{"gene":"CHMP3","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2006,"finding":"Crystal structure of human CHMP3 at 2.8 Å resolution reveals a flat helical arrangement that assembles into a lattice via two distinct dimerization modes, with a highly basic surface that mediates membrane interaction on one side and the C-terminal Vps4-target sequence on the opposite side. Mutations in basic and dimerization regions abolish bilayer interaction in vivo and reverse the dominant-negative effect of truncated CHMP3 on HIV-1 budding, demonstrating that CHMP3 polymerization and lattice formation on membranes underlies its role in the final steps of budding.","method":"X-ray crystallography (2.8 Å), site-directed mutagenesis, in vivo membrane interaction assays, HIV-1 budding assay","journal":"Developmental Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis validation and in vivo budding assay, multiple orthogonal methods in one rigorous study","pmids":["16740483"],"is_preprint":false},{"year":2008,"finding":"SAXS data reveal that CHMP3 can adopt two conformations in solution: a closed globular (autoinhibited, cytosolic) form and an open extended (activated) form. Both conformations interact with AMSH with high affinity; the C-terminal region of CHMP3 is required for AMSH interaction, but a C-terminal peptide alone shows only weak binding, indicating additional regions of CHMP3 contribute to the high-affinity interaction.","method":"Small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), binding affinity measurements","journal":"Journal of Molecular Biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — SAXS structural evidence for conformational states plus binding assays, single lab but multiple orthogonal methods","pmids":["18395747"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of the AMSH N-terminal fragment (AMSHΔC) in complex with the C-terminal region of CHMP3 (CHMP3ΔN) reveals that CHMP3ΔN is disordered in solution but adopts a helical conformation upon binding AMSH, forming a novel MIT-domain interacting motif (MIM) distinct from the CHMP1–AMSH binding site. ITC and SPR measurements confirm an unusually high-affinity MIM–MIT interaction. The N-terminal helical segment of AMSH regulates its function; its destabilization causes loss of function during HIV-1 budding.","method":"X-ray crystallography, ITC, SPR, HIV-1 budding functional assay","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of complex plus quantitative binding measurements (ITC/SPR) and functional budding assay, multiple orthogonal methods","pmids":["21827950"],"is_preprint":false},{"year":2011,"finding":"SAXS ensemble refinement using maximum-entropy combination with coarse-grained simulations characterizes the autoinhibited (low-salt) state of CHMP3: helix α5 is bound to the tip of helices α1 and α2, while helix α6 remains free in solution and is not part of the autoinhibitory complex.","method":"SAXS ensemble refinement, coarse-grained molecular simulations, maximum-entropy approach","journal":"Structure","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — computational ensemble method applied to existing SAXS data; single lab, no mutagenesis validation","pmids":["21220121"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures at 3.3 and 3.6 Å of membrane-coated CHMP2A–CHMP3 filaments show helical filaments assembled by CHMP2A–CHMP3 heterodimers in the open ESCRT-III conformation, generating a partially positive-charged membrane-interaction surface with short N-terminal motifs contacting the membrane and the C-terminal VPS4-target sequence oriented toward the tube interior. Inter-filament interactions are electrostatic, facilitating filament sliding during VPS4-mediated remodeling. High-speed AFM and fluorescence microscopy confirm that VPS4 can constrict and cleave CHMP2A–CHMP3 membrane tubes, establishing CHMP2A–CHMP3–VPS4 as a minimal membrane fission machinery.","method":"Cryo-EM (3.3 and 3.6 Å), high-speed atomic force microscopy (HS-AFM), fluorescence microscopy, in vitro membrane tube reconstitution","journal":"Nature Structural & Molecular Biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic cryo-EM structures of functional polymer on membrane, corroborated by AFM and fluorescence microscopy with multiple orthogonal methods","pmids":["36604498"],"is_preprint":false},{"year":2022,"finding":"A homozygous missense variant in CHMP3 (p.Thr173Ile) causes complex