{"gene":"CHMP6","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2005,"finding":"CHMP6 is N-myristoylated, as demonstrated by metabolic labelling with [3H]myristate incorporation into CHMP6-GFP in HEK-293 cells.","method":"Metabolic labelling with [3H]myristate in HEK-293 cells","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical labelling experiment, single lab but unambiguous method","pmids":["15511219"],"is_preprint":false},{"year":2005,"finding":"CHMP6 directly binds the ESCRT-II component EAP20 (human Vps25) and the ESCRT-III component CHMP4b, with the interaction mediated by the N-terminal basic half of CHMP6, as shown by co-immunoprecipitation and in vitro pull-down with recombinant proteins.","method":"Co-immunoprecipitation of epitope-tagged proteins in HEK-293 cells; in vitro pull-down with recombinant proteins purified from E. coli","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro reconstitution plus co-IP, two orthogonal methods, domain mapping performed","pmids":["15511219"],"is_preprint":false},{"year":2005,"finding":"Overexpressed CHMP6-GFP localizes to perinuclear puncta overlapping with LBPA-positive MVB membranes in HeLa cells, and causes accumulation of transferrin receptors in the cytoplasm (reduced surface expression), as well as accumulation of ubiquitinated proteins and endocytosed EGF, consistent with a role in endosomal cargo sorting.","method":"Fluorescence microscopy, immunofluorescence for LBPA, surface transferrin receptor assay, EGF uptake assay in HeLa cells","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple fluorescence-based functional readouts, single lab","pmids":["15511219"],"is_preprint":false},{"year":2004,"finding":"Human Vps20 (CHMP6) shows an endosomal membrane-staining pattern by immunofluorescence, and co-expression with hSnf7-1 disperses the large Snf7-staining vesicular structures, indicating functional interaction between the two ESCRT-III subunits.","method":"Immunofluorescence microscopy and co-expression in mammalian cells","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — localization and co-expression phenotype, single lab, no direct binding assay for hVps20 specifically","pmids":["14583093"],"is_preprint":false},{"year":2006,"finding":"The C-terminal domain of Vps28 (ESCRT-I subunit) employs a strictly conserved surface to interact with ESCRT-III factor Vps20 (CHMP6), as shown by mutagenesis of Vps28-CTD abolishing Vps20 binding in vitro; this interaction is required for EIAV Gag late domain rescue, suggesting Vps28-CTD recruits Vps20/ESCRT-III.","method":"Crystal structure of Vps28-CTD at 3.05 Å; mutagenesis; in vitro binding assay; EIAV Gag late domain rescue assay","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis plus functional rescue, single lab but multiple orthogonal methods","pmids":["16749904"],"is_preprint":false},{"year":2011,"finding":"Purified ESCRT-II binds Vps20 (CHMP6) on membranes in a curvature-dependent manner; together, ESCRT-II and Vps20 nucleate flexible Vps32 (Snf7) filaments that polymerize along highly curved membranes as a single string of monomers, and these filaments modulate membrane dynamics in vitro.","method":"Liposome co-flotation assays, fluorescence-based liposome interaction studies, high-resolution atomic force microscopy with purified recombinant proteins","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins, multiple orthogonal methods (liposome flotation, fluorescence, AFM), single lab","pmids":["21835927"],"is_preprint":false},{"year":2013,"finding":"The ubiquitin hydrolase Doa4 (yeast) directly binds a MIM1-like sequence in the Vps20 (CHMP6) subunit of ESCRT-III via a putative MIT domain at its N-terminus; disrupting this interaction enhances the ILV cargo deubiquitination defect and rescues ILV budding in bro1Δ cells independently of Doa4 catalytic activity.","method":"Direct binding assay (in vitro), yeast genetics (epistasis/rescue in bro1Δ mutant), mutagenesis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding demonstrated in vitro plus genetic epistasis rescue, multiple methods","pmids":["23444383"],"is_preprint":false},{"year":2015,"finding":"C. elegans VPS-20 (CHMP6 ortholog) adopts an open, extended conformation in