{"gene":"BTBD9","run_date":"2026-06-09T22:02:45","timeline":{"discoveries":[{"year":2020,"finding":"BTBD9 functions as a substrate-recognition adaptor of the CRL3 (Cul3-ROC1) E3 ubiquitin ligase complex, directly binding TNFAIP1 and promoting its polyubiquitination and proteasomal degradation; loss of BTBD9 stabilizes TNFAIP1 and enhances lung cancer cell migration.","method":"Label-free quantitative proteomics, co-immunoprecipitation, ubiquitination assays, BTBD9 knockdown with TNFAIP1 deletion rescue in lung cancer cells","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, in-cell ubiquitination assay, and genetic rescue (TNFAIP1 deletion abrogating migration phenotype), all in one study with multiple orthogonal methods","pmids":["32327643"],"is_preprint":false},{"year":2022,"finding":"BTBD9 acts as an adaptor of the CUL3-RING E3 ubiquitin ligase complex to ubiquitylate substrates; proteomic and ubiquitinome analyses identified IMPDH2 as a novel substrate targeted for degradation by BTBD9-mediated ubiquitination.","method":"Stable BTBD9-overexpressing SH-SY5Y cell line, quantitative proteomics combined with ubiquitinome (diGly remnant) profiling","journal":"ACS omega","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — two orthogonal mass-spec approaches (proteome + ubiquitinome) in a single lab; no in vitro reconstitution or mutagenesis to confirm direct ubiquitination","pmids":["35449961"],"is_preprint":false},{"year":2025,"finding":"CUL3-BTBD9 E3 ubiquitin ligase binds and ubiquitylates CAV1 (caveolin-1), the central component of caveolae, and this activity is required for insulin-dependent AKT kinase activation in myoblasts and for myogenesis in vitro.","method":"In vitro ubiquitylation assay, co-immunoprecipitation of CUL3-BTBD9 with CAV1, CUL3/BTBD9 loss-of-function with AKT signaling readout, myogenesis differentiation assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro ubiquitylation assay plus Co-IP plus functional rescue; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.05.15.654347"],"is_preprint":true},{"year":2012,"finding":"Btbd9 knockout mice display enhanced hippocampal long-term potentiation (LTP) at CA3-CA1 synapses and impaired pre-synaptic activity, with elevated dynamin 1 (an endocytosis enzyme) protein levels, indicating BTBD9 regulates synaptic plasticity.","method":"Electrophysiological recordings (LTP, input-output, PPF) in hippocampal slices; western blot for dynamin 1 in Btbd9 gene-trap mutant mice; fear-memory behavioral assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO model with electrophysiological and biochemical readouts; single lab, no in vitro reconstitution","pmids":["22536397"],"is_preprint":false},{"year":2020,"finding":"Loss of hpo-9 (BTBD9 C. elegans homolog) increases DOP-3 (D2-like dopamine receptor) expression; correspondingly, Btbd9 knockout mice show increased D2R mRNA in striatum but decreased striatal D2R protein and increased Dynamin I, while the peripheral D1R pathway is potentiated, placing BTBD9 in the dopaminergic signaling pathway.","method":"Reporter assays in C. elegans for dop-1 and dop-3; genetic interaction analysis with dopamine receptor mutants; qRT-PCR and western blot for D2R and Dynamin I in Btbd9 KO mouse striatum; electrophysiology in dopamine neurons","journal":"Brain structure & function","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (genetic epistasis in worm, western blot, electrophysiology in mouse) across two model organisms; single lab","pmids":["32468214"],"is_preprint":false},{"year":2019,"finding":"Loss of BTBD9 specifically in the cerebral cortex (cortex-specific cKO) is sufficient to produce rest-phase motor restlessness, decreased thermal sensation, reduced S1HL/M1 cortical thickness, and enhanced short-term plasticity at corticostriatal terminals onto D1 medium spiny neurons, establishing a cell-autonomous role of BTBD9 in cortical function and the corticostriatal pathway.","method":"Cortex-specific conditional Btbd9 knockout mice; in vivo manganese-enhanced MRI; cortical morphometry; ex vivo electrophysiological recordings at corticostriatal synapses; behavioral assays","journal":"Experimental neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific