probable physiological substrate in this context.\",\n      \"method\": \"Comprehensive TBC/Rab-GAP siRNA screen, overexpression, GTP-Rab22A pulldown assay, fluorescence microscopy of tubular endosome structures\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic knockdown/overexpression screen plus active Rab pulldown, single lab; mechanistic link to Rab22A supported by GTPase assay\",\n      \"pmids\": [\"40241313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"siRNA-mediated depletion of TBC1D10B in human epithelial cells resulted in increased Salmonella typhimurium replication, identifying TBC1D10B as a host factor that restricts intracellular Salmonella infection.\",\n      \"method\": \"SILAC-based quantitative proteomics of Golgi fractions plus siRNA knockdown with Salmonella replication readout\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single siRNA knockdown with phenotypic readout, no mechanistic pathway placement, single lab\",\n      \"pmids\": [\"21919203\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TBC1D10B (EPI64B) is a broad-specificity Rab-GTPase-activating protein (RabGAP) that inactivates multiple Rab isoforms—including Rab3A, Rab22A, Rab27A/B, and Rab35—via its catalytic TBC domain; it localizes to clathrin-coated pits and apical microvilli, where it operates downstream of ARF6 and upstream of Rab35 to control endocytic recycling, cytokinesis bridge stability, post-scission phosphoinositide remodeling (via Rab35-OCRL), tubular endosome formation, and epithelial apical morphology; additionally, a BAG3-HSP70 complex sequesters TBC1D10B to sustain RAB35 activity and ESCRT-dependent tau clearance, and TBC1D10B is released from phagocytic cups in a Rit1 GTPase-dependent manner to regulate FcγR-mediated phagocytosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TBC1D10B (EPI64B) is a broad-specificity Rab-GTPase-activating protein that uses its catalytic TBC domain to accelerate GTP hydrolysis on multiple Rab isoforms—including Rab3A, Rab22A, Rab27A, and Rab35—thereby switching these traffic regulators off; an active-site R134K substitution abolishes this activity, defining the TBC domain as the catalytic unit [#0]. Its best-defined cellular role is in endocytic membrane trafficking, where it functions as an effector of ARF6 that is recruited to clathrin-coated pits to restrain Rab35; the spatially controlled disappearance of TBC1D10B from newly formed clathrin-coated vesicles after scission permits switch-like Rab35 activation and downstream OCRL recruitment, and loss of this regulation produces endocytic recycling and cytokinesis defects [#1, #3]. Through its GAP activity it also shapes endosomal architecture, being required for tubular endosome formation via inactivation of Rab22A [#8], and it controls regulated secretion as a Rab27B GAP that enhances amylase release in pancreatic acinar cells [#2]. At epithelial apical surfaces TBC1D10B localizes to microvilli through a domain spanning its RabGAP region and supports apical microvillar density, acting redundantly with the paralog EPI64A [#5]. Beyond core trafficking, a BAG3–HSP70 complex sequesters TBC1D10B to attenuate its inactivation of RAB35, sustaining ESCRT-dependent endosomal tau clearance—a function whose colocalization is reduced in Alzheimer's disease brains [#6], and TBC1D10B is released from phagocytic cups in a Rit1 GTPase-dependent manner to regulate FcγR-mediated phagocytosis [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established that TBC1D10B is a catalytically active, broad-specificity RabGAP rather than an orphan TBC-domain protein, defining its molecular activity and substrate range.\",\n      \"evidence\": \"In vitro GTPase assays across multiple Rabs plus R134K active-site mutant and Rab3A exclusion assay in PC12 cells\",\n      \"pmids\": [\"19077034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish which Rab is the physiological substrate in any given cell type\", \"No structural basis for substrate selectivity\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed TBC1D10B in a signaling cascade as an ARF6 effector that negatively regulates Rab35 at clathrin-coated pits, linking its GAP activity to endocytic recycling and cytokinesis.\",\n      \"evidence\": \"Epistasis with dominant GTPase mutants, Co-IP, and live-cell imaging in human cells\",\n      \"pmids\": [\"22226746\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of ARF6-dependent recruitment not resolved at structural level\", \"Did not address other Rab substrates in this context\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showed TBC1D10B acts as a Rab27B GAP controlling regulated secretion, demonstrating a context-specific physiological substrate distinct from Rab35.