{"gene":"CHMP6","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2005,"finding":"CHMP6 is N-myristoylated at its N-terminus, localizes to endosomal membranes in the perinuclear area, directly interacts with ESCRT-II component EAP20/Vps25 and ESCRT-III component CHMP4b/Snf7, with interactions mediated by the N-terminal basic half of CHMP6; overexpression blocks cargo sorting, causing accumulation of transferrin receptors, ubiquitinated proteins, and endocytosed EGF in cytoplasmic compartments, suggesting CHMP6 acts as an acceptor for ESCRT-II on endosomal membranes.","method":"Metabolic labelling with [3H]myristate, co-immunoprecipitation of epitope-tagged proteins, in vitro pull-down with recombinant proteins, fluorescence microscopy, LBPA co-localization, cell surface transferrin receptor assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro reconstitution with recombinant proteins plus multiple orthogonal cell-based assays, moderate evidence","pmids":["15511219"],"is_preprint":false},{"year":2004,"finding":"Human Vps20 (CHMP6) shows an endosomal membrane-staining pattern and co-expression with hSnf7-1 disperses large Snf7-staining vesicles; overexpression of both induces a post-endosomal defect in cholesterol sorting.","method":"Immunofluorescence microscopy, co-expression experiments in mammalian cells","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 3 — localization and co-expression with functional phenotype, single lab","pmids":["14583093"],"is_preprint":false},{"year":2006,"finding":"The C-terminal domain of ESCRT-I subunit Vps28 (Vps28-CTD) directly interacts with ESCRT-III subunit Vps20 (CHMP6 ortholog) through a conserved surface; mutagenesis of this surface abolishes Vps20 interaction in vitro and prevents rescue of EIAV Gag late domain mutant budding, indicating Vps28 recruits Vps20/ESCRT-III downstream.","method":"Crystal structure of Vps28-CTD, mutagenesis, in vitro binding assays, viral budding rescue assay","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis plus functional viral budding assay in single study","pmids":["16749904"],"is_preprint":false},{"year":2011,"finding":"Purified ESCRT-II binds to Vps20 (CHMP6 ortholog) on membranes in a curvature-dependent manner; this complex nucleates Vps32/Snf7 filaments that polymerize along highly curved membranes as a single string of monomers and modulate membrane dynamics in vitro, providing spatial regulation of ESCRT-III assembly.","method":"Liposome co-flotation assays, fluorescence-based liposome interaction studies, high-resolution atomic force microscopy, in vitro reconstitution","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified proteins and multiple biophysical readouts","pmids":["21835927"],"is_preprint":false},{"year":2013,"finding":"Yeast Vps20 (CHMP6 ortholog) contains a MIM1-like sequence that directly binds the MIT domain at the N-terminus of Doa4 ubiquitin hydrolase; this interaction restricts a non-catalytic function of Doa4 that promotes ILV formation, and disrupting the interaction rescues ILV budding in bro1Δ cells.","method":"Direct binding assay with recombinant proteins, yeast genetics (bro1Δ epistasis), ILV biogenesis assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 — direct binding reconstituted plus genetic epistasis with defined phenotype","pmids":["23444383"],"is_preprint":false},{"year":2014,"finding":"CHMP6 and ESCRT-II form highly ordered structures at the intercellular bridge during cytokinetic abscission; a truncated CHMP6 N-terminal fragment (CHMP6-N, first 52 aa) localizes to the intercellular bridge, blocks abscission, and leads to cell death; a mutation preventing CHMP6-N binding to ESCRT-II abolishes this phenotype, and deletion of the first 10 aa of CHMP6-N prevents abscission failure without affecting bridge localization.","method":"High-resolution imaging, expression of truncated CHMP6 constructs, site-directed mutagenesis, live-cell abscission assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — high-resolution imaging with structure-function mutagenesis and defined cytokinesis phenotype","pmids":["25232011"],"is_preprint":false},{"year":2012,"finding":"CHMP6 directly binds H-Ras and N-Ras (but not K-Ras) in endosomes, with binding favored when H-Ras has a functional effector-binding loop, is GTP-bound, and ubiquitylated; repressing CHMP6 blocks Ras recycling to the plasma membrane and EGFR recycling, impairing Ras-induced transformation.","method":"cDNA library screen, direct binding assay, cell fractionation, photobleaching (FRAP), RNAi knockdown with transformation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding plus multiple cell-based assays (fractionation, FRAP, knockdown phenotype), single lab","pmids":["22231449"],"is_preprint":false},{"year":2015,"finding":"C. elegans VPS-20 (CHMP6 ortholog) adopts