KO with multiple orthogonal readouts (MRI, morphometry, electrophysiology, behavior); single lab","pmids":["31715135"],"is_preprint":false},{"year":2022,"finding":"In Btbd9 knockout mice, cerebellar Purkinje cells are hyperactive due to increased BK (large-conductance Ca2+-activated K+) channel currents and elevated BK protein levels, alongside decreased SK (small-conductance Ca2+-activated K+) channel currents; PC-specific Btbd9 KO recapitulates motor coordination deficits, demonstrating a cell-autonomous role. BTBD9 protein associates with SYNGAP1, and SYNGAP1 levels are decreased in knockout cerebellum.","method":"Dissociated and brain-slice patch-clamp recordings; TEA/BK/SK channel pharmacology; western blot for BK and SYNGAP1 protein; PC-specific conditional Btbd9 KO behavioral assays","journal":"Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology with pharmacological dissection plus protein quantification plus cell-type-specific KO; single lab, multiple orthogonal methods","pmids":["36244636"],"is_preprint":false},{"year":2022,"finding":"In C. elegans, BTBD9 homolog hpo-9 protects against Mn-induced oxidative stress and dopaminergic neurotoxicity via regulation of the insulin/IGF signaling pathway: hpo-9 overexpression upregulates FOXO and decreases AKT (protein kinase B) levels, and the protection is abolished by FOXO mutation.","method":"C. elegans hpo-9 knockout and overexpression; FOXO loss-of-function epistasis; oxidative stress and mitochondrial function assays; dopamine level measurement; dopaminergic morphology imaging; AKT/FOXO western blot","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (FOXO mutant rescue experiment) plus biochemical readouts; single lab, multiple orthogonal methods in C. elegans model","pmids":["35134179"],"is_preprint":false},{"year":2020,"finding":"MEIS1 and BTBD9 do not regulate each other: Meis1 protein level is unaffected by Btbd9 deficiency, and Btbd9 transcription is unaffected by Meis1 haploinsufficiency in mice. However, in C. elegans, hyperactive egg-laying caused by hpo-9 (BTBD9 homolog) loss is counteracted by knockdown of the MEIS1 homolog, suggesting functional interaction without mutual transcriptional regulation.","method":"Western blot for Meis1 protein in Btbd9 KO mice; RT-PCR for Btbd9 mRNA in Meis1 haploinsufficient mice; C. elegans RNAi knockdown epistasis; double KO mouse behavioral assays","journal":"Experimental results","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic epistasis in worm plus protein/mRNA measurements in mouse; single lab; negative molecular finding (no mutual regulation) is well-supported but functional interaction is from a single C. elegans epistasis experiment","pmids":["34268480"],"is_preprint":false}],"current_model":"BTBD9 functions as a substrate-recognition adaptor of the CUL3-RING E3 ubiquitin ligase complex, targeting substrates including TNFAIP1 (suppressing cancer cell migration), IMPDH2 (linked to sleep regulation), and CAV1 (enabling insulin-AKT signaling during myogenesis) for ubiquitination and degradation; in neurons, BTBD9 regulates synaptic plasticity (hippocampal LTP, Purkinje cell excitability via BK/SK channels, corticostriatal transmission), dopaminergic signaling (D1R/D2R pathway balance), and protection against Mn-induced neurotoxicity through the insulin/IGF-FOXO pathway, with loss of BTBD9 in mice producing restless-legs-syndrome-like motor restlessness, cortical thinning, and enhanced fear memory."},"narrative":{"mechanistic_narrative":"BTBD9 is a substrate-recognition adaptor of the CUL3-RING (CRL3) E3 ubiquitin ligase complex that selects specific proteins for polyubiquitination and proteasomal degradation, linking it to processes ranging from cancer cell migration to insulin signaling and neuronal excitability [PMID:32327643, PMID:bio_10.1101_2025.05.15.654347]. As a CRL3 adaptor it directly binds and ubiquitylates TNFAIP1, and loss of BTBD9 stabilizes TNFAIP1 and enhances lung cancer cell migration [PMID:32327643]; proteomic and ubiquitinome profiling identified IMPDH2 as an additional degradation substrate [PMID:35449961], and CUL3-BTBD9 ubiquitylates CAV1 in a step required for insulin-dependent AKT activation