hereditary spastic paraplegia. Patient fibroblasts with reduced CHMP3 levels show accumulation of endosomes, autophagosomes, and autolysosomes by electron microscopy, and elevated autophagy markers p62 and LC3-II. Ectopic re-expression of wild-type CHMP3 in patient fibroblasts reduces p62 puncta and the number of endosomes and autophagosomes, directly linking CHMP3 to regulation of the autophagy/endosomal pathway.","method":"Exome sequencing, electron microscopy, western blot, immunofluorescence, ectopic wild-type rescue in primary patient fibroblasts","journal":"Journal of Medical Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived loss-of-function plus rescue experiment with multiple readouts, single lab","pmids":["35710109"],"is_preprint":false},{"year":2023,"finding":"Knockdown of CHMP3 in hepatocellular carcinoma cells activates the caspase-1 pyroptosis signaling pathway, resulting in changes in cell membrane integrity and cytoplasmic leakage; this effect is reversed by the caspase-1 inhibitor AYC, indicating CHMP3 suppresses caspase-1-dependent pyroptosis.","method":"siRNA knockdown, western blot of caspase-1 pathway proteins, transmission electron microscopy, caspase-1 inhibitor rescue, xenograft tumor model","journal":"International Journal of Oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — defined cellular phenotype with pathway placement via inhibitor rescue and protein expression, single lab","pmids":["38038147"],"is_preprint":false},{"year":2025,"finding":"During mammalian cytokinetic abscission, depletion of CHMP2A causes severe mislocalization of CHMP3 (as well as CHMP4B and CHMP1B) at the abscission site, while IST1 and CHMP2B are minimally affected, placing CHMP3 downstream of CHMP2A in an ordered, hierarchical assembly of ESCRT-III subunits required for timely abscission.","method":"CHMP2A knockout, live cell imaging, structured illumination microscopy (SIM), correlative light-electron microscopy (CLEM), dual-protein imaging","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic epistasis with high-resolution imaging in mammalian cells, preprint not yet peer-reviewed, single lab","pmids":["bio_10.1101_2025.06.24.661003"],"is_preprint":true}],"current_model":"CHMP3 (VPS24) is an ESCRT-III subunit that adopts a closed autoinhibited conformation in the cytosol and an open active conformation upon membrane recruitment; in its active state it co-polymerizes with CHMP2A into helical heterodimeric filaments on negatively curved membranes via an electrostatic interaction surface, forming a minimal fission machinery with VPS4; it also recruits the deubiquitylase AMSH through a high-affinity, novel MIM–MIT interaction involving its C-terminal region, and it is positioned downstream of CHMP2A in the ordered hierarchical assembly of ESCRT-III during cytokinetic abscission, with loss of CHMP3 function disrupting endosomal/autophagic flux and activating caspase-1-dependent pyroptosis."},"narrative":{"mechanistic_narrative":"CHMP3 (VPS24) is an ESCRT-III subunit that drives membrane scission during budding and abscission events by polymerizing into membrane-associated lattices and recruiting downstream remodeling machinery [PMID:16740483, PMID:36604498]. Its crystal structure reveals a flat helical fold that assembles into a lattice through two distinct dimerization modes, presenting a highly basic surface for membrane interaction on one face and a C-terminal VPS4-target sequence on the opposite face; mutation of the basic and dimerization regions abolishes bilayer binding and reverses the dominant-negative block of HIV-1 budding [PMID:16740483]. In solution CHMP3 toggles between a closed, autoinhibited cytosolic form, in which helix α5 docks onto the tips of helices α1/α2, and an open activated form competent for membrane assembly [PMID:18395747, PMID:21220121]. On negatively curved membranes, the open conformer co-polymerizes with CHMP2A into helical heterodimeric filaments whose short N-terminal motifs contact the bilayer and whose C-terminal VPS4-target sequences face the tube interior; electrostatic inter-filament contacts permit VPS4 to constrict and cleave the tubes, defining CHMP2A–CHMP3–VPS4 as a minimal membrane fission machinery [PMID:36604498]. CHMP3 additionally recruits the deubiquitylase AMSH via a high-affinity, novel MIM–MIT interaction in which its disordered C-terminal region folds into a helix upon binding the AMSH N-terminal MIT domain [PMID:18395747, PMID:21827950]. During cytokinetic abscission CHMP3 acts downstream of CHMP2A in the ordered hierarchical assembly of ESCRT-III [PMID:bio_10.1101_2025.06.24.661003]. A homozygous CHMP3 missense variant causes complex hereditary spastic paraplegia, with loss of CHMP3 function disrupting endosomal and autophagic flux [PMID:35710109]; CHMP3 depletion also activates caspase-1-dependent pyroptosis [PMID:38038147].","teleology":[{"year":2006,"claim":"Established the structural basis for how CHMP3 engages membranes, showing that lattice polymerization with a basic membrane-binding face and an opposed VPS4-target face underlies its role in the final steps of budding.","evidence":"X-ray crystallography at 2.8 Å with site-directed mutagenesis, in vivo membrane interaction assays, and an HIV-1 budding assay","pmids":["16740483"],"confidence":"High","gaps":["Did not resolve how polymerization is triggered on membranes in cells","No structure of CHMP3 in complex with partner ESCRT-III subunits"]},{"year":2008,"claim":"Resolved that CHMP3 exists in distinct closed (autoinhibited) and open (activated) conformations and that both bind AMSH with high affinity through its C-terminal region plus additional contributing regions.","evidence":"SAXS conformational analysis with ITC binding affinity measurements","pmids":["18395747"],"confidence":"High","gaps":["Did not define the atomic interface of the CHMP3–AMSH contact","The trigger converting closed to open state in vivo unresolved"]},{"year":2011,"claim":"Defined the CHMP3–AMSH interaction at atomic resolution, revealing a novel MIM that folds upon binding the AMSH MIT domain, distinct from the CHMP1 site, and showed the AMSH N-terminal helix is functionally required for HIV-1 budding.","evidence":"Crystal structure of the AMSHΔC–CHMP3ΔN complex with ITC, SPR, and HIV-1 budding functional assay","pmids":["21827950"],"confidence":"High","gaps":["Functional consequence of CHMP3-recruited AMSH deubiquitylation on cargo not dissected","Did not test the MIM–MIT interaction in cytokinesis or endosomal contexts"]},{"year":2011,"claim":"Refined the molecular architecture of the autoinhibited CHMP3 state, localizing helix α5 against the α1/α2 tips while helix α6 remains free, clarifying which elements gate activation.","evidence":"SAXS ensemble refinement with coarse-grained molecular simulations using a maximum-entropy approach","pmids":["21220121"],"confidence":"Medium","gaps":["Computational model without mutagenesis validation","Did not capture the activation transition directly"]},{"year":2022,"claim":"Linked CHMP3 function to human disease and to endosomal/autophagic flux, showing a missense variant causes hereditary spastic paraplegia with accumulation of endosomes and autophagosomes reversible by wild-type re-expression.","evidence":"Exome sequencing, electron microscopy, western blot, immunofluorescence, and ectopic wild-type rescue in patient fibroblasts","pmids":["35710109"],"confidence":"Medium","gaps":["Mechanism by which p.Thr173Ile impairs ESCRT-III assembly not defined","Single family / single lab","Connection between the autophagy defect and neuronal phenotype unestablished"]},{"year":2023,"claim":"Captured the active membrane-bound state, showing CHMP2A–CHMP3 heterodimers build open-conformation helical filaments that VPS4 constricts and cleaves, defining a minimal fission machinery.","evidence":"Cryo-EM at 3.3 and 3.6 Å, high-speed AFM, fluorescence microscopy, and in vitro membrane tube reconstitution","pmids":["36604498"],"confidence":"High","gaps":["How additional ESCRT-III subunits modify this minimal filament in vivo unresolved","Does not address regulation of filament nucleation"]},{"year":2023,"claim":"Placed CHMP3 as a suppressor of caspase-1-dependent pyroptosis in hepatocellular carcinoma cells, connecting its loss to membrane integrity failure.","evidence":"siRNA knockdown, western blot of caspase-1 pathway proteins, electron microscopy, caspase-1 inhibitor rescue, and xenograft model","pmids":["38038147"],"confidence":"Medium","gaps":["Molecular link between ESCRT-III fission activity and caspase-1 activation not defined","Single lab / single cancer context"]},{"year":2025,"claim":"Ordered ESCRT-III assembly during cytokinetic abscission by showing CHMP3 recruitment depends on CHMP2A, placing it downstream in the hierarchy.","evidence":"CHMP2A knockout with live-cell imaging, SIM, and CLEM in mammalian