{"gene":"RHOBTB1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2019,"finding":"RhoBTB1 acts as a substrate adaptor delivering phosphodiesterase 5 (PDE5) to the Cullin-3 (CUL3) E3 RING ubiquitin ligase complex, leading to PDE5 ubiquitination and inhibition, thereby augmenting cGMP responses to nitric oxide in vascular smooth muscle cells.","method":"Genetic complementation (inducible smooth muscle-specific RhoBTB1 transgene), Co-immunoprecipitation, functional vasodilation assays in vivo","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, genetic complementation in two hypertension models, functional readout (cGMP, vasodilation), replicated across models","pmids":["30896450"],"is_preprint":false},{"year":2023,"finding":"The C-terminal half of RhoBTB1 (B1B2C region, comprising both BTB domains and C-terminal domain) is the minimal region required for PDE5 recruitment and subsequent proteasomal degradation via CUL3; the C-terminal domain is essential for PDE5 binding, and Pro353 and Ser363 are key residues required for CUL3 binding (mutation impairs CUL3 binding and PDE5 degradation without disrupting PDE5 binding).","method":"Domain truncation, Co-immunoprecipitation, site-directed mutagenesis, APEX2 proximity labeling/mass spectrometry","journal":"Function (Oxford, England)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis, domain truncation, proximity labeling with multiple orthogonal methods in single study","pmids":["37575477"],"is_preprint":false},{"year":2023,"finding":"SETD2 is a binding partner of RhoBTB1 (validated by Co-IP), and SETD2 levels increase upon proteasome inhibition, Cullin complex inhibition, CUL3 deletion, or RhoBTB1 siRNA knockdown, suggesting SETD2 is a substrate regulated by the RhoBTB1-CUL3 ubiquitin-proteasome axis.","method":"APEX2 proximity labeling/mass spectrometry, Co-immunoprecipitation, siRNA knockdown, pharmacological proteasome/Cullin inhibition","journal":"Function (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and proximity labeling with functional validation (proteasome inhibition), single lab, single study","pmids":["37575477"],"is_preprint":false},{"year":2022,"finding":"RhoBTB1 reverses established arterial stiffness by promoting actin depolymerization in vascular smooth muscle; angiotensin II-induced actin polymerization in the aorta is reversed by RhoBTB1 restoration, with consistent changes in levels of cofilin and vasodilator-stimulated phosphoprotein (VASP), two regulators of actin polymerization.","method":"Inducible smooth muscle-specific RhoBTB1 transgene, measurement of actin polymerization, immunoblotting for cofilin and VASP, arterial stiffness measurements in vivo","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic complementation with multiple mechanistic readouts (actin polymerization, cofilin, VASP) in a defined in vivo model, single lab","pmids":["35358093"],"is_preprint":false},{"year":2026,"finding":"RhoBTB1 interacts with and promotes CUL3-dependent ubiquitination and proteasomal degradation of RbFox2 in smooth muscle cells; loss of RhoBTB1 (by siRNA or angiotensin II) elevates RbFox2, and RbFox2 regulates actin cytoskeletal integrity and arterial stiffness.","method":"Co-immunoprecipitation, proximity labeling/mass spectrometry, siRNA knockdown, CUL3 deletion, RbFox2 floxed mouse model, ubiquitination assay","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ubiquitination assay, genetic knockout model with arterial stiffness readout, multiple orthogonal methods in single study","pmids":["41926228"],"is_preprint":false},{"year":2019,"finding":"RhoBTB1 associates with ROCK1 and ROCK2; the interaction with ROCK1 is mediated by the Rho domain of RhoBTB1 binding to the coiled-coil region of ROCK1 near its kinase domain. Two amino acids in the Rho domain alter RhoBTB1-ROCK1 association. RhoBTB1 is a substrate for ROCK1, and mutation of putative ROCK1 phosphorylation sites on RhoBTB1 reduces its association with Cullin3.","method":"Co-immunoprecipitation, pulldown, site-directed mutagenesis, in vitro kinase assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — Co-IP, mutagenesis of interaction interface, and in vitro kinase assay, multiple orthogonal methods in single study","pmids":["31431478"],"is_preprint":false},{"year":2019,"finding":"RhoBTB1 depletion increases prostate cancer cell invasion and induces elongation in Matrigel, a phenotype similar to that induced by depletion of ROCK1 and ROCK2, suggesting