and myogenesis [PMID:bio_10.1101_2025.05.15.654347]. In the nervous system, BTBD9 shapes synaptic plasticity and excitability: knockout mice show enhanced hippocampal CA3-CA1 LTP with impaired presynaptic function and elevated dynamin 1 [PMID:22536397], cerebellar Purkinje cell hyperactivity through increased BK and decreased SK channel currents together with reduced SYNGAP1 [PMID:36244636], and altered corticostriatal transmission, where cortex-specific deletion alone produces rest-phase motor restlessness and cortical thinning [PMID:31715135]. BTBD9 also regulates dopaminergic signaling, balancing the D1R and D2R pathways [PMID:32468214], and its C. elegans homolog hpo-9 protects dopaminergic neurons from manganese-induced oxidative stress by acting through the insulin/IGF-FOXO axis [PMID:35134179].","teleology":[{"year":2012,"claim":"Established the first mechanistic role for BTBD9 in the nervous system by asking whether it influences synaptic function, showing it constrains hippocampal plasticity and presynaptic machinery.","evidence":"Electrophysiology (LTP, PPF) and dynamin 1 western blot in Btbd9 gene-trap mutant mice with fear-memory behavior","pmids":["22536397"],"confidence":"Medium","gaps":["Molecular link between BTBD9 and dynamin 1 regulation not defined","No biochemical mechanism connecting BTBD9 loss to LTP enhancement"]},{"year":2019,"claim":"Tested whether BTBD9 function in a single brain region drives RLS-like phenotypes, demonstrating a cell-autonomous cortical role in motor behavior and corticostriatal transmission.","evidence":"Cortex-specific conditional Btbd9 KO with MRI, morphometry, corticostriatal electrophysiology, and behavior","pmids":["31715135"],"confidence":"Medium","gaps":["Molecular substrates underlying cortical thinning unknown","Mechanism linking cortical BTBD9 loss to motor restlessness not resolved"]},{"year":2020,"claim":"Identified the founding biochemical activity of BTBD9 as a CRL3 substrate-recognition adaptor, answering what BTBD9 does at the molecular level by linking it to targeted ubiquitination and a cancer migration phenotype.","evidence":"Quantitative proteomics, reciprocal Co-IP, in-cell ubiquitination assay, and TNFAIP1 deletion rescue in lung cancer cells","pmids":["32327643"],"confidence":"High","gaps":["Degron/binding determinants on TNFAIP1 not mapped","Relationship between ubiquitin ligase activity and neuronal phenotypes not established"]},{"year":2020,"claim":"Addressed how BTBD9 connects to dopamine signaling, placing it upstream of dopamine receptor expression and pathway balance across worm and mouse.","evidence":"C. elegans dopamine receptor reporter and epistasis assays plus qRT-PCR, western blot, and electrophysiology in Btbd9 KO mouse striatum","pmids":["32468214"],"confidence":"Medium","gaps":["Direct ubiquitination targets in the dopaminergic pathway not identified","Mechanism of opposing D2R mRNA increase and protein decrease unexplained"]},{"year":2022,"claim":"Expanded the BTBD9 substrate repertoire by asking what else CRL3-BTBD9 degrades, identifying IMPDH2 through unbiased ubiquitinome profiling.","evidence":"Quantitative proteomics plus diGly ubiquitinome profiling in BTBD9-overexpressing SH-SY5Y cells","pmids":["35449961"],"confidence":"Medium","gaps":["No in vitro reconstitution or mutagenesis confirming direct ubiquitination","Physiological consequence of IMPDH2 degradation not tested"]},{"year":2022,"claim":"Resolved a cell-autonomous mechanism for cerebellar dysfunction by linking BTBD9 loss to altered Ca2+-activated K+ channel currents and a candidate protein partner.","evidence":"Patch-clamp with BK/SK pharmacology, BK and SYNGAP1 western blot, and Purkinje-cell-specific conditional Btbd9 KO behavior","pmids":["36244636"],"confidence":"Medium","gaps":["Whether BK/SYNGAP1 changes reflect direct ubiquitination is untested","BTBD9-SYNGAP1 association from single-lab Co-IP without reciprocal validation"]},{"year":2022,"claim":"Defined a neuroprotective pathway for BTBD9 by asking how its homolog buffers metal-induced toxicity, showing it acts through the insulin/IGF-FOXO axis.","evidence":"C. elegans hpo-9 KO/overexpression, FOXO epistasis, oxidative stress and dopamine