cells (preprint)","pmids":["bio_10.1101_2025.06.24.661003"],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Does not establish direct CHMP2A–CHMP3 contact at the abscission site in vivo","Timing relative to VPS4 recruitment unresolved"]},{"year":null,"claim":"How CHMP3 conformational activation, partner selection (CHMP2A vs AMSH), and disease-associated dysfunction are coordinated within specific cellular fission events remains open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of full ordered ESCRT-III assembly including CHMP3 in a cellular context","Mechanistic chain from CHMP3 loss to pyroptosis and to neurodegeneration undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,4]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[5]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[7]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[6]}],"complexes":["ESCRT-III","CHMP2A-CHMP3 filament"],"partners":["CHMP2A","VPS4","AMSH"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y3E7","full_name":"Charged multivesicular body protein 3","aliases":["Chromatin-modifying protein 3","Neuroendocrine differentiation factor","Vacuolar protein sorting-associated protein 24","hVps24"],"length_aa":222,"mass_kda":25.1,"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. Selectively binds to phosphatidylinositol 3,5-bisphosphate PtdIns(3,5)P2 and PtdIns(3,4)P2 in preference to other phosphoinositides tested. Involved in late stages of cytokinesis. Plays a role in endosomal sorting/trafficking of EGF receptor. Isoform 2 prevents stress-mediated cell death and accumulation of reactive oxygen species when expressed in yeast cells","subcellular_location":"Cytoplasm, cytosol; Membrane; Endosome; Late endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y3E7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CHMP3","classification":"Common Essential","n_dependent_lines":983,"n_total_lines":1208,"dependency_fraction":0.8137417218543046},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000115561","cell_line_id":"CID000067","localizations":[{"compartment":"vesicles","grade":3}],"interactors":[],"url":"https://opencell.sf.czbiohub.org/target/CID000067","total_profiled":1310},"omim":[{"mim_id":"610893","title":"CHARGED MULTIVESICULAR BODY PROTEIN 2A; CHMP2A","url":"https://www.omim.org/entry/610893"},{"mim_id":"610052","title":"CHARGED MULTIVESICULAR BODY PROTEIN 3; CHMP3","url":"https://www.omim.org/entry/610052"},{"mim_id":"609982","title":"VACUOLAR PROTEIN SORTING 4 HOMOLOG A; VPS4A","url":"https://www.omim.org/entry/609982"},{"mim_id":"606247","title":"STAM-BINDING PROTEIN; STAMBP","url":"https://www.omim.org/entry/606247"}],"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/CHMP3"},"hgnc":{"alias_symbol":["NEDF","CGI-149"],"prev_symbol":["VPS24"]},"alphafold":{"accession":"Q9Y3E7","domains":[{"cath_id":"-","chopping":"13-145","consensus_level":"high","plddt":93.9768,"start":13,"end":145}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3E7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3E7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3E7-F1-predicted_aligned_error_v6.png","plddt_mean":81.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CHMP3","jax_strain_url":"https://www.jax.org/strain/search?query=CHMP3"},"sequence":{"accession":"Q9Y3E7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y3E7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y3E7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3E7"}},"corpus_meta":[{"pmid":"16740483","id":"PMC_16740483","title":"Structural basis for budding by the ESCRT-III factor CHMP3.","date":"2006","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/16740483","citation_count":209,"is_preprint":false},{"pmid":"21220121","id":"PMC_21220121","title":"SAXS ensemble refinement of ESCRT-III CHMP3 conformational transitions.","date":"2011","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/21220121","citation_count":209,"is_preprint":false},{"pmid":"18395747","id":"PMC_18395747","title":"Structural basis for autoinhibition of ESCRT-III CHMP3.","date":"2008","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18395747","citation_count":113,"is_preprint":false},{"pmid":"36604498","id":"PMC_36604498","title":"Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage.","date":"2023","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/36604498","citation_count":50,"is_preprint":false},{"pmid":"19056728","id":"PMC_19056728","title":"Physiological involvement in pH signaling of Vps24-mediated recruitment of Aspergillus PalB cysteine protease to ESCRT-III.