{"gene":"VPS4A","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2008,"finding":"VPS4 binds on the inside of helical tubular structures assembled in vitro by ESCRT-III proteins CHMP2A and CHMP3, and disassembles these tubes upon ATP hydrolysis, demonstrating its role in disassembling ESCRT-III helical assemblies.","method":"In vitro reconstitution with purified proteins, electron microscopy, ATPase assay","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with ATP hydrolysis assay and structural imaging, replicated concept across multiple labs","pmids":["18687924"],"is_preprint":false},{"year":2007,"finding":"The MIT domain of human VPS4A binds conserved C-terminal MIM1 amphipathic helices of CHMP1-3 class ESCRT-III proteins; crystal structures of VPS4A MIT-CHMP1A complex reveal the CHMP motif binds in a groove between helices α2 and α3 of the TPR-like repeat in a reversed orientation relative to canonical TPR interactions, with three conserved leucines docking into distinct MIT pockets.","method":"Crystal structure, NMR, mutagenesis, in vitro binding assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis validated in both yeast and human systems, replicated by two simultaneous Nature papers","pmids":["17928862","17928861"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of the yeast Vps4 MIT domain bound to the Vps2 C-terminus reveals that MIT helices α2 and α3 recognize a (D/E)xxLxxRLxxL(K/R) motif; only Vps2 and Did2 among six yeast ESCRT-III-related subunits bind the Vps4 MIT domain, and mutations in the motif cause sorting defects in yeast.","method":"Crystal structure, yeast two-hybrid, mutagenesis, vacuolar sorting assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis with functional readout, independently corroborated by Stuchell-Brereton et al. 2007","pmids":["17928861"],"is_preprint":false},{"year":2005,"finding":"The N-terminal MIT domain of VPS4A forms an asymmetric three-helix bundle resembling the first three helices of a TPR motif; it binds the C-terminal half of ESCRT-III protein CHMP1B (Kd ~20 µM); a conserved Leu-64 on helix 3 mediates CHMP1B binding by completing the TPR motif.","method":"NMR solution structure, mutagenesis, in vitro binding assay","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with mutagenesis and quantitative binding data, single lab","pmids":["16174732"],"is_preprint":false},{"year":2008,"finding":"VPS4 MIT domains bind a second MIM2 motif found in a subset of ESCRT-III subunits (e.g., CHMP6); the NMR solution structure of VPS4 MIT-CHMP6 MIM2 complex shows MIM2 elements bind in extended conformations along the groove between helices 1 and 3 of the MIT domain, distinct from the MIM1 binding site.","method":"NMR solution structure, mutagenesis, HIV-1 budding assay, lysosomal protein targeting assay","journal":"Developmental Cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure plus mutagenesis with two orthogonal functional assays (HIV budding and lysosomal sorting), single lab","pmids":["18606141"],"is_preprint":false},{"year":2000,"finding":"ATPase-defective human VPS4 (unable to bind or hydrolyze ATP) localizes to membranes and specifically induced endocytic vacuoles, whereas wild-type hVPS4 is cytosolic; expression of mutant hVPS4 causes a kinetic block in postendosomal cholesterol sorting without affecting recycling to the TGN or recycling compartment.","method":"Transient expression of wild-type and ATPase-defective hVPS4 mutants in cultured cells, subcellular fractionation, fluorescence microscopy, cholesterol staining","journal":"Molecular Biology of the Cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with dominant-negative approach and defined phenotypic readouts, multiple orthogonal methods","pmids":["10637304"],"is_preprint":false},{"year":2001,"finding":"Mammalian cells express two non-allelic VPS4 paralogs, VPS4-A and VPS4-B (80% identity); both associate with endosomal compartments; VPS4-A and VPS4-B form heteromeric complexes as shown by two-hybrid analysis; expression of dominant-negative VPS4-A(E228Q) or VPS4-B(E235Q) induces vacuolar protein sorting defects in yeast.","method":"Yeast complementation, two-hybrid interaction, subcellular localization (fluorescence microscopy), dominant-negative expression","journal":"Journal of Molecular Biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (yeast complementation, two-hybrid) in single lab","pmids":["11563910"],"is_preprint":false},{"year":2006,"finding":"Vta1 (human SBP1/LIP5) positively regulates Vps4 by promoting proper assembly and stimulating its ATPase activity through the conserved VSL (Vta1/SBP1/LIP5) region, representing an evolutionarily conserved mechanism for regulating ESCRT disassembly.","method":"In vitro ATPase assay, yeast genetic analysis, in vivo ESCRT sorting assay","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ATPase reconstitution plus in vivo genetic assay, single lab with two orthogonal methods","pmids":["16505166"],"is_preprint":false},{"year":2008,"finding":"Two distinct mechanisms stimulate Vps4 ATPase activity: Vps2 directly stimulates Vps4 via its MIT domain, whereas Vps60 stimulates Vps4 via Vta1; Did2 can stimulate Vps4 by both mechanisms in distinct contexts.","method":"In vitro ATPase assay, protein interaction studies, yeast genetics","journal":"Developmental Cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro ATPase assay with multiple substrates and genetic validation, single lab","pmids":["18194652"],"is_preprint":false},{"year":2007,"finding":"Ist1 has a dual role in Vps4 regulation: it positively regulates Vps4 recruitment to ESCRT-III via Did2, and negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer in which Vps4 cannot bind ESCRT machinery.","method":"Yeast genetic analysis, protein interaction assays, localization studies","journal":"Molecular Biology of the Cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (genetics and interaction studies) in single lab","pmids":["18032582"],"is_preprint":false},{"year":2010,"finding":"Assembly of active Vps4 on ESCRT-III requires a network of at least 12 distinct interactions; the most critical interactions are with Vta1, and ESCRT-III subunits Vps2 and Snf7; a recruitment complex of Did2-Ist1-Vps4 forms in the cytoplasm and upon binding to ESCRT-III recruits Vta1, which triggers assembly of the active Vps4 oligomer.","method":"Systematic in vivo interaction network analysis, yeast genetics, localization studies","journal":"Molecular Biology of the Cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic genetic dissection with multiple mutants, single lab","pmids":["20110351"],"is_preprint":false},{"year":2010,"finding":"Depletion of VPS4A or VPS4B, or any of the 11 human ESCRT-III (CHMP) proteins, inhibits cytokinetic abscission; VPS4 proteins concentrate at spindle poles during mitosis and at midbodies during cytokinesis; depletion also alters centrosome and spindle pole numbers, producing multipolar or monopolar spindles.","method":"siRNA knockdown, live-cell fluorescence microscopy, immunofluorescence, cell division assays","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic RNAi depletion of multiple ESCRT-III and VPS4 proteins with defined phenotypic readouts and direct localization, replicated across protein family members","pmids":["20616062"],"is_preprint":false},{"year":2014,"finding":"ANCHR (ZFYVE19) associates with VPS4 and CHMP4C at the midbody ring in an Aurora-B-dependent manner to delay abscission; this association prevents VPS4 relocalization from the midbody ring to the abscission zone; Aurora B inactivation relieves this constraint to allow abscission.","method":"Co-immunoprecipitation, fluorescence microscopy, siRNA knockdown, Aurora-B kinase inhibition, cell division assays","journal":"Nature Cell Biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional localization studies with Aurora-B manipulation, single lab with multiple orthogonal methods","pmids":["24814515"],"is_preprint":false},{"year":2014,"finding":"Coordinated binding of Vps4 to ESCRT-III subunits Vps2 and Snf7 is coupled to membrane neck constriction during intraluminal vesicle (ILV) formation; Vps4 alone (not ESCRT-III alone) is required to complete ILV biogenesis and membrane scission.","method":"Yeast genetics, biochemistry, electron tomography","journal":"Journal of Cell Biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — electron tomography combined with genetics and biochemistry showing mechanistic step, single lab with multiple orthogonal methods","pmids":["24711499"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structure