assays, AKT/FOXO western blot","pmids":["35134179"],"confidence":"Medium","gaps":["Direct molecular substrate connecting BTBD9 to FOXO/AKT not identified","Conservation of this pathway in mammalian neurons not demonstrated"]},{"year":2025,"claim":"Connected BTBD9 ubiquitin ligase activity to insulin signaling and muscle differentiation by identifying CAV1 as a substrate required for AKT activation.","evidence":"In vitro ubiquitylation assay, CUL3-BTBD9/CAV1 Co-IP, loss-of-function AKT readout, and myogenesis assay (preprint)","pmids":["bio_10.1101_2025.05.15.654347"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","In vivo relevance to muscle physiology not established"]},{"year":null,"claim":"It remains unknown which ubiquitination substrates underlie BTBD9's neuronal and behavioral phenotypes, linking its defined CRL3 enzymatic activity to its roles in synaptic plasticity and motor control.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No substrate has been shown to mediate the LTP, Purkinje, or corticostriatal phenotypes","Structural basis of substrate selection not determined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[3,5,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,4,7]}],"complexes":["CUL3-RING (CRL3) E3 ubiquitin ligase"],"partners":["TNFAIP1","IMPDH2","CAV1","CUL3","SYNGAP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96Q07","full_name":"BTB/POZ domain-containing protein 9","aliases":[],"length_aa":612,"mass_kda":69.2,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q96Q07/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/BTBD9","classification":"Not Classified","n_dependent_lines":46,"n_total_lines":1208,"dependency_fraction":0.0380794701986755},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/BTBD9","total_profiled":1310},"omim":[{"mim_id":"612853","title":"RESTLESS LEGS SYNDROME, SUSCEPTIBILITY TO, 7; RLS7","url":"https://www.omim.org/entry/612853"},{"mim_id":"611237","title":"BTB/POZ DOMAIN-CONTAINING PROTEIN 9; BTBD9","url":"https://www.omim.org/entry/611237"},{"mim_id":"611185","title":"RESTLESS LEGS SYNDROME, SUSCEPTIBILITY TO, 6; RLS6","url":"https://www.omim.org/entry/611185"},{"mim_id":"137580","title":"GILLES DE LA TOURETTE SYNDROME; GTS","url":"https://www.omim.org/entry/137580"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/BTBD9"},"hgnc":{"alias_symbol":["KIAA1880","dJ322I12.1"],"prev_symbol":[]},"alphafold":{"accession":"Q96Q07","domains":[{"cath_id":"3.30.710.10","chopping":"16-136","consensus_level":"medium","plddt":93.016,"start":16,"end":136},{"cath_id":"2.60.120.260","chopping":"279-412","consensus_level":"medium","plddt":95.0216,"start":279,"end":412},{"cath_id":"2.60.120.260","chopping":"429-554","consensus_level":"high","plddt":95.3814,"start":429,"end":554}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96Q07","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96Q07-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96Q07-F1-predicted_aligned_error_v6.png","plddt_mean":88.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=BTBD9","jax_strain_url":"https://www.jax.org/strain/search?query=BTBD9"},"sequence":{"accession":"Q96Q07","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96Q07.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96Q07/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96Q07"}},"corpus_meta":[{"pmid":"19822783","id":"PMC_19822783","title":"Association of intronic variants of the BTBD9 gene with Tourette syndrome.","date":"2009","source":"Archives of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/19822783","citation_count":35,"is_preprint":false},{"pmid":"22536397","id":"PMC_22536397","title":"Enhanced hippocampal long-term potentiation and fear memory in Btbd9 mutant mice.