{"gene":"RHOBTB3","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2009,"finding":"RhoBTB3 binds directly to Rab9 GTPase and functions with Rab9 in protein transport from endosomes to the trans-Golgi network. Biochemical analysis revealed that RhoBTB3 binds and hydrolyzes ATP (not GTP), and Rab9 binding opens the autoinhibited RhoBTB3 protein to permit maximal ATP hydrolysis. RhoBTB3 also interacts with TIP47 on membranes, suggesting a role in releasing this cargo selection protein from vesicles for efficient docking and fusion at the Golgi. Gene replacement experiments showed that both the N-terminal Rho-related domain and C-terminal sequences (important for Rab9 interaction) are required for function.","method":"Direct binding assays, biochemical ATPase activity assays, co-immunoprecipitation, gene replacement experiments in cultured cells","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods: direct biochemical ATPase assay, binding assays, domain-replacement genetics, and interaction studies in a single rigorous study","pmids":["19490898"],"is_preprint":false},{"year":2013,"finding":"RhoBTB3 is a Golgi-associated ATPase that targets cyclin E for ubiquitylation as part of a CUL3-dependent RING-E3 ubiquitin ligase complex (RhoBTB3–CUL3–RBX1) at the Golgi. Depletion of RhoBTB3 arrests cells in S phase, triggers Golgi fragmentation, and elevates cyclin E levels. Golgi association of the complex is required for its ability to catalyze cyclin E ubiquitylation and allow normal S/G2 cell cycle progression.","method":"RNAi depletion, co-immunoprecipitation, ubiquitylation assays, cell cycle analysis, Golgi localization experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ubiquitylation assay, defined cellular phenotype upon KD, Golgi localization tied to functional consequence, multiple orthogonal methods","pmids":["24145166"],"is_preprint":false},{"year":2015,"finding":"RHOBTB3 directly interacts with the prolyl hydroxylase PHD2 to promote HIFα hydroxylation, and directly interacts with VHL (an E3 ubiquitin ligase complex component) to facilitate HIFα ubiquitination. RHOBTB3 dimerizes with LIMD1 and assembles a RHOBTB3/LIMD1–PHD2–VHL–HIFα complex for maximal HIFα degradation. Hypoxia reduces this complex formation, causing HIFα accumulation. RHOBTB3 deficiency elevates the Warburg effect and accelerates xenograft tumor growth.","method":"Co-immunoprecipitation, ubiquitination assays, hydroxylation assays, RNAi knockdown, xenograft experiments","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, hydroxylation and ubiquitination assays, in vivo xenograft validation, multiple orthogonal methods in a single study","pmids":["26215701"],"is_preprint":false},{"year":2012,"finding":"RhoBTB3 interacts with the 5-HT7a serotonin receptor (binding involves both the C-terminal tail and the third intracellular loop of the receptor), co-localizes at the plasma membrane and ER, and inhibits proteasomal degradation of the 5-HT7a receptor. Notably, although RhoBTB3 interacts with CUL3, it does not recruit CUL3/ROC1 to the 5-HT7a receptor and does not mediate receptor ubiquitination.","method":"Yeast two-hybrid screen, co-immunoprecipitation in HEK293T cells, domain mapping, immunofluorescence microscopy, proteasome inhibition assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid confirmed by Co-IP and localization, functional degradation assay, single lab with multiple methods","pmids":["22245496"],"is_preprint":false},{"year":2020,"finding":"Depletion of RhoBTB3 affects Golgi complex morphology and causes changes in the trafficking speeds of membrane carriers operating at the Golgi–ER interface. RhoBTB3 was found to be present on these Golgi–ER carriers, indicating a direct role in this trafficking step.","method":"RNA interference, high-content image-based screening, live-cell imaging of carrier trafficking, fluorescence microscopy","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — RNAi phenotype with live imaging of carriers and localization data, single lab, multiple imaging-based methods","pmids":["32354068"],"is_preprint":false},{"year":2024,"finding":"RhoBTB3 forms a complex with SHIP164 and the retromer subunit Vps26B at Golgi–early endosome (EE) contact sites to promote EE bud formation. Vps26B acts as a novel Rab14 effector, and Rab14 activity regulates SHIP164 association with EEs. Suppression of RhoBTB3 phenocopies SHIP164 depletion (enlarged Rab14+ EEs without buds), revealing a lipid-transfer-dependent