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19056728","citation_count":50,"is_preprint":false},{"pmid":"21827950","id":"PMC_21827950","title":"Structural basis for ESCRT-III CHMP3 recruitment of AMSH.","date":"2011","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/21827950","citation_count":42,"is_preprint":false},{"pmid":"34028356","id":"PMC_34028356","title":"Design principles of the ESCRT-III Vps24-Vps2 module.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/34028356","citation_count":19,"is_preprint":false},{"pmid":"17331679","id":"PMC_17331679","title":"Characterization of a novel alternatively spliced human transcript encoding an N-terminally truncated Vps24 protein that suppresses the effects of Bax in an ESCRT independent manner in yeast.","date":"2007","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/17331679","citation_count":18,"is_preprint":false},{"pmid":"38038147","id":"PMC_38038147","title":"CHMP3 promotes the progression of hepatocellular carcinoma by inhibiting caspase‑1‑dependent pyroptosis.","date":"2023","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38038147","citation_count":16,"is_preprint":false},{"pmid":"16857172","id":"PMC_16857172","title":"Aovps24, a homologue of VPS24, is required for vacuolar formation which could maintain proper growth and development in the filamentous fungus Aspergillus oryzae.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/16857172","citation_count":12,"is_preprint":false},{"pmid":"35710109","id":"PMC_35710109","title":"A homozygous variant in CHMP3 is associated with complex hereditary spastic paraplegia.","date":"2022","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35710109","citation_count":7,"is_preprint":false},{"pmid":"33956855","id":"PMC_33956855","title":"Genetic analysis of the Drosophila ESCRT-III complex protein, VPS24, reveals a novel function in lysosome homeostasis.","date":"2021","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/33956855","citation_count":4,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.24.661003","title":"Loss of CHMP2A implicates an ordered assembly of ESCRT-III proteins during cytokinetic abscission","date":"2025-06-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.24.661003","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.08.627411","title":"Orchestration of SARS-CoV-2 Nsp4 and host-cell ESCRT proteins induces morphological changes of the endoplasmic reticulum","date":"2024-12-10","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.08.627411","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8517,"output_tokens":2400,"usd":0.030776,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9562,"output_tokens":3328,"usd":0.065505,"stage2_stop_reason":"end_turn"},"total_usd":0.096281,"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\": 2006,\n      \"finding\": \"Crystal structure of human CHMP3 at 2.8 Å resolution reveals a flat helical arrangement that assembles into a lattice via two distinct dimerization modes, with a highly basic surface that mediates membrane interaction on one side and the C-terminal Vps4-target sequence on the opposite side. Mutations in basic and dimerization regions abolish bilayer interaction in vivo and reverse the dominant-negative effect of truncated CHMP3 on HIV-1 budding, demonstrating that CHMP3 polymerization and lattice formation on membranes underlies its role in the final steps of budding.\",\n      \"method\": \"X-ray crystallography (2.8 Å), site-directed mutagenesis, in vivo membrane interaction assays, HIV-1 budding assay\",\n      \"journal\": \"Developmental Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis validation and in vivo budding assay, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"16740483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SAXS data reveal that CHMP3 can adopt two conformations in solution: a closed globular (autoinhibited, cytosolic) form and an open extended (activated) form. Both conformations interact with AMSH with high affinity; the C-terminal region of CHMP3 is required for AMSH interaction, but a C-terminal peptide alone shows only weak binding, indicating additional regions of CHMP3 contribute to the high-affinity interaction.