\",\n      \"evidence\": \"Adenoviral overexpression, GTP-Rab pulldown, amylase secretion assays, GAP-dead rescue, Rab27A-knockout mice in pancreatic acinar cells\",\n      \"pmids\": [\"23671284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether endogenous TBC1D10B levels limit secretion not shown\", \"Selectivity for Rab27B over Rab3D mechanism unexplained\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved how a constitutive Rab35 inhibitor permits Rab35 activation, showing spatial removal of TBC1D10B from newborn vesicles generates a switch-like Rab35-OCRL response.\",\n      \"evidence\": \"Live-cell imaging of CCV dynamics with depletion/overexpression and Rab35/OCRL localization (independent lab)\",\n      \"pmids\": [\"26725203\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger for TBC1D10B disappearance from vesicles not defined\", \"Direct OCRL link is downstream of Rab35, not of TBC1D10B itself\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked TBC1D10B to VEGFR2 signaling and angiogenesis, contrasting its action with paralog TBC1D10A.\",\n      \"evidence\": \"Overexpression in endothelial cells with ERK1/2 and p38 phospho-Western, immunofluorescence, and tube formation assay\",\n      \"pmids\": [\"31527750\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrate identification or GAP-dead control\", \"Effect shown only by overexpression, not loss-of-function\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a role at epithelial apical surfaces, showing TBC1D10B supports microvillar density redundantly with EPI64A and localizes via its RabGAP-spanning domain without binding EBP50.\",\n      \"evidence\": \"CRISPR/Cas9 single and double knockouts, immunofluorescence, and domain mapping in Jeg-3 cells\",\n      \"pmids\": [\"34757852\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal Rab substrate (Rab8/Rab35) at microvilli inferred, not directly demonstrated\", \"Functional consequence of microvillar loss not assessed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified a regulatory mechanism in which BAG3-HSP70 sequesters TBC1D10B to sustain RAB35 activity and ESCRT-dependent tau clearance, connecting its GAP regulation to neurodegeneration.\",\n      \"evidence\": \"Mass spectrometry, reciprocal Co-IP, GTP-RAB35 assays, live imaging, P301S mouse and human AD brain immunohistochemistry\",\n      \"pmids\": [\"35000752\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether reduced BAG3-TBC1D10B colocalization is cause or consequence of AD pathology unresolved\", \"Stoichiometry of the BAG3-HSP70-TBC1D10B complex not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed TBC1D10B downstream of Rit1 in macrophage phagocytosis, showing GTP-Rit1-driven dissociation from phagocytic cups regulates FcγR-mediated phagosome formation in both GAP-dependent and -independent ways.\",\n      \"evidence\": \"Live-cell imaging, Rit1 and TBC1D10B knockouts, GTP/GDP-locked Rit1 mutants in RAW264 macrophages\",\n      \"pmids\": [\"39084876\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GAP-independent mechanism of phagocytosis regulation undefined\", \"Direct Rit1-TBC1D10B physical interaction not biochemically established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified Rab22A as a probable physiological substrate by showing TBC1D10B is required for tubular endosome formation through Rab22A inactivation.\",\n      \"evidence\": \"TBC/Rab-GAP siRNA screen, overexpression, GTP-Rab22A pulldown, and tubular endosome imaging in HeLa cells\",\n      \"pmids\": [\"40241313\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why both knockdown and overexpression reduce tubular endosomes mechanistically unclear\", \"Relationship to Rab35-based functions not integrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TBC1D10B's broad in vitro substrate range is restricted to specific Rabs in specific compartments, and what governs its recruitment versus removal, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of substrate selectivity\", \"Determinants of compartment-specific recruitment unknown\", \"Integration of secretory, endosomal, phagocytic, and neurodegenerative roles into one regulatory logic missing\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 3, 8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 3, 8]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"BAG3-HSP70-TBC1D10B complex\"],\n    \"partners\": [\"ARF6\", \"RAB35\", \"RAB27B\", \"RAB22A\", \"BAG3\", \"HSP70\", \"RIT1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}