RhoBTB1 suppresses cancer cell invasion through interaction with ROCKs.","method":"siRNA knockdown, Matrigel invasion assay, cell morphology imaging","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss-of-function with defined invasion phenotype, genetic epistasis-like comparison with ROCK depletion, single lab","pmids":["31431478"],"is_preprint":false},{"year":2017,"finding":"RhoBTB1 regulates the integrity of the Golgi complex through control of METTL7B expression; silencing of either RhoBTB1 or METTL7B leads to Golgi fragmentation, and restoration of RhoBTB1 expression rescues Golgi morphology and inhibits breast cancer cell invasion.","method":"Gene silencing (RNAi), transcriptome analysis, Q-PCR, cell imaging (Golgi morphology), rescue experiments","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — RNAi knockdown plus rescue, imaging phenotype, and transcriptional target validation, single lab","pmids":["28219369"],"is_preprint":false},{"year":2020,"finding":"RhoBTB1 localizes to early endosomal intermediates, and changes in RhoBTB1 levels cause disturbances to Golgi architecture, profound changes to organisation and distribution of endosomes and lysosomes, and defects in delivery of two classes of cargo molecules to downstream compartments, linking RhoBTB1 to endocytosis and retrograde traffic pathways.","method":"RNA interference screen, high-content image-based analysis, fluorescence microscopy, cargo trafficking assays","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — systematic RNAi, localization imaging, multiple trafficking readouts, single lab","pmids":["32354068"],"is_preprint":false},{"year":2017,"finding":"RhoBTB1 is a target gene of miR-31a-5p; miR-31a-5p promotes postnatal cardiomyocyte proliferation by repressing RhoBTB1, and RhoBTB1 knockdown phenocopies miR-31a-5p overexpression in promoting cardiomyocyte proliferation markers.","method":"miRNA arrays, luciferase reporter assay (implied by target validation), immunofluorescence, EdU/Ki-67/PHH3 proliferation assays, antagomir injection in neonatal rats","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — in vivo antagomir model, target knockdown phenocopy, multiple proliferation markers, single lab","pmids":["29053138"],"is_preprint":false},{"year":2024,"finding":"Exosomal miR-31 from cutaneous squamous cell carcinoma (CSCC) cells directly targets the 3'UTR of RhoBTB1 (validated by dual luciferase reporter assay), suppressing RhoBTB1 expression and enhancing CSCC cell proliferation, migration, and invasion.","method":"Dual luciferase reporter assay, immunoblotting, qPCR, MTT assay, Transwell assay, differential ultracentrifugation for exosome isolation","journal":"Archives of dermatological research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — luciferase reporter directly validates 3'UTR targeting, functional assays, single lab","pmids":["39673615"],"is_preprint":false}],"current_model":"RhoBTB1 is an atypical Rho GTPase that functions primarily as a substrate adaptor for the Cullin-3 E3 ubiquitin ligase complex, recruiting substrates including PDE5 (via its C-terminal BTB/C-terminal domain) and RbFox2 for ubiquitination and proteasomal degradation, thereby regulating vascular smooth muscle cGMP signaling, actin cytoskeletal dynamics, arterial stiffness, and vasodilation; it also interacts with ROCK1/2 (via its Rho domain) to suppress cancer cell invasion, regulates Golgi integrity through METTL7B, and participates in endosomal/retrograde trafficking, while being post-translationally regulated by ROCK1-mediated phosphorylation that modulates its Cullin3 association."},"narrative":{"mechanistic_narrative":"RHOBTB1 is an atypical Rho GTPase that functions principally as a substrate adaptor for the Cullin-3 (CUL3) E3 ubiquitin ligase, coupling targeted protein degradation to the control of vascular smooth muscle tone and actin cytoskeletal dynamics [PMID:30896450, PMID:41926228]. It delivers phosphodiesterase 5 (PDE5) to CUL3 for ubiquitination and inhibition, thereby augmenting nitric oxide-driven cGMP signaling and promoting vasodilation [PMID:30896450], and it likewise targets the splicing regulator RbFox2 for CUL3-dependent degradation to maintain actin cytoskeletal integrity and reverse arterial stiffness [PMID:35358093, PMID:41926228]. Substrate recruitment is partitioned across the protein: the C-terminal domain mediates PDE5 binding while Pro353 and Ser363 are required for CUL3 association, and