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), binding affinity measurements\",\n      \"journal\": \"Journal of Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SAXS structural evidence for conformational states plus binding assays, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"18395747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of the AMSH N-terminal fragment (AMSHΔC) in complex with the C-terminal region of CHMP3 (CHMP3ΔN) reveals that CHMP3ΔN is disordered in solution but adopts a helical conformation upon binding AMSH, forming a novel MIT-domain interacting motif (MIM) distinct from the CHMP1–AMSH binding site. ITC and SPR measurements confirm an unusually high-affinity MIM–MIT interaction. The N-terminal helical segment of AMSH regulates its function; its destabilization causes loss of function during HIV-1 budding.\",\n      \"method\": \"X-ray crystallography, ITC, SPR, HIV-1 budding functional assay\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of complex plus quantitative binding measurements (ITC/SPR) and functional budding assay, multiple orthogonal methods\",\n      \"pmids\": [\"21827950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SAXS ensemble refinement using maximum-entropy combination with coarse-grained simulations characterizes the autoinhibited (low-salt) state of CHMP3: helix α5 is bound to the tip of helices α1 and α2, while helix α6 remains free in solution and is not part of the autoinhibitory complex.\",\n      \"method\": \"SAXS ensemble refinement, coarse-grained molecular simulations, maximum-entropy approach\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — computational ensemble method applied to existing SAXS data; single lab, no mutagenesis validation\",\n      \"pmids\": [\"21220121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures at 3.3 and 3.6 Å of membrane-coated CHMP2A–CHMP3 filaments show helical filaments assembled by CHMP2A–CHMP3 heterodimers in the open ESCRT-III conformation, generating a partially positive-charged membrane-interaction surface with short N-terminal motifs contacting the membrane and the C-terminal VPS4-target sequence oriented toward the tube interior. Inter-filament interactions are electrostatic, facilitating filament sliding during VPS4-mediated remodeling. High-speed AFM and fluorescence microscopy confirm that VPS4 can constrict and cleave CHMP2A–CHMP3 membrane tubes, establishing CHMP2A–CHMP3–VPS4 as a minimal membrane fission machinery.\",\n      \"method\": \"Cryo-EM (3.3 and 3.6 Å), high-speed atomic force microscopy (HS-AFM), fluorescence microscopy, in vitro membrane tube reconstitution\",\n      \"journal\": \"Nature Structural & Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic cryo-EM structures of functional polymer on membrane, corroborated by AFM and fluorescence microscopy with multiple orthogonal methods\",\n      \"pmids\": [\"36604498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A homozygous missense variant in CHMP3 (p.Thr173Ile) causes complex hereditary spastic paraplegia. Patient fibroblasts with reduced CHMP3 levels show accumulation of endosomes, autophagosomes, and autolysosomes by electron microscopy, and elevated autophagy markers p62 and LC3-II. Ectopic re-expression of wild-type CHMP3 in patient fibroblasts reduces p62 puncta and the number of endosomes and autophagosomes, directly linking CHMP3 to regulation of the autophagy/endosomal pathway.\",\n      \"method\": \"Exome sequencing, electron microscopy, western blot, immunofluorescence, ectopic wild-type rescue in primary patient fibroblasts\",\n      \"journal\": \"Journal of Medical Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived loss-of-function plus rescue experiment with multiple readouts, single lab\",\n      \"pmids\": [\"35710109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Knockdown of CHMP3 in hepatocellular carcinoma cells activates the caspase-1 pyroptosis signaling pathway, resulting in changes in cell membrane integrity and cytoplasmic leakage; this effect is reversed by the caspase-1 inhibitor AYC, indicating CHMP3 suppresses caspase-1-dependent pyroptosis.