pathway for EE budding.","method":"Co-immunoprecipitation, RNAi knockdown, fluorescence microscopy, rescue experiments with lipid-transfer-defective mutants","journal":"Cell discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, functional rescue with domain mutants, live imaging, single lab with multiple orthogonal methods","pmids":["38565878"],"is_preprint":false},{"year":2024,"finding":"RhoBTB3 negatively regulates autophagy by acting as an E3 ubiquitin ligase that ubiquitinates AMBRA1 (at K45 via K27-linked ubiquitin chains), promoting its proteasome-mediated degradation. RhoBTB3 deficiency induces autophagy and elevates AMBRA1 protein levels (without affecting AMBRA1 mRNA), while RhoBTB3 overexpression inhibits autophagy induction. AMBRA1 knockdown blocks RhoBTB3-depletion-induced autophagy.","method":"Immunoprecipitation, mass spectrometry, ubiquitination assays with site-specific mutants (K45), proteasome inhibition assays, RNAi knockdown, overexpression","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS identification of substrate, site-specific ubiquitination mapped, epistasis by AMBRA1 knockdown rescue, single lab with multiple orthogonal methods","pmids":["39404422"],"is_preprint":false},{"year":2025,"finding":"Diosmetin reduces formation of the RhoBTB3–PHD2 complex in UV-irradiated keratinocytes, thereby limiting HIF-1α hydroxylation and ubiquitination, and stabilizing HIF-1α. Overexpression of RhoBTB3 in mice enriches RhoBTB3, PHD2, and HIF-1α in the epidermis, consistent with RhoBTB3 promoting PHD2-mediated HIF-1α hydroxylation in skin.","method":"Co-immunoprecipitation, RNA sequencing, rAAV-mediated overexpression in mice, immunohistochemistry, in vivo UV sunburn model","journal":"Phytomedicine","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP confirmed RhoBTB3–PHD2 complex, in vivo overexpression model, corroborates prior mechanistic data (PMID 26215701), single lab","pmids":["40311591"],"is_preprint":false},{"year":2019,"finding":"Genetic deletion of RhoBTB3 in mice leads to increased alpha-granule secretion in platelets in response to thrombin, CRP, and U46619/ADP, and reduces platelet accrual on collagen under flow conditions, without affecting platelet count, granule numbers, surface receptors, aggregation, dense granule secretion, or tail bleeding time.","method":"Knockout mouse model, flow cytometry, aggregometry, granule secretion assays, collagen adhesion under flow","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined platelet functional phenotype, single lab, single study","pmids":["30754723"],"is_preprint":false}],"current_model":"RhoBTB3 is an atypical Rho-family ATPase (not a GTPase) that localizes to the Golgi and endosomes, where it functions in multiple membrane trafficking and protein degradation pathways: it binds Rab9 (with Rab9 relieving autoinhibition to stimulate ATP hydrolysis) to mediate endosome-to-TGN transport; it acts as a CUL3-RBX1 E3 ubiquitin ligase scaffold at the Golgi to ubiquitinate cyclin E for cell cycle-dependent degradation; it assembles a RHOBTB3–SHIP164–Vps26B complex at Golgi–endosome contacts to drive early endosome budding; it scaffolds a RHOBTB3/LIMD1–PHD2–VHL complex to promote HIFα hydroxylation and ubiquitin-proteasomal degradation; and it ubiquitinates AMBRA1 via K27-linked chains at K45 to suppress autophagy."},"narrative":{"mechanistic_narrative":"RHOBTB3 is an atypical Rho-family ATPase that hydrolyzes ATP rather than GTP and acts as a Golgi- and endosome-associated organizer of membrane trafficking and substrate-targeted protein degradation [PMID:19490898]. In its founding role it binds the Rab9 GTPase—which relieves RHOBTB3 autoinhibition to permit maximal ATP hydrolysis—and engages TIP47 on membranes to mediate cargo transport from endosomes to the trans-Golgi network, a function requiring both its N-terminal Rho-related domain and C-terminal Rab9-interaction sequences [PMID:19490898]. RHOBTB3 also serves as a substrate-recognition scaffold for CUL3-RBX1 RING E3 ubiquitin ligase activity at the Golgi, ubiquitylating cyclin E to drive its degradation and enable normal S/G2 cell cycle progression; its loss arrests cells in S phase and fragments the Golgi [PMID:24145166]. Beyond cyclin E, RHOBTB3 ubiquitinates AMBRA1 through K27-linked chains at K45 to promote its proteasomal degradation and thereby suppress autophagy [PMID:39404422], and it scaffolds a RHOBTB3/LIMD1–PHD2–VHL complex that promotes HIFα prolyl hydroxylation