the full C-terminal B1B2C region is the minimal degradation-competent module [PMID:37575477]. Through its N-terminal Rho domain, RHOBTB1 binds the coiled-coil region of ROCK1/ROCK2 and is itself a ROCK1 phosphorylation substrate, with phosphorylation modulating its CUL3 association — linking it to suppression of cancer cell invasion [PMID:31431478]. Beyond degradation, RHOBTB1 maintains Golgi integrity via control of METTL7B expression and localizes to early endosomes where it governs endosomal/lysosomal organization and retrograde cargo trafficking [PMID:28219369, PMID:32354068]. Its expression is repressed by miR-31/miR-31a-5p, linking its loss to cardiomyocyte proliferation and to enhanced tumor cell migration and invasion [PMID:29053138, PMID:39673615].","teleology":[{"year":2017,"claim":"Established the first cellular role for RhoBTB1 by linking it to organelle integrity, addressing whether this atypical GTPase has a defined cellular function.","evidence":"RNAi silencing, transcriptome analysis, and Golgi-morphology rescue experiments in breast cancer cells","pmids":["28219369"],"confidence":"Medium","gaps":["Mechanism by which RhoBTB1 controls METTL7B expression unknown","Does not establish direct molecular partners or enzymatic activity"]},{"year":2017,"claim":"Identified an upstream regulatory mechanism, showing RhoBTB1 is a repressed target of miR-31a-5p that restrains cardiomyocyte proliferation.","evidence":"miRNA arrays, target knockdown phenocopy, and antagomir injection in neonatal rats","pmids":["29053138"],"confidence":"Medium","gaps":["Downstream effectors mediating proliferation control not defined","Direct 3'UTR binding inferred rather than fully dissected here"]},{"year":2019,"claim":"Defined RhoBTB1's core molecular function as a CUL3 substrate adaptor for PDE5, explaining how it augments cGMP signaling and vasodilation in vivo.","evidence":"Smooth muscle-specific inducible transgene, reciprocal Co-IP, and functional vasodilation/cGMP assays in two hypertension models","pmids":["30896450"],"confidence":"High","gaps":["Precise domain requirements for PDE5 and CUL3 binding not yet mapped","Other substrates beyond PDE5 unknown at this stage"]},{"year":2019,"claim":"Connected the Rho domain to ROCK kinases and revealed a feedback loop, showing RhoBTB1 binds ROCK1/2, is phosphorylated by ROCK1, and that this phosphorylation controls its CUL3 association.","evidence":"Co-IP, pulldown, interface mutagenesis, and in vitro kinase assay; Matrigel invasion phenotyping in prostate cancer cells","pmids":["31431478"],"confidence":"High","gaps":["Functional consequence of ROCK binding for ROCK activity not resolved","Invasion suppression role (Medium-confidence) lacks in vivo validation"]},{"year":2020,"claim":"Localized RhoBTB1 to early endosomes and broadened its role to membrane trafficking, showing its dysregulation disrupts endosome/lysosome organization and retrograde cargo delivery.","evidence":"RNAi screen, high-content imaging, and cargo trafficking assays","pmids":["32354068"],"confidence":"Medium","gaps":["Whether trafficking role is CUL3-dependent unknown","Direct trafficking machinery partners not identified"]},{"year":2022,"claim":"Demonstrated RhoBTB1 can reverse established arterial stiffness via actin depolymerization, establishing a therapeutically relevant phenotype downstream of its function.","evidence":"Inducible smooth muscle transgene, actin polymerization measurement, cofilin/VASP immunoblotting, in vivo arterial stiffness measurements","pmids":["35358093"],"confidence":"High","gaps":["Direct substrate linking RhoBTB1 to cofilin/VASP changes not yet identified","Mechanistic coupling to CUL3 degradation not established in this study"]},{"year":2023,"claim":"Mapped the substrate-adaptor architecture and identified additional candidate substrates, defining the minimal degradation module and key CUL3-binding residues.","evidence":"Domain truncation, site-directed mutagenesis (Pro353, Ser363), APEX2 proximity labeling/MS, and Co-IP","pmids":["37575477"],"confidence":"High","gaps":["SETD2 as substrate is Medium-confidence and needs ubiquitination/degradation kinetics","Physiological context of SETD2 regulation not defined"]},{"year":2026,"claim":"Identified RbFox2 as a CUL3-dependent RhoBTB1 substrate, providing a molecular link from the adaptor function to actin integrity and arterial stiffness.","evidence":"Reciprocal Co-IP, ubiquitination assay, proximity