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22536397","citation_count":27,"is_preprint":false},{"pmid":"32468214","id":"PMC_32468214","title":"BTBD9 and dopaminergic dysfunction in the pathogenesis of restless legs syndrome.","date":"2020","source":"Brain structure & function","url":"https://pubmed.ncbi.nlm.nih.gov/32468214","citation_count":20,"is_preprint":false},{"pmid":"32327643","id":"PMC_32327643","title":"The CRL3BTBD9 E3 ubiquitin ligase complex targets TNFAIP1 for degradation to suppress cancer cell migration.","date":"2020","source":"Signal transduction and targeted therapy","url":"https://pubmed.ncbi.nlm.nih.gov/32327643","citation_count":19,"is_preprint":false},{"pmid":"22914617","id":"PMC_22914617","title":"Analysis of the BTBD9 and HTR2C variants in Chinese Han patients with Tourette syndrome.","date":"2012","source":"Psychiatric genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22914617","citation_count":15,"is_preprint":false},{"pmid":"31715135","id":"PMC_31715135","title":"The role of BTBD9 in the cerebral cortex and the pathogenesis of restless legs syndrome.","date":"2019","source":"Experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/31715135","citation_count":14,"is_preprint":false},{"pmid":"35134179","id":"PMC_35134179","title":"BTBD9 attenuates manganese-induced oxidative stress and neurotoxicity by regulating insulin growth factor signaling pathway.","date":"2022","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35134179","citation_count":11,"is_preprint":false},{"pmid":"23361623","id":"PMC_23361623","title":"The BTBD9 gene may be associated with antipsychotic-induced restless legs syndrome in schizophrenia.","date":"2013","source":"Human psychopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/23361623","citation_count":10,"is_preprint":false},{"pmid":"24993631","id":"PMC_24993631","title":"The BTBD9 gene polymorphisms in Polish patients with Gilles de la Tourette syndrome.","date":"2014","source":"Acta neurobiologiae experimentalis","url":"https://pubmed.ncbi.nlm.nih.gov/24993631","citation_count":8,"is_preprint":false},{"pmid":"36244636","id":"PMC_36244636","title":"Further Studies on the Role of BTBD9 in the Cerebellum, Sleep-like Behaviors and the Restless Legs Syndrome.","date":"2022","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/36244636","citation_count":6,"is_preprint":false},{"pmid":"35449961","id":"PMC_35449961","title":"Integrative Proteome and Ubiquitinome Analyses Reveal the Substrates of BTBD9 and Its Underlying Mechanism in Sleep Regulation.","date":"2022","source":"ACS omega","url":"https://pubmed.ncbi.nlm.nih.gov/35449961","citation_count":5,"is_preprint":false},{"pmid":"34268480","id":"PMC_34268480","title":"Probe the relationship between BTBD9 and MEIS1 in C. elegans and mouse.","date":"2020","source":"Experimental results","url":"https://pubmed.ncbi.nlm.nih.gov/34268480","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.15.654347","title":"Control of myogenesis by the E3 ubiquitin ligase 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co-immunoprecipitation, ubiquitination assays, BTBD9 knockdown with TNFAIP1 deletion rescue in lung cancer cells\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, in-cell ubiquitination assay, and genetic rescue (TNFAIP1 deletion abrogating migration phenotype), all in one study with multiple orthogonal methods\",\n      \"pmids\": [\"32327643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"BTBD9 acts as an adaptor of the CUL3-RING E3 ubiquitin ligase complex to ubiquitylate substrates; proteomic and ubiquitinome analyses identified IMPDH2 as a novel substrate targeted for degradation by BTBD9-mediated ubiquitination.\",\n      \"method\": \"Stable BTBD9-overexpressing SH-SY5Y cell line, quantitative proteomics combined with ubiquitinome (diGly remnant) profiling\",\n      \"journal\": \"ACS omega\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — two orthogonal mass-spec approaches (proteome + ubiquitinome) in a single lab; no in vitro reconstitution or mutagenesis to confirm direct ubiquitination\",\n      \"pmids\": [\"35449961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CUL3-BTBD9 E3 ubiquitin ligase binds and ubiquitylates CAV1 (caveolin-1), the central component of caveolae, and this activity is required for insulin-dependent AKT kinase activation in myoblasts and for myogenesis in vitro.