and ubiquitin-proteasomal degradation, restraining the Warburg effect and tumor growth [PMID:26215701, PMID:40311591]. Its trafficking activities extend to assembly of a RHOBTB3–SHIP164–Vps26B complex at Golgi–early endosome contact sites that drives lipid-transfer-dependent endosome bud formation [PMID:38565878] and to maintenance of Golgi morphology and Golgi–ER carrier trafficking [PMID:32354068].","teleology":[{"year":2009,"claim":"Established the biochemical identity of RhoBTB3 as an ATP-hydrolyzing (not GTP-hydrolyzing) Rho-family protein and placed it in endosome-to-TGN transport, answering what kind of enzyme it is and what regulates it.","evidence":"Direct binding and ATPase assays, co-IP, and domain-replacement genetics in cultured cells showing Rab9 binding relieves autoinhibition and TIP47 interaction on membranes","pmids":["19490898"],"confidence":"High","gaps":["Structural basis of the autoinhibited-to-open transition not resolved","How ATP hydrolysis is coupled mechanically to cargo release at the Golgi not defined"]},{"year":2012,"claim":"Tested whether RhoBTB3's CUL3 association extends to all binding partners by examining the 5-HT7a serotonin receptor, showing it stabilizes the receptor without ubiquitinating it—revealing CUL3-independent functions.","evidence":"Yeast two-hybrid, co-IP, domain mapping, and proteasome-inhibition assays in HEK293T cells","pmids":["22245496"],"confidence":"Medium","gaps":["Mechanism by which RhoBTB3 protects 5-HT7a from proteasomal degradation unknown","Physiological context of the interaction not established"]},{"year":2013,"claim":"Defined RhoBTB3 as the substrate-recognition subunit of a Golgi-localized CUL3-RBX1 E3 ligase targeting cyclin E, linking its trafficking compartment to cell cycle control.","evidence":"RNAi depletion, reciprocal co-IP, ubiquitylation assays, cell cycle analysis, and Golgi localization experiments","pmids":["24145166"],"confidence":"High","gaps":["Why a Golgi-localized ligase targets a nuclear/cytoplasmic cyclin not mechanistically resolved","Whether ATPase activity is required for ligase function not established"]},{"year":2015,"claim":"Extended RhoBTB3 into oxygen sensing by showing it scaffolds PHD2 and VHL with LIMD1 to maximize HIFα hydroxylation and degradation, defining a tumor-suppressive metabolic role.","evidence":"Co-IP, hydroxylation and ubiquitination assays, RNAi knockdown, and xenograft experiments","pmids":["26215701"],"confidence":"High","gaps":["How hypoxia disassembles the complex molecularly not defined","Relationship between this scaffolding role and ATPase activity unknown"]},{"year":2019,"claim":"Addressed the in vivo organismal function via knockout mice, revealing a role in platelet alpha-granule secretion and collagen-dependent thrombus formation distinct from its trafficking phenotypes.","evidence":"Knockout mouse model with flow cytometry, aggregometry, granule secretion assays, and collagen adhesion under flow","pmids":["30754723"],"confidence":"Medium","gaps":["Molecular link between RhoBTB3 and granule secretion machinery not identified","Whether the platelet phenotype reflects trafficking or ligase functions unknown"]},{"year":2020,"claim":"Resolved RhoBTB3's direct presence on Golgi–ER carriers and its requirement for normal carrier trafficking and Golgi morphology, reinforcing a direct trafficking role at the Golgi interface.","evidence":"RNAi, high-content image-based screening, and live-cell imaging of carrier trafficking","pmids":["32354068"],"confidence":"Medium","gaps":["Molecular cargo carried by these carriers not defined","Whether RhoBTB3 ATPase cycle drives carrier dynamics not tested"]},{"year":2024,"claim":"Identified a RHOBTB3–SHIP164–Vps26B complex at Golgi–early endosome contacts driving lipid-transfer-dependent endosome budding, and a separate E3 ligase activity toward AMBRA1 that suppresses autophagy.","evidence":"Co-IP, RNAi, fluorescence/live imaging, rescue with lipid-transfer-defective and K45 ubiquitination-site mutants, and IP-MS substrate identification","pmids":["38565878","39404422"],"confidence":"Medium","gaps":["How RHOBTB3 coordinates contact-site scaffolding with bud formation not resolved","Whether the AMBRA1 ligase activity uses CUL3 not specified","Relationship between the trafficking and autophagy roles unknown"]},{"year":2025,"claim":"Corroborated the RhoBTB3–PHD2–HIF-1α axis in skin in vivo and showed it is pharmacologically targetable, with diosmetin reducing complex