labeling, siRNA, CUL3 deletion, and RbFox2 floxed mouse model","pmids":["41926228"],"confidence":"High","gaps":["How RbFox2 levels translate mechanistically to actin dynamics not fully resolved","Interplay between PDE5 and RbFox2 substrate streams unknown"]},{"year":null,"claim":"How RhoBTB1's distinct functional arms — CUL3-dependent substrate degradation, ROCK binding/phosphorylation, and endosomal/Golgi trafficking — are integrated and tissue-specifically deployed remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking trafficking role to CUL3 adaptor function","Full substrate repertoire incomplete","No structural model of the RhoBTB1-CUL3-substrate complex"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[8]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[7,8]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[8]}],"complexes":["Cullin-3 (CUL3) E3 ubiquitin ligase"],"partners":["CUL3","PDE5A","RBFOX2","ROCK1","ROCK2","SETD2","METTL7B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O94844","full_name":"Rho-related BTB domain-containing protein 1","aliases":[],"length_aa":696,"mass_kda":79.4,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/O94844/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RHOBTB1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RHOBTB1","total_profiled":1310},"omim":[{"mim_id":"618753","title":"LEUCINE-RICH REPEAT-CONTAINING PROTEIN 41; LRRC41","url":"https://www.omim.org/entry/618753"},{"mim_id":"607353","title":"RHO-RELATED BTB DOMAIN-CONTAINING PROTEIN 3; RHOBTB3","url":"https://www.omim.org/entry/607353"},{"mim_id":"607352","title":"RHO-RELATED BTB DOMAIN-CONTAINING PROTEIN 2; RHOBTB2","url":"https://www.omim.org/entry/607352"},{"mim_id":"607351","title":"RHO-RELATED BTB DOMAIN-CONTAINING PROTEIN 1; RHOBTB1","url":"https://www.omim.org/entry/607351"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skeletal muscle","ntpm":135.1}],"url":"https://www.proteinatlas.org/search/RHOBTB1"},"hgnc":{"alias_symbol":["KIAA0740"],"prev_symbol":[]},"alphafold":{"accession":"O94844","domains":[{"cath_id":"3.40.50.300","chopping":"12-220","consensus_level":"high","plddt":90.3673,"start":12,"end":220}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O94844","model_url":"https://alphafold.ebi.ac.uk/files/AF-O94844-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O94844-F1-predicted_aligned_error_v6.png","plddt_mean":82.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RHOBTB1","jax_strain_url":"https://www.jax.org/strain/search?query=RHOBTB1"},"sequence":{"accession":"O94844","fasta_url":"https://rest.uniprot.org/uniprotkb/O94844.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O94844/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O94844"}},"corpus_meta":[{"pmid":"28219369","id":"PMC_28219369","title":"The tumor suppressor RhoBTB1 controls Golgi integrity and breast cancer cell invasion through METTL7B.","date":"2017","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/28219369","citation_count":70,"is_preprint":false},{"pmid":"23258531","id":"PMC_23258531","title":"The tumor suppressor gene RhoBTB1 is a novel target of miR-31 in human colon cancer.","date":"2012","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/23258531","citation_count":51,"is_preprint":false},{"pmid":"29053138","id":"PMC_29053138","title":"miR-31a-5p promotes postnatal cardiomyocyte proliferation by targeting RhoBTB1.","date":"2017","source":"Experimental & molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/29053138","citation_count":38,"is_preprint":false},{"pmid":"30896450","id":"PMC_30896450","title":"RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity.","date":"2019","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/30896450","citation_count":37,"is_preprint":false},{"pmid":"16170569","id":"PMC_16170569","title":"Identification of a candidate tumor suppressor gene RHOBTB1 located at a novel allelic loss region 10q21 in head and neck cancer.","date":"2005","source":"Journal of cancer research and clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/16170569","citation_count":33,"is_preprint":false},{"pmid":"35358093","id":"PMC_35358093","title":"RhoBTB1 reverses established arterial stiffness in angiotensin II-induced hypertension by promoting actin depolymerization.","date":"2022","source":"JCI insight","url":"https://pubmed.ncbi.nlm.nih.gov/35358093","citation_count":21,"is_preprint":false},{"pmid":"31789920","id":"PMC_31789920","title":"PPARγ and RhoBTB1 in hypertension.","date":"2020","source":"Current opinion in nephrology and hypertension","url":"https://pubmed.ncbi.nlm.nih.gov/31789920","citation_count":19,"is_preprint":false},{"pmid":"31431478","id":"PMC_31431478","title":"RhoBTB1 interacts with ROCKs and inhibits invasion.","date":"2019","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/31431478","citation_count":13,"is_preprint":false},{"pmid":"32354068","id":"PMC_32354068","title":"RNA Interference Screening Identifies Novel Roles for RhoBTB1 and RhoBTB3 in Membrane Trafficking Events in Mammalian Cells.