an open extended conformation in solution — unlike the closed auto-inhibited conformation of VPS-24 — and interacts directly with ESCRT-II both in cytosolic extracts and with recombinant proteins in vitro, indicating VPS-20 does not require membrane-associated ESCRT-II for activation.","method":"In vitro binding with recombinant proteins, cytosolic extract co-immunoprecipitation, biophysical conformational analysis","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro reconstitution with purified proteins, single lab, C. elegans ortholog","pmids":["25588614"],"is_preprint":false},{"year":2022,"finding":"In yeast, Bro1 directly binds the Vps20 (CHMP6 ortholog) subunit of ESCRT-III; this interaction suppresses the ability of Vps20 to antagonize Doa4 binding to Snf7, placing Bro1 as a regulator that relieves Vps20-mediated restriction of Doa4 at ESCRT-III.","method":"Direct binding assay (recombinant proteins), yeast genetics with constitutively Doa4-binding Vps20 mutant allele","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding plus genetic epistasis, single lab","pmids":["34908216"],"is_preprint":false},{"year":2022,"finding":"The N-terminal myristoylation sequence of CHMP6 (Myr(CHMP6)) is sufficient to increase small extracellular vesicle (sEV) production when overexpressed, and N-myristoylation alone is necessary but not sufficient for efficient protein packaging into sEVs, indicating additional sequences beyond the myristoylation motif contribute to CHMP6-mediated EV biogenesis.","method":"NanoGlo luciferase assay, nanoparticle tracking analysis, transmission electron microscopy, Western blotting, fusion protein expression","journal":"Bioengineered","confidence":"Low","confidence_rationale":"Tier 3 — functional overexpression assay without identification of direct molecular partner, single lab","pmids":["35188876"],"is_preprint":false},{"year":2025,"finding":"CEP55 binds directly to CHMP6 and promotes its expression; this interaction facilitates ferroptosis inhibition and malignant progression of triple-negative breast cancer cells.","method":"Co-immunoprecipitation (co-IP), knockdown/overexpression with ferroptosis markers (Fe2+, MDA, GSH, ROS), xenograft tumor model","journal":"Clinical breast cancer","confidence":"Low","confidence_rationale":"Tier 3 — single co-IP with functional knockdown but no direct mechanistic dissection of how CEP55-CHMP6 interaction modulates ESCRT function","pmids":["40925844"],"is_preprint":false}],"current_model":"CHMP6 (human Vps20) is an N-myristoylated ESCRT-III subunit that localizes to endosomal membranes where its N-terminal domain directly recruits ESCRT-II (via EAP20/Vps25 and the Vps28 C-terminal domain) and nucleates ESCRT-III filaments (via CHMP4b/Snf7) in a membrane-curvature-dependent manner to drive intralumenal vesicle formation, endosomal cargo sorting, EGFR/Ras recycling to the plasma membrane, and cytokinetic abscission, while its interaction with Doa4/Bro1 at Vps20 provides an additional regulatory layer controlling ESCRT-III disassembly kinetics."},"narrative":{"teleology":[{"year":2004,"claim":"Establishing that human Vps20/CHMP6 localizes to endosomes and functionally cooperates with Snf7-1 answered the basic question of where this ESCRT-III subunit acts and provided the first evidence it participates in post-endosomal sorting.","evidence":"Immunofluorescence and co-expression experiments in mammalian cells showing endosomal staining and cholesterol sorting defects","pmids":["14583093"],"confidence":"Medium","gaps":["No direct binding partners identified","Mechanism of cholesterol sorting defect unexplored"]},{"year":2005,"claim":"Identifying CHMP6 as N-myristoylated and showing it directly bridges ESCRT-II (via EAP20) to ESCRT-III (via CHMP4b) through its N-terminal basic domain defined CHMP6 as the critical adaptor linking ESCRT-II to ESCRT-III on endosomal membranes.","evidence":"Metabolic [3H]myristate labelling, co-immunoprecipitation, in vitro pull-down with recombinant proteins, and cargo sorting assays in mammalian cells","pmids":["15511219"],"confidence":"High","gaps":["Structural basis of the EAP20–CHMP6 interface not resolved","Contribution of myristoylation to membrane targeting versus protein–protein interaction not separated"]},{"year":2006,"claim":"Crystallography of the Vps28 C-terminal domain and mutagenesis revealed a second, ESCRT-I–mediated route for Vps20/CHMP6 recruitment, and functional assays showed this interface is required for ESCRT-dependent viral budding.","evidence":"Crystal structure of Vps28-CTD, mutagenesis abolishing Vps20 binding in vitro, and EIAV Gag late-domain budding rescue assay","pmids":["16749904"],"confidence":"High","gaps":["Relative contributions of ESCRT-I versus ESCRT-II recruitment of CHMP6 in physiological MVB biogenesis not