formation to stabilize HIF-1α.","evidence":"Co-IP, RNA-seq, rAAV overexpression in mice, immunohistochemistry, and a UV sunburn model","pmids":["40311591"],"confidence":"Medium","gaps":["Direct binding site of diosmetin and whether it acts on RhoBTB3 or PHD2 not established","Tissue specificity of the skin phenotype not explained"]},{"year":null,"claim":"How RhoBTB3's single ATPase scaffold protein selects among its diverse functions—endosome-TGN transport, CUL3 ligase activity toward distinct substrates, contact-site lipid-transfer scaffolding, and HIF degradation—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating the ATPase cycle with substrate/partner selection","Determinants directing RhoBTB3 to one complex versus another not identified","Whether ATP hydrolysis is required across all of its roles untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,6]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,5]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1,4,5]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,5]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,4,5]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,6]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[6]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[2]}],"complexes":["RHOBTB3–CUL3–RBX1 E3 ubiquitin ligase","RHOBTB3/LIMD1–PHD2–VHL–HIFα complex","RHOBTB3–SHIP164–Vps26B complex"],"partners":["RAB9A","CUL3","RBX1","PHD2","VHL","LIMD1","AMBRA1","SHIP164"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O94955","full_name":"Rho-related BTB domain-containing protein 3","aliases":[],"length_aa":611,"mass_kda":69.4,"function":"Rab9-regulated ATPase required for endosome to Golgi transport. Involved in transport vesicle docking at the Golgi complex, possibly by participating in release M6PRBP1/TIP47 from vesicles to permit their efficient docking and fusion at the Golgi. Specifically binds Rab9, but not other Rab proteins. Has low intrinsic ATPase activity due to autoinhibition, which is relieved by Rab9","subcellular_location":"Golgi apparatus","url":"https://www.uniprot.org/uniprotkb/O94955/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RHOBTB3","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RHOBTB3","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"},{"mim_id":"603575","title":"NME/NM23 FAMILY, MEMBER 5; NME5","url":"https://www.omim.org/entry/603575"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Vesicles","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RHOBTB3"},"hgnc":{"alias_symbol":["KIAA0878"],"prev_symbol":[]},"alphafold":{"accession":"O94955","domains":[{"cath_id":"3.40.50.300","chopping":"3-43_50-140_155-206","consensus_level":"high","plddt":73.3604,"start":3,"end":206},{"cath_id":"3.30.710.10","chopping":"240-313_324-388","consensus_level":"high","plddt":89.6767,"start":240,"end":388},{"cath_id":"3.30.710.10","chopping":"412-592","consensus_level":"medium","plddt":91.3071,"start":412,"end":592}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O94955","model_url":"https://alphafold.ebi.ac.uk/files/AF-O94955-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O94955-F1-predicted_aligned_error_v6.png","plddt_mean":78.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RHOBTB3","jax_strain_url":"https://www.jax.org/strain/search?query=RHOBTB3"},"sequence":{"accession":"O94955","fasta_url":"https://rest.uniprot.org/uniprotkb/O94955.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O94955/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O94955"}},"corpus_meta":[{"pmid":"19490898","id":"PMC_19490898","title":"RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport.","date":"2009","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/19490898","citation_count":79,"is_preprint":false},{"pmid":"26215701","id":"PMC_26215701","title":"RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.","date":"2015","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/26215701","citation_count":48,"is_preprint":false},{"pmid":"24145166","id":"PMC_24145166","title":"Golgi-associated RhoBTB3 targets cyclin E for ubiquitylation and promotes cell cycle progression.","date":"2013","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/24145166","citation_count":39,"is_preprint":false},{"pmid":"35698915","id":"PMC_35698915","title":"RhoBTB3 Regulates Proliferation and Invasion of Breast Cancer Cells via Col1a1.","date":"2022","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/35698915","citation_count":20,"is_preprint":false},{"pmid":"22245496","id":"PMC_22245496","title":"RhoBTB3 