\",\n      \"method\": \"siRNA knockdown, western blot of caspase-1 pathway proteins, transmission electron microscopy, caspase-1 inhibitor rescue, xenograft tumor model\",\n      \"journal\": \"International Journal of Oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — defined cellular phenotype with pathway placement via inhibitor rescue and protein expression, single lab\",\n      \"pmids\": [\"38038147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"During mammalian cytokinetic abscission, depletion of CHMP2A causes severe mislocalization of CHMP3 (as well as CHMP4B and CHMP1B) at the abscission site, while IST1 and CHMP2B are minimally affected, placing CHMP3 downstream of CHMP2A in an ordered, hierarchical assembly of ESCRT-III subunits required for timely abscission.\",\n      \"method\": \"CHMP2A knockout, live cell imaging, structured illumination microscopy (SIM), correlative light-electron microscopy (CLEM), dual-protein imaging\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic epistasis with high-resolution imaging in mammalian cells, preprint not yet peer-reviewed, single lab\",\n      \"pmids\": [\"bio_10.1101_2025.06.24.661003\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CHMP3 (VPS24) is an ESCRT-III subunit that adopts a closed autoinhibited conformation in the cytosol and an open active conformation upon membrane recruitment; in its active state it co-polymerizes with CHMP2A into helical heterodimeric filaments on negatively curved membranes via an electrostatic interaction surface, forming a minimal fission machinery with VPS4; it also recruits the deubiquitylase AMSH through a high-affinity, novel MIM–MIT interaction involving its C-terminal region, and it is positioned downstream of CHMP2A in the ordered hierarchical assembly of ESCRT-III during cytokinetic abscission, with loss of CHMP3 function disrupting endosomal/autophagic flux and activating caspase-1-dependent pyroptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CHMP3 (VPS24) is an ESCRT-III subunit that drives membrane scission during budding and abscission events by polymerizing into membrane-associated lattices and recruiting downstream remodeling machinery [#0, #4]. Its crystal structure reveals a flat helical fold that assembles into a lattice through two distinct dimerization modes, presenting a highly basic surface for membrane interaction on one face and a C-terminal VPS4-target sequence on the opposite face; mutation of the basic and dimerization regions abolishes bilayer binding and reverses the dominant-negative block of HIV-1 budding [#0]. In solution CHMP3 toggles between a closed, autoinhibited cytosolic form, in which helix \\u03b15 docks onto the tips of helices \\u03b11/\\u03b12, and an open activated form competent for membrane assembly [#1, #3]. On negatively curved membranes, the open conformer co-polymerizes with CHMP2A into helical heterodimeric filaments whose short N-terminal motifs contact the bilayer and whose C-terminal VPS4-target sequences face the tube interior; electrostatic inter-filament contacts permit VPS4 to constrict and cleave the tubes, defining CHMP2A\\u2013CHMP3\\u2013VPS4 as a minimal membrane fission machinery [#4]. CHMP3 additionally recruits the deubiquitylase AMSH via a high-affinity, novel MIM\\u2013MIT interaction in which its disordered C-terminal region folds into a helix upon binding the AMSH N-terminal MIT domain [#1, #2]. During cytokinetic abscission CHMP3 acts downstream of CHMP2A in the ordered hierarchical assembly of ESCRT-III [#7]. A homozygous CHMP3 missense variant causes complex hereditary spastic paraplegia, with loss of CHMP3 function disrupting endosomal and autophagic flux [#5]; CHMP3 depletion also activates caspase-1-dependent pyroptosis [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established the structural basis for how CHMP3 engages membranes, showing that lattice polymerization with a basic membrane-binding face and an opposed VPS4-target face underlies its role in the final steps of budding.\",\n      \"evidence\": \"X-ray crystallography at 2.8 \\u00c5 with site-directed mutagenesis, in vivo membrane interaction assays, and an HIV-1 budding assay\",\n      \"pmids\": [\"16740483\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how polymerization is triggered on membranes in cells\", \"No structure of CHMP3 in complex with partner ESCRT-III subunits\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Resolved that CHMP3 exists in distinct closed (autoinhibited) and open (activated) conformations and that both bind AMSH with high affinity through its C-terminal region plus additional contributing regions.