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/32354068","citation_count":7,"is_preprint":false},{"pmid":"37575477","id":"PMC_37575477","title":"Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination.","date":"2023","source":"Function (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/37575477","citation_count":5,"is_preprint":false},{"pmid":"40287984","id":"PMC_40287984","title":"Machine learning analysis of FOSL2 and RHoBTB1 as central immunological regulators in knee osteoarthritis synovium.","date":"2025","source":"The Journal of international medical research","url":"https://pubmed.ncbi.nlm.nih.gov/40287984","citation_count":2,"is_preprint":false},{"pmid":"38196837","id":"PMC_38196837","title":"The 2023 Walter B. Cannon Award Lecture: Mechanisms Regulating Vascular Function and Blood Pressure by the PPARγ-RhoBTB1-CUL3 Pathway.","date":"2024","source":"Function (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/38196837","citation_count":1,"is_preprint":false},{"pmid":"39673615","id":"PMC_39673615","title":"Cutaneous squamous cell carcinoma-derived exosomal MicroRNA-31 acts as an oncogene by targeting the tumor suppressor RhoBTB1.","date":"2024","source":"Archives of dermatological research","url":"https://pubmed.ncbi.nlm.nih.gov/39673615","citation_count":1,"is_preprint":false},{"pmid":"41926228","id":"PMC_41926228","title":"Vascular smooth muscle RbFox2 regulates the cytoskeleton and arterial stiffness by a RhoBTB1/Cullin-3 mechanism.","date":"2026","source":"JCI insight","url":"https://pubmed.ncbi.nlm.nih.gov/41926228","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.20.25340682","title":"Identification and validation of biomarkers  <i>FAM107A</i>  ,  <i>RHOBTB1</i>  and  <i>ZBTB16</i>  associated with dietary restriction in osteoarthritis","date":"2025-11-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.20.25340682","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10269,"output_tokens":2875,"usd":0.036966,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10150,"output_tokens":3073,"usd":0.063787,"stage2_stop_reason":"end_turn"},"total_usd":0.100753,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"RhoBTB1 acts as a substrate adaptor delivering phosphodiesterase 5 (PDE5) to the Cullin-3 (CUL3) E3 RING ubiquitin ligase complex, leading to PDE5 ubiquitination and inhibition, thereby augmenting cGMP responses to nitric oxide in vascular smooth muscle cells.\",\n      \"method\": \"Genetic complementation (inducible smooth muscle-specific RhoBTB1 transgene), Co-immunoprecipitation, functional vasodilation assays in vivo\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, genetic complementation in two hypertension models, functional readout (cGMP, vasodilation), replicated across models\",\n      \"pmids\": [\"30896450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The C-terminal half of RhoBTB1 (B1B2C region, comprising both BTB domains and C-terminal domain) is the minimal region required for PDE5 recruitment and subsequent proteasomal degradation via CUL3; the C-terminal domain is essential for PDE5 binding, and Pro353 and Ser363 are key residues required for CUL3 binding (mutation impairs CUL3 binding and PDE5 degradation without disrupting PDE5 binding).\",\n      \"method\": \"Domain truncation, Co-immunoprecipitation, site-directed mutagenesis, APEX2 proximity labeling/mass spectrometry\",\n      \"journal\": \"Function (Oxford, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis, domain truncation, proximity labeling with multiple orthogonal methods in single study\",\n      \"pmids\": [\"37575477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SETD2 is a binding partner of RhoBTB1 (validated by Co-IP), and SETD2 levels increase upon proteasome inhibition, Cullin complex inhibition, CUL3 deletion, or RhoBTB1 siRNA knockdown, suggesting SETD2 is a substrate regulated by the RhoBTB1-CUL3 ubiquitin-proteasome axis.