interacts with the 5-HT7a receptor and inhibits its proteasomal degradation.","date":"2012","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/22245496","citation_count":15,"is_preprint":false},{"pmid":"24923387","id":"PMC_24923387","title":"Expression analysis of mouse Rhobtb3 using a LacZ reporter and preliminary characterization of a knockout strain.","date":"2014","source":"Histochemistry and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/24923387","citation_count":10,"is_preprint":false},{"pmid":"37045834","id":"PMC_37045834","title":"MicroRNA-142-3p promotes renal cell carcinoma progression by targeting RhoBTB3 to regulate HIF-1 signaling and GGT/GSH pathways.","date":"2023","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/37045834","citation_count":9,"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":"34093823","id":"PMC_34093823","title":"High Expression of RhoBTB3 Predicts Favorable Chemothrapy Outcomes in non-M3 Acute Myeloid Leukemia.","date":"2021","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/34093823","citation_count":7,"is_preprint":false},{"pmid":"38565878","id":"PMC_38565878","title":"Biogenesis of Rab14-positive endosome buds at Golgi-endosome contacts by the RhoBTB3-SHIP164-Vps26B complex.","date":"2024","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/38565878","citation_count":5,"is_preprint":false},{"pmid":"30754723","id":"PMC_30754723","title":"Alterations in Platelet Alpha-Granule Secretion and Adhesion on Collagen under Flow in Mice Lacking the Atypical Rho GTPase RhoBTB3.","date":"2019","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/30754723","citation_count":4,"is_preprint":false},{"pmid":"39404422","id":"PMC_39404422","title":"RhoBTB3 Functions as a Novel Regulator of Autophagy by Suppressing AMBRA1 Stability.","date":"2024","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/39404422","citation_count":2,"is_preprint":false},{"pmid":"40311591","id":"PMC_40311591","title":"Diosmetin attenuates the ubiquitination of epidermal hypoxia-inducible factor 1 alpha by diminishing the formation of RhoBTB3/PHD2 complex in ultraviolet radiation-induced sunburn in mice.","date":"2025","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40311591","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8687,"output_tokens":2784,"usd":0.033911,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10101,"output_tokens":3578,"usd":0.069977,"stage2_stop_reason":"end_turn"},"total_usd":0.103888,"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\": 2009,\n      \"finding\": \"RhoBTB3 binds directly to Rab9 GTPase and functions with Rab9 in protein transport from endosomes to the trans-Golgi network. Biochemical analysis revealed that RhoBTB3 binds and hydrolyzes ATP (not GTP), and Rab9 binding opens the autoinhibited RhoBTB3 protein to permit maximal ATP hydrolysis. RhoBTB3 also interacts with TIP47 on membranes, suggesting a role in releasing this cargo selection protein from vesicles for efficient docking and fusion at the Golgi. Gene replacement experiments showed that both the N-terminal Rho-related domain and C-terminal sequences (important for Rab9 interaction) are required for function.\",\n      \"method\": \"Direct binding assays, biochemical ATPase activity assays, co-immunoprecipitation, gene replacement experiments in cultured cells\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods: direct biochemical ATPase assay, binding assays, domain-replacement genetics, and interaction studies in a single rigorous study\",\n      \"pmids\": [\"19490898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RhoBTB3 is a Golgi-associated ATPase that targets cyclin E for ubiquitylation as part of a CUL3-dependent RING-E3 ubiquitin ligase complex (RhoBTB3–CUL3–RBX1) at the Golgi. Depletion of RhoBTB3 arrests cells in S phase, triggers Golgi fragmentation, and elevates cyclin E levels. Golgi association of the complex is required for its ability to catalyze cyclin E ubiquitylation and allow normal S/G2 cell cycle progression.