\",\n      \"method\": \"In vitro ubiquitylation assay, co-immunoprecipitation of CUL3-BTBD9 with CAV1, CUL3/BTBD9 loss-of-function with AKT signaling readout, myogenesis differentiation assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro ubiquitylation assay plus Co-IP plus functional rescue; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.05.15.654347\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Btbd9 knockout mice display enhanced hippocampal long-term potentiation (LTP) at CA3-CA1 synapses and impaired pre-synaptic activity, with elevated dynamin 1 (an endocytosis enzyme) protein levels, indicating BTBD9 regulates synaptic plasticity.\",\n      \"method\": \"Electrophysiological recordings (LTP, input-output, PPF) in hippocampal slices; western blot for dynamin 1 in Btbd9 gene-trap mutant mice; fear-memory behavioral assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO model with electrophysiological and biochemical readouts; single lab, no in vitro reconstitution\",\n      \"pmids\": [\"22536397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss of hpo-9 (BTBD9 C. elegans homolog) increases DOP-3 (D2-like dopamine receptor) expression; correspondingly, Btbd9 knockout mice show increased D2R mRNA in striatum but decreased striatal D2R protein and increased Dynamin I, while the peripheral D1R pathway is potentiated, placing BTBD9 in the dopaminergic signaling pathway.\",\n      \"method\": \"Reporter assays in C. elegans for dop-1 and dop-3; genetic interaction analysis with dopamine receptor mutants; qRT-PCR and western blot for D2R and Dynamin I in Btbd9 KO mouse striatum; electrophysiology in dopamine neurons\",\n      \"journal\": \"Brain structure & function\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (genetic epistasis in worm, western blot, electrophysiology in mouse) across two model organisms; single lab\",\n      \"pmids\": [\"32468214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Loss of BTBD9 specifically in the cerebral cortex (cortex-specific cKO) is sufficient to produce rest-phase motor restlessness, decreased thermal sensation, reduced S1HL/M1 cortical thickness, and enhanced short-term plasticity at corticostriatal terminals onto D1 medium spiny neurons, establishing a cell-autonomous role of BTBD9 in cortical function and the corticostriatal pathway.\",\n      \"method\": \"Cortex-specific conditional Btbd9 knockout mice; in vivo manganese-enhanced MRI; cortical morphometry; ex vivo electrophysiological recordings at corticostriatal synapses; behavioral assays\",\n      \"journal\": \"Experimental neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific KO with multiple orthogonal readouts (MRI, morphometry, electrophysiology, behavior); single lab\",\n      \"pmids\": [\"31715135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Btbd9 knockout mice, cerebellar Purkinje cells are hyperactive due to increased BK (large-conductance Ca2+-activated K+) channel currents and elevated BK protein levels, alongside decreased SK (small-conductance Ca2+-activated K+) channel currents; PC-specific Btbd9 KO recapitulates motor coordination deficits, demonstrating a cell-autonomous role. BTBD9 protein associates with SYNGAP1, and SYNGAP1 levels are decreased in knockout cerebellum.\",\n      \"method\": \"Dissociated and brain-slice patch-clamp recordings; TEA/BK/SK channel pharmacology; western blot for BK and SYNGAP1 protein; PC-specific conditional Btbd9 KO behavioral assays\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology with pharmacological dissection plus protein quantification plus cell-type-specific KO; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36244636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In C. elegans, BTBD9 homolog hpo-9 protects against Mn-induced oxidative stress and dopaminergic neurotoxicity via regulation of the insulin/IGF signaling pathway: hpo-9 overexpression upregulates FOXO and decreases AKT (protein kinase B) levels, and the protection is abolished by FOXO mutation.\",\n      \"method\": \"C. elegans hpo-9 knockout and overexpression; FOXO loss-of-function epistasis; oxidative stress and mitochondrial function assays; dopamine level measurement; dopaminergic morphology imaging; AKT/FOXO western blot\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (FOXO mutant rescue experiment) plus biochemical readouts; single lab, multiple orthogonal methods in C. elegans model\",\n      \"pmids\": [\"35134179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MEIS1 and BTBD9 do not regulate each other: Meis1 protein level is unaffected by Btbd9 deficiency, and Btbd9 transcription is unaffected by Meis1 haploinsufficiency in mice. However, in C. elegans, hyperactive egg-laying caused by hpo-9 (BTBD9 homolog) loss is counteracted by knockdown of the MEIS1 homolog, suggesting functional interaction without mutual transcriptional regulation.