determined"]},{"year":2011,"claim":"Reconstitution on liposomes demonstrated that ESCRT-II–Vps20 binding is membrane-curvature dependent and that this complex nucleates single-filament Snf7 polymers, establishing the mechanistic basis for spatially restricted ESCRT-III assembly.","evidence":"Liposome co-flotation, fluorescence-based binding, and high-resolution atomic force microscopy with purified yeast proteins","pmids":["21835927"],"confidence":"High","gaps":["Filament architecture at endogenous protein concentrations unknown","Whether mammalian CHMP6–CHMP4b polymerization follows identical curvature dependence not tested"]},{"year":2012,"claim":"Discovery that CHMP6 selectively binds GTP-loaded, ubiquitylated H-Ras and N-Ras on endosomes and that CHMP6 knockdown blocks Ras and EGFR recycling to the plasma membrane expanded CHMP6 function beyond ILV formation to signaling-relevant receptor and GTPase recycling.","evidence":"cDNA library screen, direct binding assay, FRAP, cell fractionation, RNAi with transformation assay","pmids":["22231449"],"confidence":"Medium","gaps":["Structural basis of CHMP6–Ras interaction not defined","Whether CHMP6–Ras binding is direct on native endosomes or requires additional adaptors not resolved","Single-lab finding"]},{"year":2013,"claim":"Identification of a MIM1-like motif in Vps20 that binds the Doa4 MIT domain, restricting Doa4's non-catalytic ILV-promoting function, revealed an unexpected regulatory role for Vps20 beyond filament nucleation.","evidence":"Recombinant protein binding assay and yeast genetic epistasis (bro1Δ rescue) with ILV biogenesis readout","pmids":["23444383"],"confidence":"High","gaps":["Whether mammalian CHMP6 similarly regulates a deubiquitinase untested","Molecular details of Doa4's non-catalytic ILV function unclear"]},{"year":2014,"claim":"Demonstrating that CHMP6 and ESCRT-II form ordered structures at the intercellular bridge and that a dominant-negative CHMP6 N-terminal fragment blocks abscission in an ESCRT-II-binding-dependent manner established a direct role for CHMP6 in cytokinesis.","evidence":"High-resolution imaging, truncation and point mutagenesis of CHMP6-N (first 52 aa), live-cell abscission assay","pmids":["25232011"],"confidence":"High","gaps":["Mechanism by which the first 10 residues of CHMP6 contribute to abscission function beyond bridge localization not elucidated"]},{"year":2015,"claim":"Biophysical analysis of C. elegans VPS-20 showed it adopts an open, extended conformation in solution — unlike VPS-24 — indicating CHMP6/Vps20 is constitutively competent for ESCRT-II engagement without requiring membrane-dependent activation.","evidence":"Recombinant protein binding and biophysical conformational analysis of C. elegans VPS-20","pmids":["25588614"],"confidence":"Medium","gaps":["Whether mammalian CHMP6 shares this open conformation not directly tested","Single-lab study on ortholog"]},{"year":2022,"claim":"Showing that Bro1 binds Vps20 and relieves Vps20-mediated suppression of Doa4–Snf7 interaction completed a regulatory circuit in which Bro1 coordinates deubiquitinase engagement with ESCRT-III disassembly.","evidence":"Recombinant protein binding assay, yeast genetics with constitutively Doa4-binding Vps20 mutant","pmids":["34908216"],"confidence":"Medium","gaps":["Kinetics of Bro1-mediated relief in vivo not measured","Mammalian ALIX–CHMP6 regulatory axis not validated"]},{"year":null,"claim":"Key unresolved questions include the high-resolution structure of the CHMP6–ESCRT-II interface in human, whether CHMP6's Ras-binding function is mechanistically coupled to ESCRT-III filament dynamics, and the physiological relevance of CHMP6 in extracellular vesicle biogenesis beyond overexpression systems.","evidence":"","pmids":[],"confidence":"Low","gaps":["No atomic-resolution structure of human CHMP6 in complex with ESCRT-II","CHMP6–Ras interaction awaits structural and independent validation","Role in EV biogenesis demonstrated only by overexpression of myristoylation peptide"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,5]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[3,5]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,9]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6]}],"complexes":["ESCRT-III"],"partners":["EAP20","CHMP4B","VPS28","HRAS","NRAS","BRO1","DOA4","CEP55"],"other_free_text":[]},"mechanistic_narrative":"CHMP6 (also known as human Vps20) is an N-myristoylated ESCRT-III subunit that bridges ESCRT-II to ESCRT-III on endosomal membranes, nucleating Snf7/CHMP4b filament assembly to drive intralumenal vesicle formation, cargo sorting, and cytokinetic abscission. CHMP6 is recruited to membranes via N-terminal myristoylation and directly engages ESCRT-II through both the EAP20/Vps25 subunit and the Vps28 C-terminal domain, with ESCRT-II–Vps20 binding enhanced on curved membranes to provide spatial regulation of ESCRT-III polymerization [PMID:15511219, PMID:16749904, PMID:21835927]. At the intercellular bridge during cytokinesis, CHMP6 and ESCRT-II form ordered assemblies whose disruption by dominant-negative CHMP6 fragments blocks abscission, and on endosomes CHMP6 additionally binds H-Ras and N-Ras to facilitate their recycling to the plasma membrane [PMID:25232011, PMID:22231449]. The Bro1/ALIX–Vps20 interaction modulates Doa4 deubiquitinase access to Snf7, establishing a regulatory checkpoint that coordinates ubiquitin recycling with ESCRT-III disassembly [PMID:23444383, PMID:34908216]."