at 4.3 Å of active Vps4 hexamer with cofactor Vta1, ADP·BeFx, and ESCRT-III substrate peptide shows four Vps4 subunits forming a helix that binds substrate; Vta1 stabilizes the helix; the structure supports a 'walking' model in which ATP binding propagates helix growth at one end while hydrolysis promotes disassembly at the other, translocating ESCRT-III through the pore.","method":"Cryo-EM structure (4.3 Å resolution), biochemical assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — near-atomic resolution cryo-EM structure of the active complex with substrate, single lab","pmids":["28379137"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structure at 3.2 Å of Vps4 bound to ESCRT-III peptide substrate reveals the peptide adopts a β-strand conformation with helical symmetry matching the five Vps4 subunits it contacts; adjacent Vps4 subunits make equivalent interactions with successive substrate dipeptides through two classes of pore loop 1 side-chain binding pockets, supporting a 'conveyor belt' translocation model.","method":"Cryo-EM structure (3.2 Å resolution)","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with detailed mechanistic interpretation, builds on prior structure from same lab","pmids":["29165244"],"is_preprint":false},{"year":2015,"finding":"Vps4 completely unfolds ESCRT-III substrates during disassembly (EX1 hydrogen/deuterium exchange behavior); Vps4 hexamers containing single pore-loop cysteines cross-link to cysteines in the folded core domain of ESCRT-III subunits, supporting a mechanism in which Vps4 disassembles ESCRT-III by globally unfolding substrates and threading them through the central pore.","method":"Hydrogen/deuterium exchange mass spectrometry, cysteine cross-linking, in vitro disassembly assay","journal":"Nature Structural & Molecular Biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — two orthogonal biophysical methods (HDX and cross-linking) establishing the unfolding mechanism, single lab","pmids":["25938660"],"is_preprint":false},{"year":2017,"finding":"Quantitative lattice light-sheet microscopy shows that productive MVB budding events require at least two Vps4 hexamers during ESCRT-III assembly, and that membrane budding involves continuous stochastic exchange of Vps4 and ESCRT-III components dependent on Vps4 ATPase activity; acute disruption of Vps4 recruitment by tomographic EM stalled membrane budding.","method":"Quantitative fluorescence lattice light-sheet microscopy, tomographic electron microscopy","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative live imaging with orthogonal EM validation, single lab with multiple methods","pmids":["29019322"],"is_preprint":false},{"year":2019,"finding":"VPS4 constricts and cleaves CHMP2A-CHMP3 helical filaments in vitro as observed by high-speed atomic force microscopy and EM; constriction starts asymmetrically and progressively decreases filament diameter, coiling filaments into dome-like end caps before complete disassembly.","method":"High-speed atomic force microscopy, electron microscopy, in vitro reconstitution with purified proteins","journal":"Science Advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct real-time visualization of VPS4-driven constriction with two orthogonal structural methods, single lab","pmids":["30989108"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structures of the ATP-bound Vps4E233Q hexamer (3.9 Å) and its complex with Vta1 (4.2 Å) reveal six subunits in a spiral-shaped ring; Vta1 dimer bridges two adjacent Vps4 subunits via two different interaction modes at the ring periphery to promote active Vps4 hexamer formation.","method":"Cryo-EM structure determination, single-molecule analysis, biochemical assays","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — near-atomic cryo-EM structures with biochemical validation, single lab","pmids":["28714467"],"is_preprint":false},{"year":2013,"finding":"Active wild-type yeast and archaeal Vps4 enzymes form hexamers (not dodecamers) in the presence of ATP/ADP; Vta1 binds the hexameric Vps4 without changing its oligomeric state; hexamerization interface mutations abolish ATPase activity and block Vps4p function in yeast.","method":"Size-exclusion chromatography, equilibrium analytical ultracentrifugation, crystal