solution rather than the auto-inhibited closed conformation typical of other ESCRT-III subunits, and interacts directly with ESCRT-II both in cytosolic extracts and with recombinant proteins in vitro, independent of membranes.","method":"In vitro binding assay with recombinant proteins; structural analysis of purified VPS-20 conformation; cell extract co-precipitation","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical and conformational analysis, single lab, C. elegans ortholog","pmids":["25588614"],"is_preprint":false},{"year":2014,"finding":"ESCRT-II and CHMP6 form highly ordered structures at the intercellular bridge during cytokinetic abscission; a truncated CHMP6 fragment (first 52 aa, CHMP6-N) blocks abscission and causes cell death, an effect abolished by mutation preventing CHMP6-N binding to ESCRT-II, and deleting the first 10 aa of CHMP6-N abolishes the abscission-failure phenotype without preventing bridge localization.","method":"High-resolution imaging of endogenous proteins at cytokinetic bridge; dominant-negative truncation overexpression; structure-function mutagenesis; cell death assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — high-resolution imaging plus structure-function mutagenesis plus functional phenotype, single lab but multiple orthogonal approaches","pmids":["25232011"],"is_preprint":false},{"year":2012,"finding":"CHMP6 directly binds H-Ras and N-Ras (but not K-Ras) in endosomes, with binding most efficient when H-Ras is GTP-bound and ubiquitylated; silencing CHMP6 blocks Ras recycling to the plasma membrane (as measured by cell fractionation and photobleaching) and blocks EGFR recycling, implicating CHMP6 in a positive feedback loop for growth factor signaling.","method":"cDNA library screen for H-Ras-binding proteins; direct binding assay; cell fractionation; FRAP (photobleaching); RNA knockdown with transformation and EGFR recycling assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding plus fractionation plus FRAP plus functional KD readouts, single lab","pmids":["22231449"],"is_preprint":false},{"year":2022,"finding":"Bro1 binds directly to the Vps20 (CHMP6) subunit of ESCRT-III; this interaction suppresses the antagonistic effect of Vps20 on Doa4–Snf7 binding, thereby enabling Doa4-mediated regulation of ESCRT-III.","method":"In vitro direct binding assay; yeast genetics (epistasis with vps20 alleles); co-immunoprecipitation in yeast","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding demonstrated plus genetic epistasis, two orthogonal methods, builds on prior replicated work","pmids":["34908216"],"is_preprint":false},{"year":2022,"finding":"The myristoylated N-terminal peptide of CHMP6 (Myr-CHMP6) is sufficient to increase small extracellular vesicle (sEV) production and to load cargo proteins into sEVs; N-myristoylation alone is necessary but not sufficient for effective packaging, indicating additional sequences in the CHMP6 N-terminus contribute to EV biogenesis.","method":"NanoGlo luciferase cargo-loading assay; nanoparticle tracking analysis; transmission electron microscopy; Western blotting; fluorescence reporter gene editing assay in Lenti-X 293T cells","journal":"Bioengineered","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal assays (NTA, TEM, luciferase, WB, functional editing), single lab","pmids":["35188876"],"is_preprint":false},{"year":2025,"finding":"CEP55 binds CHMP6 (co-IP), and CEP55 overexpression promotes CHMP6 expression, which in turn suppresses ferroptosis and facilitates malignant progression of triple-negative breast cancer cells.","method":"Co-immunoprecipitation; Western blot; cell proliferation, invasion, ferroptosis assays (Fe2+, MDA, GSH, ROS); xenograft tumor model","journal":"Clinical breast cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP plus downstream phenotypic assays, no mechanistic detail on how CEP55 promotes CHMP6 expression, single lab","pmids":["40925844"],"is_preprint":false}],"current_model":"CHMP6 (VPS20) is an N-myristoylated ESCRT-III subunit that adopts an open conformation enabling direct binding to ESCRT-II (via EAP20/Vps25 and the Vps28 C-terminal domain) and to CHMP4b/Snf7; at curved endosomal membranes the ESCRT-II–CHMP6 complex nucleates Snf7 filaments to drive intralumenal vesicle scission, and this same pathway operates during cytokinetic abscission; additionally, CHMP6 binds ubiquitylated, GTP-loaded H-Ras and N-Ras to promote their recycling and that of EGFR back to the plasma membrane, while its activity is modulated by Doa4 (which binds a MIM1-like motif in CHMP6) and by Bro1 (which relieves Vps20-mediated inhibition of Doa4–Snf7 interaction)."