\",\n      \"method\": \"Western blot for Meis1 protein in Btbd9 KO mice; RT-PCR for Btbd9 mRNA in Meis1 haploinsufficient mice; C. elegans RNAi knockdown epistasis; double KO mouse behavioral assays\",\n      \"journal\": \"Experimental results\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic epistasis in worm plus protein/mRNA measurements in mouse; single lab; negative molecular finding (no mutual regulation) is well-supported but functional interaction is from a single C. elegans epistasis experiment\",\n      \"pmids\": [\"34268480\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"BTBD9 functions as a substrate-recognition adaptor of the CUL3-RING E3 ubiquitin ligase complex, targeting substrates including TNFAIP1 (suppressing cancer cell migration), IMPDH2 (linked to sleep regulation), and CAV1 (enabling insulin-AKT signaling during myogenesis) for ubiquitination and degradation; in neurons, BTBD9 regulates synaptic plasticity (hippocampal LTP, Purkinje cell excitability via BK/SK channels, corticostriatal transmission), dopaminergic signaling (D1R/D2R pathway balance), and protection against Mn-induced neurotoxicity through the insulin/IGF-FOXO pathway, with loss of BTBD9 in mice producing restless-legs-syndrome-like motor restlessness, cortical thinning, and enhanced fear memory.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"BTBD9 is a substrate-recognition adaptor of the CUL3-RING (CRL3) E3 ubiquitin ligase complex that selects specific proteins for polyubiquitination and proteasomal degradation, linking it to processes ranging from cancer cell migration to insulin signaling and neuronal excitability [#0, #2]. As a CRL3 adaptor it directly binds and ubiquitylates TNFAIP1, and loss of BTBD9 stabilizes TNFAIP1 and enhances lung cancer cell migration [#0]; proteomic and ubiquitinome profiling identified IMPDH2 as an additional degradation substrate [#1], and CUL3-BTBD9 ubiquitylates CAV1 in a step required for insulin-dependent AKT activation and myogenesis [#2]. In the nervous system, BTBD9 shapes synaptic plasticity and excitability: knockout mice show enhanced hippocampal CA3-CA1 LTP with impaired presynaptic function and elevated dynamin 1 [#3], cerebellar Purkinje cell hyperactivity through increased BK and decreased SK channel currents together with reduced SYNGAP1 [#6], and altered corticostriatal transmission, where cortex-specific deletion alone produces rest-phase motor restlessness and cortical thinning [#5]. BTBD9 also regulates dopaminergic signaling, balancing the D1R and D2R pathways [#4], and its C. elegans homolog hpo-9 protects dopaminergic neurons from manganese-induced oxidative stress by acting through the insulin/IGF-FOXO axis [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established the first mechanistic role for BTBD9 in the nervous system by asking whether it influences synaptic function, showing it constrains hippocampal plasticity and presynaptic machinery.\",\n      \"evidence\": \"Electrophysiology (LTP, PPF) and dynamin 1 western blot in Btbd9 gene-trap mutant mice with fear-memory behavior\",\n      \"pmids\": [\"22536397\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between BTBD9 and dynamin 1 regulation not defined\", \"No biochemical mechanism connecting BTBD9 loss to LTP enhancement\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Tested whether BTBD9 function in a single brain region drives RLS-like phenotypes, demonstrating a cell-autonomous cortical role in motor behavior and corticostriatal transmission.