structures, mutagenesis, yeast functional assay","journal":"Journal of Molecular Biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures combined with solution biophysics and functional mutagenesis, multiple orthogonal approaches","pmids":["24161953"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM of hydrolysis-deficient Vps4 with ATP shows an asymmetric hexameric ring with a mobile subunit whose substrate-binding loop moves 33 Å from the top to the bottom of the central pore upon a conformational transition; mutant-doping experiments support a sequential and processive ATP hydrolysis mechanism.","method":"Cryo-EM, mutant-doping biochemical assay, ATPase activity assay","journal":"Science Advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM with functional biochemical validation, single lab","pmids":["28439563"],"is_preprint":false},{"year":2015,"finding":"Substrate engagement to the central pore of Vps4 is autoinhibited by the MIT domain and relieved by binding of either Type 1 (MIM1) or Type 2 (MIM2) MIT-interacting motifs; residues from helix 5 of Vps2 bind the central pore of an asymmetric Vps4 hexamer in a nucleotide-state-dependent manner.","method":"Quantitative binding assays, cross-linking, mutagenesis","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative binding studies with mutagenesis revealing autoinhibitory mechanism, single lab with two orthogonal methods","pmids":["25833946"],"is_preprint":false},{"year":2010,"finding":"Purified human VPS4A is essentially inactive but is stimulated to hydrolyze ATP by ESCRT-III proteins (CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, CHMP5) in a reaction requiring their MIT-interacting motifs plus ~50 adjacent residues; the MIT domain and adjacent linker autoinhibit VPS4A; pore loop mutations alter the response to ESCRT-III, consistent with substrate engagement inside the pore.","method":"In vitro ATPase assay with purified proteins, mutagenesis, liposome-based oligomerization assay","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ATPase reconstitution with multiple substrates and mutagenesis, single lab","pmids":["20805225"],"is_preprint":false},{"year":2015,"finding":"The N-terminal domain of LIP5 (LIP5NTD) stimulates VPS4 ATPase activity; CHMP5 binding to LIP5NTD strongly inhibits this stimulation via insertion of a conserved Tyr182 residue into the core of LIP5NTD; crystal structure at 1 Å of LIP5NTD bound to CHMP5 and CHMP1B MIMs reveals ESCRT-III binding-induced conformational change in LIP5NTD.","method":"Crystal structure (1 Å resolution), ATPase assay, mutagenesis","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — atomic-resolution crystal structure with ATPase assay and mutagenesis validating the mechanism, single lab","pmids":["25637630"],"is_preprint":false},{"year":2008,"finding":"LIP5 binds to CHMP5, CHMP1B, CHMP2A, and CHMP3 but not CHMP4A or CHMP6; LIP5 binds a different region in CHMP5 than in other ESCRT-III proteins; a second VPS4 binding site exists in CHMP2A and CHMP1B allowing simultaneous binding of LIP5 and VPS4; LIP5 preferentially binds soluble CHMP5 but polymerized CHMP2A.","method":"Co-immunoprecipitation, in vitro binding assays, competition binding studies","journal":"Molecular Biology of the Cell","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple binding assays but no structural or functional validation, single lab","pmids":["18385515"],"is_preprint":false},{"year":2010,"finding":"VPS4/SKD1 regulates endosomal cholesterol transport independently of ESCRT-III: VPS4 knockdown in HeLa cells causes LDL-cholesterol accumulation in late endosomes/lysosomes, while depletion of any ESCRT-III component has no significant effect on endosomal cholesterol transport.","method":"siRNA knockdown, fluorescence cholesterol staining, NPC1/NPC2 localization assays","journal":"Traffic","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional separation of VPS4 from ESCRT-III by systematic knockdown with defined phenotypic readout, single lab","pmids":["23009658"],"is_preprint":false},{"year":2011,"finding":"Dominant-negative VPS4 impairs MVB biogenesis and blocks lysosomal targeting of α-synuclein while facilitating its extracellular secretion; hypersecretion of α-synuclein in VPS4-defective cells is restored by functional disruption of