},"narrative":{"mechanistic_narrative":"CHMP6 (VPS20) is an N-myristoylated ESCRT-III subunit that bridges ESCRT-II to the downstream ESCRT-III polymer to drive membrane scission [PMID:15511219, PMID:21835927]. It binds directly to the ESCRT-II subunit EAP20/Vps25 through its N-terminal basic half and engages the ESCRT-III subunit CHMP4b/Snf7, and it is additionally recruited via a conserved surface on the Vps28 C-terminal domain of ESCRT-I [PMID:15511219, PMID:16749904]. Unlike autoinhibited ESCRT-III subunits, CHMP6 adopts an open, extended conformation that permits constitutive ESCRT-II engagement independent of membranes [PMID:25588614]. On highly curved membranes the ESCRT-II–CHMP6 assembly nucleates flexible Snf7/Vps32 filaments that remodel the bilayer, the molecular basis of intralumenal vesicle formation in multivesicular body sorting [PMID:21835927, PMID:14583093]; consistent with this, CHMP6 localizes to MVB membranes and is required for endosomal cargo sorting, including the downregulation of transferrin receptor and EGF [PMID:15511219]. The same ESCRT-II–CHMP6 nucleation module assembles into ordered structures at the cytokinetic intercellular bridge, where a CHMP6 N-terminal fragment acts as a dominant-negative that blocks abscission in an ESCRT-II-binding-dependent manner [PMID:25232011]. CHMP6 activity is regulated by accessory factors: it presents a MIM1-like motif bound by the deubiquitinase Doa4, and Bro1 binding relieves CHMP6-mediated antagonism of the Doa4–Snf7 interaction [PMID:23444383, PMID:34908216]. Beyond canonical scission, CHMP6 binds GTP-loaded, ubiquitylated H-Ras and N-Ras to promote recycling of Ras and EGFR to the plasma membrane [PMID:22231449], and its myristoylated N-terminal peptide is sufficient to enhance small extracellular vesicle production and cargo loading [PMID:35188876].","teleology":[{"year":2004,"claim":"Establishing whether the human VPS20 ortholog behaved as a bona fide ESCRT-III subunit was the first step in placing CHMP6 in the endosomal machinery.","evidence":"Immunofluorescence and co-expression with hSnf7-1 in mammalian cells","pmids":["14583093"],"confidence":"Medium","gaps":["No direct binding assay for human Vps20 in this study","Functional interaction inferred from co-expression phenotype only"]},{"year":2005,"claim":"Defining CHMP6's molecular connections answered how ESCRT-II is physically linked to ESCRT-III and showed CHMP6 is lipid-modified, anchoring it as the membrane-proximal nucleator.","evidence":"Metabolic [3H]myristate labelling, co-IP and recombinant pull-down with domain mapping, and MVB localization/cargo-sorting assays in HEK-293 and HeLa cells","pmids":["15511219"],"confidence":"High","gaps":["Cargo-sorting readouts rely on overexpression","Did not resolve filament-nucleation mechanism"]},{"year":2006,"claim":"Identifying a second upstream contact resolved how the ESCRT-I subunit Vps28 helps recruit CHMP6/ESCRT-III, structurally defining the recruitment surface.","evidence":"Crystal structure of Vps28-CTD with mutagenesis, in vitro binding, and EIAV Gag late-domain rescue","pmids":["16749904"],"confidence":"High","gaps":["Relative contribution of Vps28 versus ESCRT-II to recruitment in cells not quantified"]},{"year":2011,"claim":"Reconstitution answered how ESCRT-II and CHMP6 generate membrane deformation, showing they nucleate curvature-dependent Snf7 filaments.","evidence":"Liposome co-flotation, fluorescence interaction assays, and high-resolution AFM with purified recombinant proteins","pmids":["21835927"],"confidence":"High","gaps":["Single-lab in vitro system","How filament dynamics couple to scission not directly demonstrated"]},{"year":2012,"claim":"A screen revealed a non-canonical role: CHMP6 directly engages GTP-loaded, ubiquitylated H-/N-Ras to drive Ras and EGFR