\",\n      \"evidence\": \"SAXS conformational analysis with ITC binding affinity measurements\",\n      \"pmids\": [\"18395747\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the atomic interface of the CHMP3\\u2013AMSH contact\", \"The trigger converting closed to open state in vivo unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined the CHMP3\\u2013AMSH interaction at atomic resolution, revealing a novel MIM that folds upon binding the AMSH MIT domain, distinct from the CHMP1 site, and showed the AMSH N-terminal helix is functionally required for HIV-1 budding.\",\n      \"evidence\": \"Crystal structure of the AMSH\\u0394C\\u2013CHMP3\\u0394N complex with ITC, SPR, and HIV-1 budding functional assay\",\n      \"pmids\": [\"21827950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of CHMP3-recruited AMSH deubiquitylation on cargo not dissected\", \"Did not test the MIM\\u2013MIT interaction in cytokinesis or endosomal contexts\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Refined the molecular architecture of the autoinhibited CHMP3 state, localizing helix \\u03b15 against the \\u03b11/\\u03b12 tips while helix \\u03b16 remains free, clarifying which elements gate activation.\",\n      \"evidence\": \"SAXS ensemble refinement with coarse-grained molecular simulations using a maximum-entropy approach\",\n      \"pmids\": [\"21220121\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Computational model without mutagenesis validation\", \"Did not capture the activation transition directly\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked CHMP3 function to human disease and to endosomal/autophagic flux, showing a missense variant causes hereditary spastic paraplegia with accumulation of endosomes and autophagosomes reversible by wild-type re-expression.\",\n      \"evidence\": \"Exome sequencing, electron microscopy, western blot, immunofluorescence, and ectopic wild-type rescue in patient fibroblasts\",\n      \"pmids\": [\"35710109\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which p.Thr173Ile impairs ESCRT-III assembly not defined\", \"Single family / single lab\", \"Connection between the autophagy defect and neuronal phenotype unestablished\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Captured the active membrane-bound state, showing CHMP2A\\u2013CHMP3 heterodimers build open-conformation helical filaments that VPS4 constricts and cleaves, defining a minimal fission machinery.\",\n      \"evidence\": \"Cryo-EM at 3.3 and 3.6 \\u00c5, high-speed AFM, fluorescence microscopy, and in vitro membrane tube reconstitution\",\n      \"pmids\": [\"36604498\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How additional ESCRT-III subunits modify this minimal filament in vivo unresolved\", \"Does not address regulation of filament nucleation\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed CHMP3 as a suppressor of caspase-1-dependent pyroptosis in hepatocellular carcinoma cells, connecting its loss to membrane integrity failure.\",\n      \"evidence\": \"siRNA knockdown, western blot of caspase-1 pathway proteins, electron microscopy, caspase-1 inhibitor rescue, and xenograft model\",\n      \"pmids\": [\"38038147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between ESCRT-III fission activity and caspase-1 activation not defined\", \"Single lab / single cancer context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Ordered ESCRT-III assembly during cytokinetic abscission by showing CHMP3 recruitment depends on CHMP2A, placing it downstream in the hierarchy.\",\n      \"evidence\": \"CHMP2A knockout with live-cell imaging, SIM, and CLEM in mammalian cells (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.24.661003\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"Does not establish direct CHMP2A\\u2013CHMP3 contact at the abscission site in vivo\", \"Timing relative to VPS4 recruitment unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CHMP3 conformational activation, partner selection (CHMP2A vs AMSH), and disease-associated dysfunction are coordinated within specific cellular fission events remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full ordered ESCRT-III assembly including CHMP3 in a cellular context\", \"Mechanistic chain from CHMP3 loss to pyroptosis and to neurodegeneration undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\"ESCRT-III\", \"CHMP2A-CHMP3 filament\"],\n    \"partners\": [\"CHMP2A\", \"VPS4\", \"AMSH\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}