\",\n      \"method\": \"APEX2 proximity labeling/mass spectrometry, Co-immunoprecipitation, siRNA knockdown, pharmacological proteasome/Cullin inhibition\",\n      \"journal\": \"Function (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and proximity labeling with functional validation (proteasome inhibition), single lab, single study\",\n      \"pmids\": [\"37575477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RhoBTB1 reverses established arterial stiffness by promoting actin depolymerization in vascular smooth muscle; angiotensin II-induced actin polymerization in the aorta is reversed by RhoBTB1 restoration, with consistent changes in levels of cofilin and vasodilator-stimulated phosphoprotein (VASP), two regulators of actin polymerization.\",\n      \"method\": \"Inducible smooth muscle-specific RhoBTB1 transgene, measurement of actin polymerization, immunoblotting for cofilin and VASP, arterial stiffness measurements in vivo\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with multiple mechanistic readouts (actin polymerization, cofilin, VASP) in a defined in vivo model, single lab\",\n      \"pmids\": [\"35358093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RhoBTB1 interacts with and promotes CUL3-dependent ubiquitination and proteasomal degradation of RbFox2 in smooth muscle cells; loss of RhoBTB1 (by siRNA or angiotensin II) elevates RbFox2, and RbFox2 regulates actin cytoskeletal integrity and arterial stiffness.\",\n      \"method\": \"Co-immunoprecipitation, proximity labeling/mass spectrometry, siRNA knockdown, CUL3 deletion, RbFox2 floxed mouse model, ubiquitination assay\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ubiquitination assay, genetic knockout model with arterial stiffness readout, multiple orthogonal methods in single study\",\n      \"pmids\": [\"41926228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RhoBTB1 associates with ROCK1 and ROCK2; the interaction with ROCK1 is mediated by the Rho domain of RhoBTB1 binding to the coiled-coil region of ROCK1 near its kinase domain. Two amino acids in the Rho domain alter RhoBTB1-ROCK1 association. RhoBTB1 is a substrate for ROCK1, and mutation of putative ROCK1 phosphorylation sites on RhoBTB1 reduces its association with Cullin3.\",\n      \"method\": \"Co-immunoprecipitation, pulldown, site-directed mutagenesis, in vitro kinase assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — Co-IP, mutagenesis of interaction interface, and in vitro kinase assay, multiple orthogonal methods in single study\",\n      \"pmids\": [\"31431478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RhoBTB1 depletion increases prostate cancer cell invasion and induces elongation in Matrigel, a phenotype similar to that induced by depletion of ROCK1 and ROCK2, suggesting RhoBTB1 suppresses cancer cell invasion through interaction with ROCKs.\",\n      \"method\": \"siRNA knockdown, Matrigel invasion assay, cell morphology imaging\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss-of-function with defined invasion phenotype, genetic epistasis-like comparison with ROCK depletion, single lab\",\n      \"pmids\": [\"31431478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RhoBTB1 regulates the integrity of the Golgi complex through control of METTL7B expression; silencing of either RhoBTB1 or METTL7B leads to Golgi fragmentation, and restoration of RhoBTB1 expression rescues Golgi morphology and inhibits breast cancer cell invasion.\",\n      \"method\": \"Gene silencing (RNAi), transcriptome analysis, Q-PCR, cell imaging (Golgi morphology), rescue experiments\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — RNAi knockdown plus rescue, imaging phenotype, and transcriptional target validation, single lab\",\n      \"pmids\": [\"28219369\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RhoBTB1 localizes to early endosomal intermediates, and changes in RhoBTB1 levels cause disturbances to Golgi architecture, profound changes to organisation and distribution of endosomes and lysosomes, and defects in delivery of two classes of cargo molecules to downstream compartments, linking RhoBTB1 to endocytosis and retrograde traffic pathways.