recycling, linking ESCRT-III to growth factor signaling feedback.","evidence":"cDNA library screen, direct binding, cell fractionation, FRAP, and knockdown with EGFR-recycling and transformation assays","pmids":["22231449"],"confidence":"Medium","gaps":["Single lab","Mechanism connecting recycling to canonical scission activity unresolved"]},{"year":2013,"claim":"Mapping the Doa4 contact explained how deubiquitination is coupled to ESCRT-III, showing CHMP6 carries a MIM1-like motif bound by the Doa4 MIT domain.","evidence":"In vitro direct binding, yeast epistasis/rescue in bro1Δ, and mutagenesis","pmids":["23444383"],"confidence":"High","gaps":["Conservation of this regulatory motif in human CHMP6 not tested here"]},{"year":2014,"claim":"Imaging and structure-function dissection established that the ESCRT-II–CHMP6 module operates at the cytokinetic bridge and that its N-terminal ESCRT-II-binding activity is essential for abscission.","evidence":"High-resolution imaging of endogenous proteins, dominant-negative truncation, structure-function mutagenesis, and cell death assays","pmids":["25232011"],"confidence":"High","gaps":["Distinct contributions of the first 10 residues versus ESCRT-II binding not fully separated mechanistically"]},{"year":2015,"claim":"Conformational analysis answered why CHMP6 can constitutively bind ESCRT-II, showing it is an open, extended subunit rather than autoinhibited.","evidence":"In vitro binding with recombinant proteins, conformational analysis, and extract co-precipitation in C. elegans VPS-20","pmids":["25588614"],"confidence":"Medium","gaps":["Single lab, ortholog system","No high-resolution structure of the open state"]},{"year":2022,"claim":"Two findings extended CHMP6 regulation and function: Bro1 relieves CHMP6 antagonism of Doa4–Snf7 binding, and the CHMP6 myristoylated N-terminus is sufficient to promote extracellular vesicle biogenesis and cargo loading.","evidence":"In vitro binding plus yeast epistasis/co-IP (Bro1); NTA, TEM, luciferase cargo assays, WB, and editing reporter in Lenti-X 293T cells (Myr-CHMP6 EVs)","pmids":["34908216","35188876"],"confidence":"High","gaps":["Additional N-terminal sequences required for EV packaging not identified","Mechanistic link between Bro1 regulation and EV biogenesis not established"]},{"year":2025,"claim":"A disease-context study linked CHMP6 to CEP55 and ferroptosis suppression in triple-negative breast cancer.","evidence":"Co-IP, Western blot, ferroptosis assays, and xenograft model","pmids":["40925844"],"confidence":"Low","gaps":["Single co-IP without reciprocal validation","No mechanism for how CEP55 promotes CHMP6 expression","Connection to ESCRT scission function not established"]},{"year":null,"claim":"How CHMP6's canonical ESCRT-III nucleation role mechanistically integrates with its non-canonical Ras/EGFR recycling and tumor-associated ferroptosis functions remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of the human ESCRT-II–CHMP6 complex","Whether recycling and scission activities use the same or distinct interaction surfaces unknown","In vivo disease relevance of ferroptosis link untested beyond single study"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,4,5,8]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,11]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,3,9]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[9]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,5,11]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[8]}],"complexes":["ESCRT-III"],"partners":["VPS25","CHMP4B","VPS28","DOA4","BRO1","HRAS","NRAS","CEP55"],"other_free_text":[]}},"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":58,"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":11,"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":7,"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":9851,"output_tokens":3281,"usd":0.039384,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10682,"output_tokens":3613,"usd":0.071868,"stage2_stop_reason":"end_turn"},"total_usd":0.111252,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"CHMP6 is N-myristoylated, as demonstrated by metabolic labelling with [3H]myristate incorporation into CHMP6-GFP in HEK-293 cells.