},"prefetch_data":{"uniprot":{"accession":"Q96FZ7","full_name":"Charged multivesicular body protein 6","aliases":["Chromatin-modifying protein 6","Vacuolar protein sorting-associated protein 20","Vps20","hVps20"],"length_aa":201,"mass_kda":23.5,"function":"Probable core component of the endosomal sorting required for transport complex III (ESCRT-III) which is involved in multivesicular bodies (MVBs) formation and sorting of endosomal cargo proteins into MVBs. MVBs contain intraluminal vesicles (ILVs) that are generated by invagination and scission from the limiting membrane of the endosome and mostly are delivered to lysosomes enabling degradation of membrane proteins, such as stimulated growth factor receptors, lysosomal enzymes and lipids. The MVB pathway appears to require the sequential function of ESCRT-O, -I,-II and -III complexes. ESCRT-III proteins mostly dissociate from the invaginating membrane before the ILV is released. The ESCRT machinery also functions in topologically equivalent membrane fission events, such as the terminal stages of cytokinesis and the budding of enveloped viruses (HIV-1 and other lentiviruses). ESCRT-III proteins are believed to mediate the necessary vesicle extrusion and/or membrane fission activities, possibly in conjunction with the AAA ATPase VPS4. In the ESCRT-III complex, it probably serves as an acceptor for the ESCRT-II complex on endosomal membranes","subcellular_location":"Endomembrane system; Endosome membrane; Late endosome membrane; Membrane","url":"https://www.uniprot.org/uniprotkb/Q96FZ7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CHMP6","classification":"Common Essential","n_dependent_lines":1207,"n_total_lines":1208,"dependency_fraction":0.9991721854304636},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000176108","cell_line_id":"CID000068","localizations":[{"compartment":"membrane","grade":3},{"compartment":"vesicles","grade":3},{"compartment":"er","grade":2}],"interactors":[],"url":"https://opencell.sf.czbiohub.org/target/CID000068","total_profiled":1310},"omim":[{"mim_id":"610907","title":"VACUOLAR PROTEIN SORTING 25 HOMOLOG; VPS25","url":"https://www.omim.org/entry/610907"},{"mim_id":"610901","title":"CHARGED MULTIVESICULAR BODY PROTEIN 6; CHMP6","url":"https://www.omim.org/entry/610901"},{"mim_id":"610897","title":"CHARGED MULTIVESICULAR BODY PROTEIN 4B; CHMP4B","url":"https://www.omim.org/entry/610897"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CHMP6"},"hgnc":{"alias_symbol":["FLJ11749","VPS20"],"prev_symbol":[]},"alphafold":{"accession":"Q96FZ7","domains":[{"cath_id":"1.20.120","chopping":"14-98","consensus_level":"high","plddt":94.4391,"start":14,"end":98},{"cath_id":"1.20.5","chopping":"119-165","consensus_level":"high","plddt":84.1732,"start":119,"end":165}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96FZ7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96FZ7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96FZ7-F1-predicted_aligned_error_v6.png","plddt_mean":81.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CHMP6","jax_strain_url":"https://www.jax.org/strain/search?query=CHMP6"},"sequence":{"accession":"Q96FZ7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96FZ7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96FZ7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96FZ7"}},"corpus_meta":[{"pmid":"15511219","id":"PMC_15511219","title":"Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting.","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15511219","citation_count":103,"is_preprint":false},{"pmid":"21835927","id":"PMC_21835927","title":"Association of the endosomal sorting complex ESCRT-II with the Vps20 subunit of ESCRT-III generates a curvature-sensitive complex capable of nucleating ESCRT-III filaments.