recycling endosome regulator Rab11a.","method":"Dominant-negative VPS4 expression, secretion assays, Rab11a co-manipulation, immunostaining","journal":"PLoS ONE","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative approach with epistasis (Rab11a rescue) and multiple assay readouts, single lab","pmids":["22216284"],"is_preprint":false},{"year":2015,"finding":"Vps4A facilitates the secretion of oncogenic miRNAs into exosomes and the accumulation of tumor suppressor miRNAs within HCC cells; overexpression of Vps4A inactivates the PI3K/Akt signaling pathway in hepatoma cells.","method":"Small RNA sequencing, Vps4A overexpression/knockdown, exosome isolation, PI3K/Akt pathway analysis","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional studies with gain/loss of function and defined pathway readout, single lab","pmids":["25503676"],"is_preprint":false},{"year":2019,"finding":"Vps4A interacts with β-catenin and CHMP4B (identified by mass spectrometry of immunoprecipitated Vps4A complex); through this interaction, Vps4A promotes plasma membrane localization and exosome release of β-catenin; Vps4A overexpression decreases β-catenin signaling and inhibits EMT in HCC cells.","method":"Co-immunoprecipitation, mass spectrometry, siRNA knockdown, cell fractionation, EMT marker analysis","journal":"Cancer Letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with MS identification plus functional assays, single lab","pmids":["31059752"],"is_preprint":false},{"year":2002,"finding":"VPS4-A directly binds the Rho family GTPase Rnd2 regardless of its nucleotide-bound state (GTP- or GDP-bound); co-expression of Rnd2 with the ATPase-defective VPS4-A(E228Q) recruits Rnd2 to VPS4-A-bound early endosomes.","method":"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation, fluorescence microscopy","journal":"Biochemical Journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — three orthogonal binding assays plus localization, but no downstream functional mechanism defined, single lab","pmids":["11931639"],"is_preprint":false},{"year":2012,"finding":"VPS4A and CHMP6/VPS20 interact directly with H-Ras (but not K-Ras) in endosomes in a GTP-bound and ubiquitylated state-dependent manner; repressing CHMP6 and VPS4A blocks Ras-induced transformation and impairs Ras recycling to the plasma membrane and EGFR recycling.","method":"Co-immunoprecipitation, cell fractionation, FRAP, siRNA knockdown, transformation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus multiple functional assays establishing pathway position, single lab","pmids":["22231449"],"is_preprint":false},{"year":2020,"finding":"De novo missense variants in the ATPase domain of VPS4A cause enlarged endosomal vacuoles; proband-derived fibroblasts show enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane; VPS4A is also required for normal centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, and mitotic spindle formation.","method":"Patient-derived fibroblasts, iPSC-derived neurons, immunofluorescence, dominant-negative VPS4A overexpression as comparison","journal":"American Journal of Human Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with multiple orthogonal cellular phenotype readouts, single study","pmids":["33186545"],"is_preprint":false},{"year":2020,"finding":"VPS4A mutations cause cytokinesis and abscission defects in erythropoiesis (binucleated erythroblasts, cytoplasmic bridges); VPS4A is required for reticulocyte maturation including removal of transferrin receptor (CD71) from the red blood cell membrane; iPSC-derived erythroid cells from probands recapitulate the dyserythropoietic phenotype.","method":"Bone marrow analysis, iPSC differentiation, flow cytometry (CD71), patient-derived fibroblast studies","journal":"American Journal of Human Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived iPSC model with defined mechanistic readouts (cytokinesis, transferrin receptor trafficking), single study","pmids":["33186543"],"is_preprint":false},{"year":2018,"finding":"GFP-VPS4 is a dynamic component of both mother and daughter centrioles; ATPase-defective VPS4EQ accumulates at centrosomes and reduces γ-tubulin levels, decreases microtubule growth, eliminates centriolar satellites, and pauses ciliogenesis after ciliary vesicle formation; ESCRT-III