\",\n      \"method\": \"Metabolic labelling with [3H]myristate in HEK-293 cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical labelling experiment, single lab but unambiguous method\",\n      \"pmids\": [\"15511219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CHMP6 directly binds the ESCRT-II component EAP20 (human Vps25) and the ESCRT-III component CHMP4b, with the interaction mediated by the N-terminal basic half of CHMP6, as shown by co-immunoprecipitation and in vitro pull-down with recombinant proteins.\",\n      \"method\": \"Co-immunoprecipitation of epitope-tagged proteins in HEK-293 cells; in vitro pull-down with recombinant proteins purified from E. coli\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro reconstitution plus co-IP, two orthogonal methods, domain mapping performed\",\n      \"pmids\": [\"15511219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Overexpressed CHMP6-GFP localizes to perinuclear puncta overlapping with LBPA-positive MVB membranes in HeLa cells, and causes accumulation of transferrin receptors in the cytoplasm (reduced surface expression), as well as accumulation of ubiquitinated proteins and endocytosed EGF, consistent with a role in endosomal cargo sorting.\",\n      \"method\": \"Fluorescence microscopy, immunofluorescence for LBPA, surface transferrin receptor assay, EGF uptake assay in HeLa cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple fluorescence-based functional readouts, single lab\",\n      \"pmids\": [\"15511219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human Vps20 (CHMP6) shows an endosomal membrane-staining pattern by immunofluorescence, and co-expression with hSnf7-1 disperses the large Snf7-staining vesicular structures, indicating functional interaction between the two ESCRT-III subunits.\",\n      \"method\": \"Immunofluorescence microscopy and co-expression in mammalian cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — localization and co-expression phenotype, single lab, no direct binding assay for hVps20 specifically\",\n      \"pmids\": [\"14583093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The C-terminal domain of Vps28 (ESCRT-I subunit) employs a strictly conserved surface to interact with ESCRT-III factor Vps20 (CHMP6), as shown by mutagenesis of Vps28-CTD abolishing Vps20 binding in vitro; this interaction is required for EIAV Gag late domain rescue, suggesting Vps28-CTD recruits Vps20/ESCRT-III.\",\n      \"method\": \"Crystal structure of Vps28-CTD at 3.05 Å; mutagenesis; in vitro binding assay; EIAV Gag late domain rescue assay\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis plus functional rescue, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16749904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Purified ESCRT-II binds Vps20 (CHMP6) on membranes in a curvature-dependent manner; together, ESCRT-II and Vps20 nucleate flexible Vps32 (Snf7) filaments that polymerize along highly curved membranes as a single string of monomers, and these filaments modulate membrane dynamics in vitro.\",\n      \"method\": \"Liposome co-flotation assays, fluorescence-based liposome interaction studies, high-resolution atomic force microscopy with purified recombinant proteins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins, multiple orthogonal methods (liposome flotation, fluorescence, AFM), single lab\",\n      \"pmids\": [\"21835927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The ubiquitin hydrolase Doa4 (yeast) directly binds a MIM1-like sequence in the Vps20 (CHMP6) subunit of ESCRT-III via a putative MIT domain at its N-terminus; disrupting this interaction enhances the ILV cargo deubiquitination defect and rescues ILV budding in bro1Δ cells independently of Doa4 catalytic activity.\",\n      \"method\": \"Direct binding assay (in vitro), yeast genetics (epistasis/rescue in bro1Δ mutant), mutagenesis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding demonstrated in vitro plus genetic epistasis rescue, multiple methods\",\n      \"pmids\": [\"23444383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"C. elegans VPS-20 (CHMP6 ortholog) adopts an open, extended conformation in solution rather than the auto-inhibited closed conformation typical of other ESCRT-III subunits, and interacts directly with ESCRT-II both in cytosolic extracts and with recombinant proteins in vitro, independent of membranes.