\",\n      \"method\": \"RNA interference screen, high-content image-based analysis, fluorescence microscopy, cargo trafficking assays\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — systematic RNAi, localization imaging, multiple trafficking readouts, single lab\",\n      \"pmids\": [\"32354068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RhoBTB1 is a target gene of miR-31a-5p; miR-31a-5p promotes postnatal cardiomyocyte proliferation by repressing RhoBTB1, and RhoBTB1 knockdown phenocopies miR-31a-5p overexpression in promoting cardiomyocyte proliferation markers.\",\n      \"method\": \"miRNA arrays, luciferase reporter assay (implied by target validation), immunofluorescence, EdU/Ki-67/PHH3 proliferation assays, antagomir injection in neonatal rats\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — in vivo antagomir model, target knockdown phenocopy, multiple proliferation markers, single lab\",\n      \"pmids\": [\"29053138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Exosomal miR-31 from cutaneous squamous cell carcinoma (CSCC) cells directly targets the 3'UTR of RhoBTB1 (validated by dual luciferase reporter assay), suppressing RhoBTB1 expression and enhancing CSCC cell proliferation, migration, and invasion.\",\n      \"method\": \"Dual luciferase reporter assay, immunoblotting, qPCR, MTT assay, Transwell assay, differential ultracentrifugation for exosome isolation\",\n      \"journal\": \"Archives of dermatological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — luciferase reporter directly validates 3'UTR targeting, functional assays, single lab\",\n      \"pmids\": [\"39673615\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RhoBTB1 is an atypical Rho GTPase that functions primarily as a substrate adaptor for the Cullin-3 E3 ubiquitin ligase complex, recruiting substrates including PDE5 (via its C-terminal BTB/C-terminal domain) and RbFox2 for ubiquitination and proteasomal degradation, thereby regulating vascular smooth muscle cGMP signaling, actin cytoskeletal dynamics, arterial stiffness, and vasodilation; it also interacts with ROCK1/2 (via its Rho domain) to suppress cancer cell invasion, regulates Golgi integrity through METTL7B, and participates in endosomal/retrograde trafficking, while being post-translationally regulated by ROCK1-mediated phosphorylation that modulates its Cullin3 association.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RHOBTB1 is an atypical Rho GTPase that functions principally as a substrate adaptor for the Cullin-3 (CUL3) E3 ubiquitin ligase, coupling targeted protein degradation to the control of vascular smooth muscle tone and actin cytoskeletal dynamics [#0, #4]. It delivers phosphodiesterase 5 (PDE5) to CUL3 for ubiquitination and inhibition, thereby augmenting nitric oxide-driven cGMP signaling and promoting vasodilation [#0], and it likewise targets the splicing regulator RbFox2 for CUL3-dependent degradation to maintain actin cytoskeletal integrity and reverse arterial stiffness [#3, #4]. Substrate recruitment is partitioned across the protein: the C-terminal domain mediates PDE5 binding while Pro353 and Ser363 are required for CUL3 association, and the full C-terminal B1B2C region is the minimal degradation-competent module [#1]. Through its N-terminal Rho domain, RHOBTB1 binds the coiled-coil region of ROCK1/ROCK2 and is itself a ROCK1 phosphorylation substrate, with phosphorylation modulating its CUL3 association — linking it to suppression of cancer cell invasion [#5, #6]. Beyond degradation, RHOBTB1 maintains Golgi integrity via control of METTL7B expression and localizes to early endosomes where it governs endosomal/lysosomal organization and retrograde cargo trafficking [#7, #8]. Its expression is repressed by miR-31/miR-31a-5p, linking its loss to cardiomyocyte proliferation and to enhanced tumor cell migration and invasion [#9, #10].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established the first cellular role for RhoBTB1 by linking it to organelle integrity, addressing whether this atypical GTPase has a defined cellular function.\",\n      \"evidence\": \"RNAi silencing, transcriptome analysis, and Golgi-morphology rescue experiments in breast cancer cells\",\n      \"pmids\": [\"28219369\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which RhoBTB1 controls METTL7B expression unknown\", \"Does not establish direct molecular partners or enzymatic activity\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified an upstream regulatory mechanism, showing RhoBTB1 is a repressed target of miR-31a-5p that restrains cardiomyocyte proliferation.