\",\n      \"method\": \"RNAi depletion, co-immunoprecipitation, ubiquitylation assays, cell cycle analysis, Golgi localization experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ubiquitylation assay, defined cellular phenotype upon KD, Golgi localization tied to functional consequence, multiple orthogonal methods\",\n      \"pmids\": [\"24145166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RHOBTB3 directly interacts with the prolyl hydroxylase PHD2 to promote HIFα hydroxylation, and directly interacts with VHL (an E3 ubiquitin ligase complex component) to facilitate HIFα ubiquitination. RHOBTB3 dimerizes with LIMD1 and assembles a RHOBTB3/LIMD1–PHD2–VHL–HIFα complex for maximal HIFα degradation. Hypoxia reduces this complex formation, causing HIFα accumulation. RHOBTB3 deficiency elevates the Warburg effect and accelerates xenograft tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, hydroxylation assays, RNAi knockdown, xenograft experiments\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, hydroxylation and ubiquitination assays, in vivo xenograft validation, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"26215701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RhoBTB3 interacts with the 5-HT7a serotonin receptor (binding involves both the C-terminal tail and the third intracellular loop of the receptor), co-localizes at the plasma membrane and ER, and inhibits proteasomal degradation of the 5-HT7a receptor. Notably, although RhoBTB3 interacts with CUL3, it does not recruit CUL3/ROC1 to the 5-HT7a receptor and does not mediate receptor ubiquitination.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation in HEK293T cells, domain mapping, immunofluorescence microscopy, proteasome inhibition assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid confirmed by Co-IP and localization, functional degradation assay, single lab with multiple methods\",\n      \"pmids\": [\"22245496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Depletion of RhoBTB3 affects Golgi complex morphology and causes changes in the trafficking speeds of membrane carriers operating at the Golgi–ER interface. RhoBTB3 was found to be present on these Golgi–ER carriers, indicating a direct role in this trafficking step.\",\n      \"method\": \"RNA interference, high-content image-based screening, live-cell imaging of carrier trafficking, fluorescence microscopy\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — RNAi phenotype with live imaging of carriers and localization data, single lab, multiple imaging-based methods\",\n      \"pmids\": [\"32354068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RhoBTB3 forms a complex with SHIP164 and the retromer subunit Vps26B at Golgi–early endosome (EE) contact sites to promote EE bud formation. Vps26B acts as a novel Rab14 effector, and Rab14 activity regulates SHIP164 association with EEs. Suppression of RhoBTB3 phenocopies SHIP164 depletion (enlarged Rab14+ EEs without buds), revealing a lipid-transfer-dependent pathway for EE budding.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, fluorescence microscopy, rescue experiments with lipid-transfer-defective mutants\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, functional rescue with domain mutants, live imaging, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38565878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RhoBTB3 negatively regulates autophagy by acting as an E3 ubiquitin ligase that ubiquitinates AMBRA1 (at K45 via K27-linked ubiquitin chains), promoting its proteasome-mediated degradation. RhoBTB3 deficiency induces autophagy and elevates AMBRA1 protein levels (without affecting AMBRA1 mRNA), while RhoBTB3 overexpression inhibits autophagy induction. AMBRA1 knockdown blocks RhoBTB3-depletion-induced autophagy.\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, ubiquitination assays with site-specific mutants (K45), proteasome inhibition assays, RNAi knockdown, overexpression\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS identification of substrate, site-specific ubiquitination mapped, epistasis by AMBRA1 knockdown rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"39404422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Diosmetin reduces formation of the RhoBTB3–PHD2 complex in UV-irradiated keratinocytes, thereby limiting HIF-1α hydroxylation and ubiquitination, and stabilizing HIF-1α. Overexpression of RhoBTB3 in mice enriches RhoBTB3, PHD2, and HIF-1α in the epidermis, consistent with RhoBTB3 promoting PHD2-mediated HIF-1α hydroxylation in skin.