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21835927","citation_count":77,"is_preprint":false},{"pmid":"14583093","id":"PMC_14583093","title":"Structure and function of human Vps20 and Snf7 proteins.","date":"2004","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/14583093","citation_count":57,"is_preprint":false},{"pmid":"16749904","id":"PMC_16749904","title":"The crystal structure of the C-terminal domain of Vps28 reveals a conserved surface required for Vps20 recruitment.","date":"2006","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/16749904","citation_count":51,"is_preprint":false},{"pmid":"25232011","id":"PMC_25232011","title":"Inhibition of ESCRT-II-CHMP6 interactions impedes cytokinetic abscission and leads to cell death.","date":"2014","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/25232011","citation_count":48,"is_preprint":false},{"pmid":"22231449","id":"PMC_22231449","title":"CHMP6 and VPS4A mediate the recycling of Ras to the plasma membrane to promote growth factor signaling.","date":"2012","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/22231449","citation_count":28,"is_preprint":false},{"pmid":"23444383","id":"PMC_23444383","title":"Doa4 function in ILV budding is restricted through its interaction with the Vps20 subunit of ESCRT-III.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23444383","citation_count":20,"is_preprint":false},{"pmid":"25588614","id":"PMC_25588614","title":"The VPS-20 subunit of the endosomal sorting complex ESCRT-III exhibits an open conformation in the absence of upstream activation.","date":"2015","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/25588614","citation_count":19,"is_preprint":false},{"pmid":"35188876","id":"PMC_35188876","title":"A peptide derived from the N-terminus of charged multivesicular body protein 6 (CHMP6) promotes the secretion of gene editing proteins via small extracellular vesicle production.","date":"2022","source":"Bioengineered","url":"https://pubmed.ncbi.nlm.nih.gov/35188876","citation_count":9,"is_preprint":false},{"pmid":"19270365","id":"PMC_19270365","title":"Overexpression of CHMP6 induces cellular oncosis and apoptosis in HeLa cells.","date":"2009","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19270365","citation_count":9,"is_preprint":false},{"pmid":"34908216","id":"PMC_34908216","title":"Bro1 binds the Vps20 subunit of ESCRT-III and promotes ESCRT-III regulation by Doa4.","date":"2022","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/34908216","citation_count":6,"is_preprint":false},{"pmid":"40691598","id":"PMC_40691598","title":"Ferroptosis-disulfidptosis-related CHMP6 is a clinico-immune target in colorectal cancer.","date":"2025","source":"Biology direct","url":"https://pubmed.ncbi.nlm.nih.gov/40691598","citation_count":2,"is_preprint":false},{"pmid":"39305302","id":"PMC_39305302","title":"ELK4 targets CHMP6 to inhibit ferroptosis and enhance malignant properties of skin cutaneous melanoma cells.","date":"2024","source":"Archives of dermatological research","url":"https://pubmed.ncbi.nlm.nih.gov/39305302","citation_count":2,"is_preprint":false},{"pmid":"40925844","id":"PMC_40925844","title":"The USP8/CEP55/CHMP6 Axis Orchestrates Triple-Negative Breast Cancer Progression by Regulating Ferroptosis and Macrophage M2 Polarization.","date":"2025","source":"Clinical breast cancer","url":"https://pubmed.ncbi.nlm.nih.gov/40925844","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.06.26.600760","title":"The ataxia-telangiectasia disease protein ATM controls vesicular protein secretion via CHGA and microtubule dynamics via CRMP5","date":"2024-06-26","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.26.600760","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9088,"output_tokens":2812,"usd":0.034722},"stage2":{"model":"claude-opus-4-6","input_tokens":6149,"output_tokens":2683,"usd":0.14673},"total_usd":0.181452,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"CHMP6 is N-myristoylated at its N-terminus, localizes to endosomal membranes in the perinuclear area, directly interacts with ESCRT-II component EAP20/Vps25 and ESCRT-III component CHMP4b/Snf7, with interactions mediated by the N-terminal basic half of CHMP6; overexpression blocks cargo sorting, causing accumulation of transferrin receptors, ubiquitinated proteins, and endocytosed EGF in cytoplasmic compartments, suggesting CHMP6 acts as an acceptor for ESCRT-II on endosomal membranes.\",\n      \"method\": \"Metabolic labelling with [3H]myristate, co-immunoprecipitation of epitope-tagged proteins, in vitro pull-down with recombinant proteins, fluorescence microscopy, LBPA co-localization, cell surface transferrin receptor assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution with recombinant proteins plus multiple orthogonal cell-based assays, moderate evidence\",\n      \"pmids\": [\"15511219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human Vps20 (CHMP6) shows an endosomal membrane-staining pattern and co-expression with hSnf7-1 disperses large Snf7-staining vesicles; overexpression of both induces a post-endosomal defect in cholesterol sorting.