\",\n      \"evidence\": \"miRNA arrays, target knockdown phenocopy, and antagomir injection in neonatal rats\",\n      \"pmids\": [\"29053138\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream effectors mediating proliferation control not defined\", \"Direct 3'UTR binding inferred rather than fully dissected here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined RhoBTB1's core molecular function as a CUL3 substrate adaptor for PDE5, explaining how it augments cGMP signaling and vasodilation in vivo.\",\n      \"evidence\": \"Smooth muscle-specific inducible transgene, reciprocal Co-IP, and functional vasodilation/cGMP assays in two hypertension models\",\n      \"pmids\": [\"30896450\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise domain requirements for PDE5 and CUL3 binding not yet mapped\", \"Other substrates beyond PDE5 unknown at this stage\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected the Rho domain to ROCK kinases and revealed a feedback loop, showing RhoBTB1 binds ROCK1/2, is phosphorylated by ROCK1, and that this phosphorylation controls its CUL3 association.\",\n      \"evidence\": \"Co-IP, pulldown, interface mutagenesis, and in vitro kinase assay; Matrigel invasion phenotyping in prostate cancer cells\",\n      \"pmids\": [\"31431478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of ROCK binding for ROCK activity not resolved\", \"Invasion suppression role (Medium-confidence) lacks in vivo validation\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Localized RhoBTB1 to early endosomes and broadened its role to membrane trafficking, showing its dysregulation disrupts endosome/lysosome organization and retrograde cargo delivery.\",\n      \"evidence\": \"RNAi screen, high-content imaging, and cargo trafficking assays\",\n      \"pmids\": [\"32354068\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether trafficking role is CUL3-dependent unknown\", \"Direct trafficking machinery partners not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated RhoBTB1 can reverse established arterial stiffness via actin depolymerization, establishing a therapeutically relevant phenotype downstream of its function.\",\n      \"evidence\": \"Inducible smooth muscle transgene, actin polymerization measurement, cofilin/VASP immunoblotting, in vivo arterial stiffness measurements\",\n      \"pmids\": [\"35358093\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrate linking RhoBTB1 to cofilin/VASP changes not yet identified\", \"Mechanistic coupling to CUL3 degradation not established in this study\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Mapped the substrate-adaptor architecture and identified additional candidate substrates, defining the minimal degradation module and key CUL3-binding residues.\",\n      \"evidence\": \"Domain truncation, site-directed mutagenesis (Pro353, Ser363), APEX2 proximity labeling/MS, and Co-IP\",\n      \"pmids\": [\"37575477\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SETD2 as substrate is Medium-confidence and needs ubiquitination/degradation kinetics\", \"Physiological context of SETD2 regulation not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified RbFox2 as a CUL3-dependent RhoBTB1 substrate, providing a molecular link from the adaptor function to actin integrity and arterial stiffness.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assay, proximity labeling, siRNA, CUL3 deletion, and RbFox2 floxed mouse model\",\n      \"pmids\": [\"41926228\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How RbFox2 levels translate mechanistically to actin dynamics not fully resolved\", \"Interplay between PDE5 and RbFox2 substrate streams unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RhoBTB1's distinct functional arms — CUL3-dependent substrate degradation, ROCK binding/phosphorylation, and endosomal/Golgi trafficking — are integrated and tissue-specifically deployed remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking trafficking role to CUL3 adaptor function\", \"Full substrate repertoire incomplete\", \"No structural model of the RhoBTB1-CUL3-substrate complex\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [7, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"Cullin-3 (CUL3) E3 ubiquitin ligase\"],\n    \"partners\": [\"CUL3\", \"PDE5A\", \"RBFOX2\", \"ROCK1\", \"ROCK2\", \"SETD2\", \"METTL7B\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"faith_supported":6,"faith_total":6,"faith_pct":100.0}}