\",\n      \"method\": \"Co-immunoprecipitation, RNA sequencing, rAAV-mediated overexpression in mice, immunohistochemistry, in vivo UV sunburn model\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP confirmed RhoBTB3–PHD2 complex, in vivo overexpression model, corroborates prior mechanistic data (PMID 26215701), single lab\",\n      \"pmids\": [\"40311591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Genetic deletion of RhoBTB3 in mice leads to increased alpha-granule secretion in platelets in response to thrombin, CRP, and U46619/ADP, and reduces platelet accrual on collagen under flow conditions, without affecting platelet count, granule numbers, surface receptors, aggregation, dense granule secretion, or tail bleeding time.\",\n      \"method\": \"Knockout mouse model, flow cytometry, aggregometry, granule secretion assays, collagen adhesion under flow\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined platelet functional phenotype, single lab, single study\",\n      \"pmids\": [\"30754723\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RhoBTB3 is an atypical Rho-family ATPase (not a GTPase) that localizes to the Golgi and endosomes, where it functions in multiple membrane trafficking and protein degradation pathways: it binds Rab9 (with Rab9 relieving autoinhibition to stimulate ATP hydrolysis) to mediate endosome-to-TGN transport; it acts as a CUL3-RBX1 E3 ubiquitin ligase scaffold at the Golgi to ubiquitinate cyclin E for cell cycle-dependent degradation; it assembles a RHOBTB3–SHIP164–Vps26B complex at Golgi–endosome contacts to drive early endosome budding; it scaffolds a RHOBTB3/LIMD1–PHD2–VHL complex to promote HIFα hydroxylation and ubiquitin-proteasomal degradation; and it ubiquitinates AMBRA1 via K27-linked chains at K45 to suppress autophagy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RHOBTB3 is an atypical Rho-family ATPase that hydrolyzes ATP rather than GTP and acts as a Golgi- and endosome-associated organizer of membrane trafficking and substrate-targeted protein degradation [#0]. In its founding role it binds the Rab9 GTPase—which relieves RHOBTB3 autoinhibition to permit maximal ATP hydrolysis—and engages TIP47 on membranes to mediate cargo transport from endosomes to the trans-Golgi network, a function requiring both its N-terminal Rho-related domain and C-terminal Rab9-interaction sequences [#0]. RHOBTB3 also serves as a substrate-recognition scaffold for CUL3-RBX1 RING E3 ubiquitin ligase activity at the Golgi, ubiquitylating cyclin E to drive its degradation and enable normal S/G2 cell cycle progression; its loss arrests cells in S phase and fragments the Golgi [#1]. Beyond cyclin E, RHOBTB3 ubiquitinates AMBRA1 through K27-linked chains at K45 to promote its proteasomal degradation and thereby suppress autophagy [#6], and it scaffolds a RHOBTB3/LIMD1–PHD2–VHL complex that promotes HIFα prolyl hydroxylation and ubiquitin-proteasomal degradation, restraining the Warburg effect and tumor growth [#2, #7]. Its trafficking activities extend to assembly of a RHOBTB3–SHIP164–Vps26B complex at Golgi–early endosome contact sites that drives lipid-transfer-dependent endosome bud formation [#5] and to maintenance of Golgi morphology and Golgi–ER carrier trafficking [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established the biochemical identity of RhoBTB3 as an ATP-hydrolyzing (not GTP-hydrolyzing) Rho-family protein and placed it in endosome-to-TGN transport, answering what kind of enzyme it is and what regulates it.\",\n      \"evidence\": \"Direct binding and ATPase assays, co-IP, and domain-replacement genetics in cultured cells showing Rab9 binding relieves autoinhibition and TIP47 interaction on membranes\",\n      \"pmids\": [\"19490898\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the autoinhibited-to-open transition not resolved\", \"How ATP hydrolysis is coupled mechanically to cargo release at the Golgi not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Tested whether RhoBTB3's CUL3 association extends to all binding partners by examining the 5-HT7a serotonin receptor, showing it stabilizes the receptor without ubiquitinating it—revealing CUL3-independent functions.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, domain mapping, and proteasome-inhibition assays in HEK293T cells\",\n      \"pmids\": [\"22245496\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which RhoBTB3 protects 5-HT7a from proteasomal degradation unknown\", \"Physiological context of the interaction not established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined RhoBTB3 as the substrate-recognition subunit of a Golgi-localized CUL3-RBX1 E3 ligase targeting cyclin E, linking its trafficking compartment to cell cycle control.