\",\n      \"method\": \"Immunofluorescence microscopy, co-expression experiments in mammalian cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization and co-expression with functional phenotype, single lab\",\n      \"pmids\": [\"14583093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The C-terminal domain of ESCRT-I subunit Vps28 (Vps28-CTD) directly interacts with ESCRT-III subunit Vps20 (CHMP6 ortholog) through a conserved surface; mutagenesis of this surface abolishes Vps20 interaction in vitro and prevents rescue of EIAV Gag late domain mutant budding, indicating Vps28 recruits Vps20/ESCRT-III downstream.\",\n      \"method\": \"Crystal structure of Vps28-CTD, mutagenesis, in vitro binding assays, viral budding rescue assay\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis plus functional viral budding assay in single study\",\n      \"pmids\": [\"16749904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Purified ESCRT-II binds to Vps20 (CHMP6 ortholog) on membranes in a curvature-dependent manner; this complex nucleates Vps32/Snf7 filaments that polymerize along highly curved membranes as a single string of monomers and modulate membrane dynamics in vitro, providing spatial regulation of ESCRT-III assembly.\",\n      \"method\": \"Liposome co-flotation assays, fluorescence-based liposome interaction studies, high-resolution atomic force microscopy, in vitro reconstitution\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified proteins and multiple biophysical readouts\",\n      \"pmids\": [\"21835927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Yeast Vps20 (CHMP6 ortholog) contains a MIM1-like sequence that directly binds the MIT domain at the N-terminus of Doa4 ubiquitin hydrolase; this interaction restricts a non-catalytic function of Doa4 that promotes ILV formation, and disrupting the interaction rescues ILV budding in bro1Δ cells.\",\n      \"method\": \"Direct binding assay with recombinant proteins, yeast genetics (bro1Δ epistasis), ILV biogenesis assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct binding reconstituted plus genetic epistasis with defined phenotype\",\n      \"pmids\": [\"23444383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CHMP6 and ESCRT-II form highly ordered structures at the intercellular bridge during cytokinetic abscission; a truncated CHMP6 N-terminal fragment (CHMP6-N, first 52 aa) localizes to the intercellular bridge, blocks abscission, and leads to cell death; a mutation preventing CHMP6-N binding to ESCRT-II abolishes this phenotype, and deletion of the first 10 aa of CHMP6-N prevents abscission failure without affecting bridge localization.\",\n      \"method\": \"High-resolution imaging, expression of truncated CHMP6 constructs, site-directed mutagenesis, live-cell abscission assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — high-resolution imaging with structure-function mutagenesis and defined cytokinesis phenotype\",\n      \"pmids\": [\"25232011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CHMP6 directly binds H-Ras and N-Ras (but not K-Ras) in endosomes, with binding favored when H-Ras has a functional effector-binding loop, is GTP-bound, and ubiquitylated; repressing CHMP6 blocks Ras recycling to the plasma membrane and EGFR recycling, impairing Ras-induced transformation.\",\n      \"method\": \"cDNA library screen, direct binding assay, cell fractionation, photobleaching (FRAP), RNAi knockdown with transformation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding plus multiple cell-based assays (fractionation, FRAP, knockdown phenotype), single lab\",\n      \"pmids\": [\"22231449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"C. elegans VPS-20 (CHMP6 ortholog) adopts an open extended conformation in solution — unlike the closed auto-inhibited conformation of VPS-24 — and interacts directly with ESCRT-II both in cytosolic extracts and with recombinant proteins in vitro, indicating VPS-20 does not require membrane-associated ESCRT-II for activation.\",\n      \"method\": \"In vitro binding with recombinant proteins, cytosolic extract co-immunoprecipitation, biophysical conformational analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution with purified proteins, single lab, C. elegans ortholog\",\n      \"pmids\": [\"25588614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In yeast, Bro1 directly binds the Vps20 (CHMP6 ortholog) subunit of ESCRT-III; this interaction suppresses the ability of Vps20 to antagonize Doa4 binding to Snf7, placing Bro1 as a regulator that relieves Vps20-mediated restriction of Doa4 at ESCRT-III.