proteins rarely localize to centrosomes and their depletion does not phenocopy VPS4EQ, indicating an ESCRT-III-independent centrosomal function.","method":"Live-cell fluorescence microscopy, FRAP, siRNA knockdown, zebrafish embryo injection, immunofluorescence","journal":"Scientific Reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct live-cell localization with functional consequence, ESCRT-III independence established by parallel knockdowns, single lab","pmids":["29463826"],"is_preprint":false},{"year":2022,"finding":"HCV infection activates ROS/JNK signaling to phosphorylate and activate the E3 ubiquitin ligase Itch, which specifically polyubiquitylates VPS4A (at K23 and K121) but not VPS4B; this polyubiquitylation enhances the VPS4A-CHMP1B interaction, increases VPS4A ATPase activity, and promotes HCV particle release.","method":"siRNA knockdown, site-directed mutagenesis, co-immunoprecipitation, ATPase activity assay, JNK inhibition, HCV infectivity titer assay","journal":"Journal of Virology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — identifies writer (Itch), modification sites (K23/K121), demonstrates functional consequence on ATPase activity and CHMP1B interaction with orthogonal methods, single lab","pmids":["35044214"],"is_preprint":false},{"year":2024,"finding":"VPS4A functions as a selective receptor for lipophagy: phosphorylation of VPS4A at Ser95 and Ser97 drives its localization to lipid droplets in response to fasting; VPS4A and lipid droplets are concomitantly degraded in lysosomes in an ATG7-sensitive manner; silencing VPS4A or blocking its LC3 binding specifically inhibits lipophagy without affecting other selective autophagy pathways.","method":"Mouse liver phosphoproteomics, 3D imaging reconstruction, co-immunoprecipitation, siRNA knockdown, phosphorylation site mutagenesis, lysosomal degradation assay","journal":"Molecular Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — identifies phosphorylation events, localization to lipid droplets, LC3 interaction, and lysosomal co-degradation with multiple orthogonal methods including in vivo and in vitro evidence","pmids":["39520981"],"is_preprint":false},{"year":2024,"finding":"VPS4A is a direct target of the compound aloperine; amino acids F153 and D263 of VPS4A are confirmed as aloperine binding sites; VPS4A knockout mimics aloperine treatment, inhibiting autophagosome-lysosome fusion and blocking autophagic flux.","method":"Target identification by drug-protein interaction assays, CRISPR knockout, autophagy flux assays","journal":"Advanced Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding site identification with CRISPR KO phenocopying drug effect, single lab","pmids":["39166458"],"is_preprint":false},{"year":2021,"finding":"The conserved V domain of Bro1 (yeast homologue of ALIX/HD-PTP) directly stimulates Vps4; this stimulatory activity is required for MVB cargo sorting; ubiquitin binding enhances V domain stimulation of Vps4 to promote intraluminal vesicle formation.","method":"In vitro ATPase assay, yeast genetics, in vivo MVB sorting assay","journal":"Journal of Cell Biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct in vitro stimulation assay combined with yeast functional genetics, single lab","pmids":["34160559"],"is_preprint":false},{"year":2015,"finding":"Ist1 can both inhibit and stimulate Vps4 ATPase activity depending on its conformation: inhibition requires both the MIM and a conserved ELYC surface; binding of the ESCRT-III partner Did2 converts Ist1 from an inhibitor to a stimulator of Vps4, coordinating Vps4 activity with the timing of ESCRT-III disassembly.","method":"In vitro ATPase assay, ESCRT-III disassembly assay, mutagenesis","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro ATPase and disassembly assays with mutagenesis, single lab","pmids":["26515066"],"is_preprint":false},{"year":2014,"finding":"The Vps4 stimulatory element (VSE) of Vta1 contacts Vps4 α-helices 7 and 9 of the small AAA domain to stimulate ATP hydrolysis; intergenic compensatory mutations between VSE and these Vps4 helices validate the interaction surface.","method":"Mutagenesis, ATPase assay, yeast complementation, structural modeling","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis with compensatory mutations and functional ATPase