\",\n      \"method\": \"In vitro binding assay with recombinant proteins; structural analysis of purified VPS-20 conformation; cell extract co-precipitation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical and conformational analysis, single lab, C. elegans ortholog\",\n      \"pmids\": [\"25588614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ESCRT-II and CHMP6 form highly ordered structures at the intercellular bridge during cytokinetic abscission; a truncated CHMP6 fragment (first 52 aa, CHMP6-N) blocks abscission and causes cell death, an effect abolished by mutation preventing CHMP6-N binding to ESCRT-II, and deleting the first 10 aa of CHMP6-N abolishes the abscission-failure phenotype without preventing bridge localization.\",\n      \"method\": \"High-resolution imaging of endogenous proteins at cytokinetic bridge; dominant-negative truncation overexpression; structure-function mutagenesis; cell death assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — high-resolution imaging plus structure-function mutagenesis plus functional phenotype, single lab but multiple orthogonal approaches\",\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 most efficient when H-Ras is GTP-bound and ubiquitylated; silencing CHMP6 blocks Ras recycling to the plasma membrane (as measured by cell fractionation and photobleaching) and blocks EGFR recycling, implicating CHMP6 in a positive feedback loop for growth factor signaling.\",\n      \"method\": \"cDNA library screen for H-Ras-binding proteins; direct binding assay; cell fractionation; FRAP (photobleaching); RNA knockdown with transformation and EGFR recycling assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding plus fractionation plus FRAP plus functional KD readouts, single lab\",\n      \"pmids\": [\"22231449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Bro1 binds directly to the Vps20 (CHMP6) subunit of ESCRT-III; this interaction suppresses the antagonistic effect of Vps20 on Doa4–Snf7 binding, thereby enabling Doa4-mediated regulation of ESCRT-III.\",\n      \"method\": \"In vitro direct binding assay; yeast genetics (epistasis with vps20 alleles); co-immunoprecipitation in yeast\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding demonstrated plus genetic epistasis, two orthogonal methods, builds on prior replicated work\",\n      \"pmids\": [\"34908216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The myristoylated N-terminal peptide of CHMP6 (Myr-CHMP6) is sufficient to increase small extracellular vesicle (sEV) production and to load cargo proteins into sEVs; N-myristoylation alone is necessary but not sufficient for effective packaging, indicating additional sequences in the CHMP6 N-terminus contribute to EV biogenesis.\",\n      \"method\": \"NanoGlo luciferase cargo-loading assay; nanoparticle tracking analysis; transmission electron microscopy; Western blotting; fluorescence reporter gene editing assay in Lenti-X 293T cells\",\n      \"journal\": \"Bioengineered\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal assays (NTA, TEM, luciferase, WB, functional editing), single lab\",\n      \"pmids\": [\"35188876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CEP55 binds CHMP6 (co-IP), and CEP55 overexpression promotes CHMP6 expression, which in turn suppresses ferroptosis and facilitates malignant progression of triple-negative breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation; Western blot; cell proliferation, invasion, ferroptosis assays (Fe2+, MDA, GSH, ROS); xenograft tumor model\",\n      \"journal\": \"Clinical breast cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP plus downstream phenotypic assays, no mechanistic detail on how CEP55 promotes CHMP6 expression, single lab\",\n      \"pmids\": [\"40925844\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CHMP6 (VPS20) is an N-myristoylated ESCRT-III subunit that adopts an open conformation enabling direct binding to ESCRT-II (via EAP20/Vps25 and the Vps28 C-terminal domain) and to CHMP4b/Snf7; at curved endosomal membranes the ESCRT-II–CHMP6 complex nucleates Snf7 filaments to drive intralumenal vesicle scission, and this same pathway operates during cytokinetic abscission; additionally, CHMP6 binds ubiquitylated, GTP-loaded H-Ras and N-Ras to promote their recycling and that of EGFR back to the plasma membrane, while its activity is modulated by Doa4 (which binds a MIM1-like motif in CHMP6) and by Bro1 (which relieves Vps20-mediated inhibition of Doa4–Snf7 interaction).