\",\n      \"evidence\": \"RNAi depletion, reciprocal co-IP, ubiquitylation assays, cell cycle analysis, and Golgi localization experiments\",\n      \"pmids\": [\"24145166\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why a Golgi-localized ligase targets a nuclear/cytoplasmic cyclin not mechanistically resolved\", \"Whether ATPase activity is required for ligase function not established\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended RhoBTB3 into oxygen sensing by showing it scaffolds PHD2 and VHL with LIMD1 to maximize HIFα hydroxylation and degradation, defining a tumor-suppressive metabolic role.\",\n      \"evidence\": \"Co-IP, hydroxylation and ubiquitination assays, RNAi knockdown, and xenograft experiments\",\n      \"pmids\": [\"26215701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How hypoxia disassembles the complex molecularly not defined\", \"Relationship between this scaffolding role and ATPase activity unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Addressed the in vivo organismal function via knockout mice, revealing a role in platelet alpha-granule secretion and collagen-dependent thrombus formation distinct from its trafficking phenotypes.\",\n      \"evidence\": \"Knockout mouse model with flow cytometry, aggregometry, granule secretion assays, and collagen adhesion under flow\",\n      \"pmids\": [\"30754723\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between RhoBTB3 and granule secretion machinery not identified\", \"Whether the platelet phenotype reflects trafficking or ligase functions unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved RhoBTB3's direct presence on Golgi–ER carriers and its requirement for normal carrier trafficking and Golgi morphology, reinforcing a direct trafficking role at the Golgi interface.\",\n      \"evidence\": \"RNAi, high-content image-based screening, and live-cell imaging of carrier trafficking\",\n      \"pmids\": [\"32354068\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular cargo carried by these carriers not defined\", \"Whether RhoBTB3 ATPase cycle drives carrier dynamics not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a RHOBTB3–SHIP164–Vps26B complex at Golgi–early endosome contacts driving lipid-transfer-dependent endosome budding, and a separate E3 ligase activity toward AMBRA1 that suppresses autophagy.\",\n      \"evidence\": \"Co-IP, RNAi, fluorescence/live imaging, rescue with lipid-transfer-defective and K45 ubiquitination-site mutants, and IP-MS substrate identification\",\n      \"pmids\": [\"38565878\", \"39404422\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How RHOBTB3 coordinates contact-site scaffolding with bud formation not resolved\", \"Whether the AMBRA1 ligase activity uses CUL3 not specified\", \"Relationship between the trafficking and autophagy roles unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Corroborated the RhoBTB3–PHD2–HIF-1α axis in skin in vivo and showed it is pharmacologically targetable, with diosmetin reducing complex formation to stabilize HIF-1α.\",\n      \"evidence\": \"Co-IP, RNA-seq, rAAV overexpression in mice, immunohistochemistry, and a UV sunburn model\",\n      \"pmids\": [\"40311591\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding site of diosmetin and whether it acts on RhoBTB3 or PHD2 not established\", \"Tissue specificity of the skin phenotype not explained\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RhoBTB3's single ATPase scaffold protein selects among its diverse functions—endosome-TGN transport, CUL3 ligase activity toward distinct substrates, contact-site lipid-transfer scaffolding, and HIF degradation—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model integrating the ATPase cycle with substrate/partner selection\", \"Determinants directing RhoBTB3 to one complex versus another not identified\", \"Whether ATP hydrolysis is required across all of its roles untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 6]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1, 4, 5]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\n      \"RHOBTB3–CUL3–RBX1 E3 ubiquitin ligase\",\n      \"RHOBTB3/LIMD1–PHD2–VHL–HIFα complex\",\n      \"RHOBTB3–SHIP164–Vps26B complex\"\n    ],\n    \"partners\": [\n      \"RAB9A\",\n      \"CUL3\",\n      \"RBX1\",\n      \"PHD2\",\n      \"VHL\",\n      \"LIMD1\",\n      \"AMBRA1\",\n      \"SHIP164\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}