\",\n      \"evidence\": \"Cortex-specific conditional Btbd9 KO with MRI, morphometry, corticostriatal electrophysiology, and behavior\",\n      \"pmids\": [\"31715135\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrates underlying cortical thinning unknown\", \"Mechanism linking cortical BTBD9 loss to motor restlessness not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified the founding biochemical activity of BTBD9 as a CRL3 substrate-recognition adaptor, answering what BTBD9 does at the molecular level by linking it to targeted ubiquitination and a cancer migration phenotype.\",\n      \"evidence\": \"Quantitative proteomics, reciprocal Co-IP, in-cell ubiquitination assay, and TNFAIP1 deletion rescue in lung cancer cells\",\n      \"pmids\": [\"32327643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degron/binding determinants on TNFAIP1 not mapped\", \"Relationship between ubiquitin ligase activity and neuronal phenotypes not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Addressed how BTBD9 connects to dopamine signaling, placing it upstream of dopamine receptor expression and pathway balance across worm and mouse.\",\n      \"evidence\": \"C. elegans dopamine receptor reporter and epistasis assays plus qRT-PCR, western blot, and electrophysiology in Btbd9 KO mouse striatum\",\n      \"pmids\": [\"32468214\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination targets in the dopaminergic pathway not identified\", \"Mechanism of opposing D2R mRNA increase and protein decrease unexplained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded the BTBD9 substrate repertoire by asking what else CRL3-BTBD9 degrades, identifying IMPDH2 through unbiased ubiquitinome profiling.\",\n      \"evidence\": \"Quantitative proteomics plus diGly ubiquitinome profiling in BTBD9-overexpressing SH-SY5Y cells\",\n      \"pmids\": [\"35449961\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution or mutagenesis confirming direct ubiquitination\", \"Physiological consequence of IMPDH2 degradation not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved a cell-autonomous mechanism for cerebellar dysfunction by linking BTBD9 loss to altered Ca2+-activated K+ channel currents and a candidate protein partner.\",\n      \"evidence\": \"Patch-clamp with BK/SK pharmacology, BK and SYNGAP1 western blot, and Purkinje-cell-specific conditional Btbd9 KO behavior\",\n      \"pmids\": [\"36244636\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether BK/SYNGAP1 changes reflect direct ubiquitination is untested\", \"BTBD9-SYNGAP1 association from single-lab Co-IP without reciprocal validation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined a neuroprotective pathway for BTBD9 by asking how its homolog buffers metal-induced toxicity, showing it acts through the insulin/IGF-FOXO axis.\",\n      \"evidence\": \"C. elegans hpo-9 KO/overexpression, FOXO epistasis, oxidative stress and dopamine assays, AKT/FOXO western blot\",\n      \"pmids\": [\"35134179\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular substrate connecting BTBD9 to FOXO/AKT not identified\", \"Conservation of this pathway in mammalian neurons not demonstrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected BTBD9 ubiquitin ligase activity to insulin signaling and muscle differentiation by identifying CAV1 as a substrate required for AKT activation.\",\n      \"evidence\": \"In vitro ubiquitylation assay, CUL3-BTBD9/CAV1 Co-IP, loss-of-function AKT readout, and myogenesis assay (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.05.15.654347\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"In vivo relevance to muscle physiology not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown which ubiquitination substrates underlie BTBD9's neuronal and behavioral phenotypes, linking its defined CRL3 enzymatic activity to its roles in synaptic plasticity and motor control.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrate has been shown to mediate the LTP, Purkinje, or corticostriatal phenotypes\", \"Structural basis of substrate selection not determined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [3, 5, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 4, 7]}\n    ],\n    \"complexes\": [\"CUL3-RING (CRL3) E3 ubiquitin ligase\"],\n    \"partners\": [\"TNFAIP1\", \"IMPDH2\", \"CAV1\", \"CUL3\", \"SYNGAP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":4,"faith_total":4,"faith_pct":100.0}}