\",\n      \"method\": \"Direct binding assay (recombinant proteins), yeast genetics with constitutively Doa4-binding Vps20 mutant allele\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding plus genetic epistasis, single lab\",\n      \"pmids\": [\"34908216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The N-terminal myristoylation sequence of CHMP6 (Myr(CHMP6)) is sufficient to increase small extracellular vesicle (sEV) production when overexpressed, and N-myristoylation alone is necessary but not sufficient for efficient protein packaging into sEVs, indicating additional sequences beyond the myristoylation motif contribute to CHMP6-mediated EV biogenesis.\",\n      \"method\": \"NanoGlo luciferase assay, nanoparticle tracking analysis, transmission electron microscopy, Western blotting, fusion protein expression\",\n      \"journal\": \"Bioengineered\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — functional overexpression assay without identification of direct molecular partner, single lab\",\n      \"pmids\": [\"35188876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CEP55 binds directly to CHMP6 and promotes its expression; this interaction facilitates ferroptosis inhibition and malignant progression of triple-negative breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation (co-IP), knockdown/overexpression with ferroptosis markers (Fe2+, MDA, GSH, ROS), xenograft tumor model\",\n      \"journal\": \"Clinical breast cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP with functional knockdown but no direct mechanistic dissection of how CEP55-CHMP6 interaction modulates ESCRT function\",\n      \"pmids\": [\"40925844\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CHMP6 (human Vps20) is an N-myristoylated ESCRT-III subunit that localizes to endosomal membranes where its N-terminal domain directly recruits ESCRT-II (via EAP20/Vps25 and the Vps28 C-terminal domain) and nucleates ESCRT-III filaments (via CHMP4b/Snf7) in a membrane-curvature-dependent manner to drive intralumenal vesicle formation, endosomal cargo sorting, EGFR/Ras recycling to the plasma membrane, and cytokinetic abscission, while its interaction with Doa4/Bro1 at Vps20 provides an additional regulatory layer controlling ESCRT-III disassembly kinetics.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CHMP6 (also known as human Vps20) is an N-myristoylated ESCRT-III subunit that bridges ESCRT-II to ESCRT-III on endosomal membranes, nucleating Snf7/CHMP4b filament assembly to drive intralumenal vesicle formation, cargo sorting, and cytokinetic abscission. CHMP6 is recruited to membranes via N-terminal myristoylation and directly engages ESCRT-II through both the EAP20/Vps25 subunit and the Vps28 C-terminal domain, with ESCRT-II–Vps20 binding enhanced on curved membranes to provide spatial regulation of ESCRT-III polymerization [PMID:15511219, PMID:16749904, PMID:21835927]. At the intercellular bridge during cytokinesis, CHMP6 and ESCRT-II form ordered assemblies whose disruption by dominant-negative CHMP6 fragments blocks abscission, and on endosomes CHMP6 additionally binds H-Ras and N-Ras to facilitate their recycling to the plasma membrane [PMID:25232011, PMID:22231449]. The Bro1/ALIX–Vps20 interaction modulates Doa4 deubiquitinase access to Snf7, establishing a regulatory checkpoint that coordinates ubiquitin recycling with ESCRT-III disassembly [PMID:23444383, PMID:34908216].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing that human Vps20/CHMP6 localizes to endosomes and functionally cooperates with Snf7-1 answered the basic question of where this ESCRT-III subunit acts and provided the first evidence it participates in post-endosomal sorting.\",\n      \"evidence\": \"Immunofluorescence and co-expression experiments in mammalian cells showing endosomal staining and cholesterol sorting defects\",\n      \"pmids\": [\"14583093\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct binding partners identified\", \"Mechanism of cholesterol sorting defect unexplored\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identifying CHMP6 as N-myristoylated and showing it directly bridges ESCRT-II (via EAP20) to ESCRT-III (via CHMP4b) through its N-terminal basic domain defined CHMP6 as the critical adaptor linking ESCRT-II to ESCRT-III on endosomal membranes.\",\n      \"evidence\": \"Metabolic [3H]myristate labelling, co-immunoprecipitation, in vitro pull-down with recombinant proteins, and cargo sorting assays in mammalian cells\",\n      \"pmids\": [\"15511219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the EAP20–CHMP6 interface not resolved\", \"Contribution of myristoylation to membrane targeting versus protein–protein interaction not separated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Crystallography of the Vps28 C-terminal domain and mutagenesis revealed a second, ESCRT-I–mediated route for Vps20/CHMP6 recruitment, and functional assays showed this interface is required for ESCRT-dependent viral budding.