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CHMP6 (VPS20) is an N-myristoylated ESCRT-III subunit that bridges ESCRT-II to the downstream ESCRT-III polymer to drive membrane scission [#0, #1, #5]. It binds directly to the ESCRT-II subunit EAP20/Vps25 through its N-terminal basic half and engages the ESCRT-III subunit CHMP4b/Snf7, and it is additionally recruited via a conserved surface on the Vps28 C-terminal domain of ESCRT-I [#1, #4]. Unlike autoinhibited ESCRT-III subunits, CHMP6 adopts an open, extended conformation that permits constitutive ESCRT-II engagement independent of membranes [#7]. On highly curved membranes the ESCRT-II–CHMP6 assembly nucleates flexible Snf7/Vps32 filaments that remodel the bilayer, the molecular basis of intralumenal vesicle formation in multivesicular body sorting [#5, #3]; consistent with this, CHMP6 localizes to MVB membranes and is required for endosomal cargo sorting, including the downregulation of transferrin receptor and EGF [#2]. The same ESCRT-II–CHMP6 nucleation module assembles into ordered structures at the cytokinetic intercellular bridge, where a CHMP6 N-terminal fragment acts as a dominant-negative that blocks abscission in an ESCRT-II-binding-dependent manner [#8]. CHMP6 activity is regulated by accessory factors: it presents a MIM1-like motif bound by the deubiquitinase Doa4, and Bro1 binding relieves CHMP6-mediated antagonism of the Doa4–Snf7 interaction [#6, #10]. Beyond canonical scission, CHMP6 binds GTP-loaded, ubiquitylated H-Ras and N-Ras to promote recycling of Ras and EGFR to the plasma membrane [#9], and its myristoylated N-terminal peptide is sufficient to enhance small extracellular vesicle production and cargo loading [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing whether the human VPS20 ortholog behaved as a bona fide ESCRT-III subunit was the first step in placing CHMP6 in the endosomal machinery.\",\n      \"evidence\": \"Immunofluorescence and co-expression with hSnf7-1 in mammalian cells\",\n      \"pmids\": [\"14583093\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No direct binding assay for human Vps20 in this study\", \"Functional interaction inferred from co-expression phenotype only\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defining CHMP6's molecular connections answered how ESCRT-II is physically linked to ESCRT-III and showed CHMP6 is lipid-modified, anchoring it as the membrane-proximal nucleator.\",\n      \"evidence\": \"Metabolic [3H]myristate labelling, co-IP and recombinant pull-down with domain mapping, and MVB localization/cargo-sorting assays in HEK-293 and HeLa cells\",\n      \"pmids\": [\"15511219\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Cargo-sorting readouts rely on overexpression\", \"Did not resolve filament-nucleation mechanism\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identifying a second upstream contact resolved how the ESCRT-I subunit Vps28 helps recruit CHMP6/ESCRT-III, structurally defining the recruitment surface.\",\n      \"evidence\": \"Crystal structure of Vps28-CTD with mutagenesis, in vitro binding, and EIAV Gag late-domain rescue\",\n      \"pmids\": [\"16749904\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Relative contribution of Vps28 versus ESCRT-II to recruitment in cells not quantified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Reconstitution answered how ESCRT-II and CHMP6 generate membrane deformation, showing they nucleate curvature-dependent Snf7 filaments.