\",\n      \"evidence\": \"Crystal structure of Vps28-CTD, mutagenesis abolishing Vps20 binding in vitro, and EIAV Gag late-domain budding rescue assay\",\n      \"pmids\": [\"16749904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of ESCRT-I versus ESCRT-II recruitment of CHMP6 in physiological MVB biogenesis not determined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Reconstitution on liposomes demonstrated that ESCRT-II–Vps20 binding is membrane-curvature dependent and that this complex nucleates single-filament Snf7 polymers, establishing the mechanistic basis for spatially restricted ESCRT-III assembly.\",\n      \"evidence\": \"Liposome co-flotation, fluorescence-based binding, and high-resolution atomic force microscopy with purified yeast proteins\",\n      \"pmids\": [\"21835927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Filament architecture at endogenous protein concentrations unknown\", \"Whether mammalian CHMP6–CHMP4b polymerization follows identical curvature dependence not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that CHMP6 selectively binds GTP-loaded, ubiquitylated H-Ras and N-Ras on endosomes and that CHMP6 knockdown blocks Ras and EGFR recycling to the plasma membrane expanded CHMP6 function beyond ILV formation to signaling-relevant receptor and GTPase recycling.\",\n      \"evidence\": \"cDNA library screen, direct binding assay, FRAP, cell fractionation, RNAi with transformation assay\",\n      \"pmids\": [\"22231449\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of CHMP6–Ras interaction not defined\", \"Whether CHMP6–Ras binding is direct on native endosomes or requires additional adaptors not resolved\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of a MIM1-like motif in Vps20 that binds the Doa4 MIT domain, restricting Doa4's non-catalytic ILV-promoting function, revealed an unexpected regulatory role for Vps20 beyond filament nucleation.\",\n      \"evidence\": \"Recombinant protein binding assay and yeast genetic epistasis (bro1Δ rescue) with ILV biogenesis readout\",\n      \"pmids\": [\"23444383\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mammalian CHMP6 similarly regulates a deubiquitinase untested\", \"Molecular details of Doa4's non-catalytic ILV function unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating that CHMP6 and ESCRT-II form ordered structures at the intercellular bridge and that a dominant-negative CHMP6 N-terminal fragment blocks abscission in an ESCRT-II-binding-dependent manner established a direct role for CHMP6 in cytokinesis.\",\n      \"evidence\": \"High-resolution imaging, truncation and point mutagenesis of CHMP6-N (first 52 aa), live-cell abscission assay\",\n      \"pmids\": [\"25232011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which the first 10 residues of CHMP6 contribute to abscission function beyond bridge localization not elucidated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Biophysical analysis of C. elegans VPS-20 showed it adopts an open, extended conformation in solution — unlike VPS-24 — indicating CHMP6/Vps20 is constitutively competent for ESCRT-II engagement without requiring membrane-dependent activation.\",\n      \"evidence\": \"Recombinant protein binding and biophysical conformational analysis of C. elegans VPS-20\",\n      \"pmids\": [\"25588614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether mammalian CHMP6 shares this open conformation not directly tested\", \"Single-lab study on ortholog\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showing that Bro1 binds Vps20 and relieves Vps20-mediated suppression of Doa4–Snf7 interaction completed a regulatory circuit in which Bro1 coordinates deubiquitinase engagement with ESCRT-III disassembly.\",\n      \"evidence\": \"Recombinant protein binding assay, yeast genetics with constitutively Doa4-binding Vps20 mutant\",\n      \"pmids\": [\"34908216\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinetics of Bro1-mediated relief in vivo not measured\", \"Mammalian ALIX–CHMP6 regulatory axis not validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the high-resolution structure of the CHMP6–ESCRT-II interface in human, whether CHMP6's Ras-binding function is mechanistically coupled to ESCRT-III filament dynamics, and the physiological relevance of CHMP6 in extracellular vesicle biogenesis beyond overexpression systems.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No atomic-resolution structure of human CHMP6 in complex with ESCRT-II\", \"CHMP6–Ras interaction awaits structural and independent validation\", \"Role in EV biogenesis demonstrated only by overexpression of myristoylation peptide\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"ESCRT-III\"\n    ],\n    \"partners\": [\n      \"EAP20\",\n      \"CHMP4B\",\n      \"VPS28\",\n      \"HRAS\",\n      \"NRAS\",\n      \"BRO1\",\n      \"DOA4\",\n      \"CEP55\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}