\",\n      \"evidence\": \"Liposome co-flotation, fluorescence interaction assays, and high-resolution AFM with purified recombinant proteins\",\n      \"pmids\": [\"21835927\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single-lab in vitro system\", \"How filament dynamics couple to scission not directly demonstrated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"A screen revealed a non-canonical role: CHMP6 directly engages GTP-loaded, ubiquitylated H-/N-Ras to drive Ras and EGFR recycling, linking ESCRT-III to growth factor signaling feedback.\",\n      \"evidence\": \"cDNA library screen, direct binding, cell fractionation, FRAP, and knockdown with EGFR-recycling and transformation assays\",\n      \"pmids\": [\"22231449\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single lab\", \"Mechanism connecting recycling to canonical scission activity unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapping the Doa4 contact explained how deubiquitination is coupled to ESCRT-III, showing CHMP6 carries a MIM1-like motif bound by the Doa4 MIT domain.\",\n      \"evidence\": \"In vitro direct binding, yeast epistasis/rescue in bro1Δ, and mutagenesis\",\n      \"pmids\": [\"23444383\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Conservation of this regulatory motif in human CHMP6 not tested here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Imaging and structure-function dissection established that the ESCRT-II–CHMP6 module operates at the cytokinetic bridge and that its N-terminal ESCRT-II-binding activity is essential for abscission.\",\n      \"evidence\": \"High-resolution imaging of endogenous proteins, dominant-negative truncation, structure-function mutagenesis, and cell death assays\",\n      \"pmids\": [\"25232011\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Distinct contributions of the first 10 residues versus ESCRT-II binding not fully separated mechanistically\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Conformational analysis answered why CHMP6 can constitutively bind ESCRT-II, showing it is an open, extended subunit rather than autoinhibited.\",\n      \"evidence\": \"In vitro binding with recombinant proteins, conformational analysis, and extract co-precipitation in C. elegans VPS-20\",\n      \"pmids\": [\"25588614\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single lab, ortholog system\", \"No high-resolution structure of the open state\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Two findings extended CHMP6 regulation and function: Bro1 relieves CHMP6 antagonism of Doa4–Snf7 binding, and the CHMP6 myristoylated N-terminus is sufficient to promote extracellular vesicle biogenesis and cargo loading.\",\n      \"evidence\": \"In vitro binding plus yeast epistasis/co-IP (Bro1); NTA, TEM, luciferase cargo assays, WB, and editing reporter in Lenti-X 293T cells (Myr-CHMP6 EVs)\",\n      \"pmids\": [\"34908216\", \"35188876\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Additional N-terminal sequences required for EV packaging not identified\", \"Mechanistic link between Bro1 regulation and EV biogenesis not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A disease-context study linked CHMP6 to CEP55 and ferroptosis suppression in triple-negative breast cancer.\",\n      \"evidence\": \"Co-IP, Western blot, ferroptosis assays, and xenograft model\",\n      \"pmids\": [\"40925844\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single co-IP without reciprocal validation\", \"No mechanism for how CEP55 promotes CHMP6 expression\", \"Connection to ESCRT scission function not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CHMP6's canonical ESCRT-III nucleation role mechanistically integrates with its non-canonical Ras/EGFR recycling and tumor-associated ferroptosis functions remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No structure of the human ESCRT-II–CHMP6 complex\", \"Whether recycling and scission activities use the same or distinct interaction surfaces unknown\", \"In vivo disease relevance of ferroptosis link untested beyond single study\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 4, 5, 8]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 3, 9]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 5, 11]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"ESCRT-III\"],\n    \"partners\": [\"VPS25\", \"CHMP4B\", \"VPS28\", \"DOA4\", \"BRO1\", \"HRAS\", \"NRAS\", \"CEP55\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}