assay, single lab","pmids":["25164817"],"is_preprint":false},{"year":2014,"finding":"Vfa1 binds to the MIT domain of Vps4 via its C-terminal 17 residues adopting a canonical MIM2 conformation (crystal structure determined); this interaction is high affinity and greatly stimulates Vps4 ATPase activity.","method":"Crystal structure of Vps4-MIT/Vfa1 complex, ATPase assay, SPR binding measurement","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with quantitative binding and ATPase stimulation assay, single lab","pmids":["24567329"],"is_preprint":false},{"year":2019,"finding":"Vps4 can bind and translocate cyclic peptides in a hairpin conformation through its central pore, with one strand making primary contacts and the second returning through the pore without intimate contacts; this reveals a general mechanism for translocation of extended chains, hairpins, and crosslinked polypeptides.","method":"Cryo-EM structure of cyclic peptide complexes, binding affinity measurements","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with binding assays revealing translocation mechanism, single lab","pmids":["31184588"],"is_preprint":false}],"current_model":"VPS4A is a hexameric AAA+ ATPase that functions as the primary energy-providing enzyme of the ESCRT pathway: its N-terminal MIT domain binds C-terminal MIM1 and MIM2 motifs on ESCRT-III subunits (CHMP proteins) to recruit the enzyme to ESCRT-III filaments, whereupon ESCRT-III binding relieves MIT-domain autoinhibition and activates the central pore, which translocates and globally unfolds ESCRT-III subunits by a processive 'conveyor belt' mechanism driven by sequential ATP hydrolysis around an asymmetric hexameric ring, thereby constricting and disassembling ESCRT-III filaments to drive membrane neck constriction and fission during MVB biogenesis, cytokinesis, nuclear envelope/NPC surveillance, and enveloped virus budding; VPS4A activity is positively regulated by the cofactor LIP5/Vta1 (which bridges adjacent subunits and contains a VSE element that contacts Vps4 α7/α9) and by direct stimulation from ESCRT-III subunits Vps2 and Snf7/CHMP4, and is negatively regulated by Ist1 unless it is partnered with Did2; additionally, VPS4A performs ESCRT-III-independent functions at centrosomes (regulating γ-tubulin, centriolar satellites, and ciliogenesis), in lipophagy (acting as a selective receptor for lipid droplet autophagy upon phosphorylation at Ser95/97 enabling LC3 binding), and in endosomal cholesterol trafficking, and its activity is modulated post-translationally by polyubiquitylation at K23/K121 by the E3 ligase Itch downstream of HCV-induced ROS/JNK signaling."},"narrative":{"mechanistic_narrative":"VPS4A is the energy-providing AAA+ ATPase of the ESCRT pathway, a hexameric enzyme that disassembles ESCRT-III filaments to drive membrane constriction and fission [PMID:18687924, PMID:24161953]. Its N-terminal MIT domain forms an asymmetric three-helix bundle that recognizes ESCRT-III subunits through two distinct interaction modes: it binds C-terminal MIM1 amphipathic helices of CHMP1-3-class subunits in a groove between MIT helices α2 and α3 [PMID:17928862, PMID:17928861, PMID:16174732], and binds a second MIM2 motif of subunits such as CHMP6 in an extended conformation along the groove between helices 1 and 3 [PMID:18606141]. Purified VPS4A is essentially inactive and autoinhibited by its MIT domain and adjacent linker; ESCRT-III binding relieves this autoinhibition and engages substrate in the central pore to stimulate ATP hydrolysis [PMID:25833946, PMID:20805225]. Active VPS4 assembles into an asymmetric spiral hexamer that processively translocates and globally unfolds ESCRT-III substrates through its central pore by a sequential, 'conveyor-belt' hydrolysis mechanism, thereby constricting and severing ESCRT-III filaments [PMID:28379137, PMID:29165244, PMID:25938660, PMID:30989108, PMID:28439563]. This activity is tuned by an extensive cofactor network: LIP5/Vta1 stabilizes the active hexamer and stimulates ATPase activity via its conserved VSE/VSL elements that bridge adjacent subunits and contact VPS4 helices α7/α9 [PMID:16505166, PMID:28714467, PMID:25637630, PMID:25164817], ESCRT-III subunits Vps2 and Snf7 and the Bro1 V domain directly stimulate the enzyme [PMID:18194652, PMID:24711499, PMID:34160559], and Ist1 acts as a context-dependent inhibitor or activator depending on its partnership with Did2 [PMID:18032582, PMID:26515066]. Through this machinery VPS4A executes membrane scission in MVB/intraluminal-vesicle biogenesis and cytokinetic abscission, where it concentrates at spindle poles and midbodies and is held in check by an Aurora-B/ANCHR checkpoint at the midbody ring [PMID:20616062, PMID:24814515, PMID:24711499, PMID:29019322]. VPS4A additionally performs ESCRT-III-independent roles: regulation of endosomal cholesterol transport [PMID:10637304, PMID:23009658], centrosome/centriole homeostasis and ciliogenesis [PMID:29463826], and lipophagy, where phosphorylation at Ser95/97 directs it to lipid droplets and enables LC3 binding for selective autophagic degradation [PMID:39520981]. De novo missense variants in the VPS4A ATPase domain cause a multisystem disorder featuring enlarged endosomal vacuoles, dyserythropoiesis with cytokinesis defects, and neurodevelopmental phenotypes [PMID:33186545, PMID:33186543].","teleology":[{"year":2000,"claim":"Establishing that VPS4 ATPase activity gates a discrete membrane-trafficking step, the first cell-based work showed catalytically dead enzyme dominantly blocks a defined sorting process rather than acting cytosolically.","evidence":"Expression of ATPase-defective hVPS4 mutants in cultured cells with fractionation and cholesterol staining","pmids":["10637304"],"confidence":"High","gaps":["Did not define the molecular substrate of the ATPase","Mechanism of membrane recruitment unknown at this stage"]},{"year":2001,"claim":"Resolving the gene architecture in mammals, two paralogs VPS4A and VPS4B were shown to associate with endosomes and form heteromeric complexes, framing later paralog-specific questions.","evidence":"Yeast complementation, two-hybrid, dominant-negative expression and localization","pmids":["11563910"],"confidence":"Medium","gaps":["Functional division of labor between VPS4A and VPS4B not resolved","Oligomeric state misassigned (later corrected to hexamer)"]},{"year":2005,"claim":"To explain how VPS4 finds its targets, the MIT domain was solved and shown to recognize the C-terminal half of an ESCRT-III subunit, identifying the substrate-recruitment module.","evidence":"NMR solution structure of VPS4A MIT with CHMP1B binding and mutagenesis","pmids":["16174732"],"confidence":"High","gaps":["Only one ESCRT-III partner tested","Did not address how binding couples to ATPase activation"]},{"year":2007,"claim":"Defining the MIM1 recognition code, crystal structures established the conserved amphipathic-helix motif and the reversed TPR-like binding mode, explaining ESCRT-III selectivity of the MIT domain across yeast and human.","evidence":"Crystal structures of VPS4 MIT-CHMP1A and yeast Vps4 MIT-Vps2 with mutagenesis and sorting assays","pmids":["17928862","17928861"],"confidence":"High","gaps":["Did not explain subunits lacking MIM1","Did not capture the assembled hexamer-substrate complex"]},{"year":2008,"claim":"Extending the recognition repertoire, a second motif class (MIM2) bound at a distinct MIT site was identified, showing VPS4 reads multiple ESCRT-III tags relevant to HIV budding and lysosomal sorting.","evidence":"NMR structure of VPS4 MIT-CHMP6 MIM2 with HIV-1 budding and lysosomal targeting assays","pmids":["18606141"],"confidence":"High","gaps":["Relative contributions of MIM1 vs MIM2 in vivo unresolved","How dual motifs coordinate on filaments not addressed"]},{"year":2008,"claim":"Demonstrating direct disassembly, reconstitution showed VPS4 binds inside ESCRT-III tubes and severs them upon ATP hydrolysis, providing the mechanistic basis for filament turnover.","evidence":"In vitro reconstitution with purified CHMP2A/CHMP3, EM, ATPase assay","pmids":["18687924"],"confidence":"High","gaps":["Atomic mechanism of substrate engagement still unknown","Cofactor requirements for full activity not yet defined"]},{"year":2008,"claim":"Beginning to map cofactor control, Vta1/LIP5 was shown to stimulate ATPase activity and promote assembly, and distinct stimulation routes (Vps2 direct vs Vps60/Did2 via Vta1) were distinguished.","evidence":"In vitro ATPase assays, yeast genetics, binding studies; LIP5-CHMP co-IP/competition assays","pmids":["16505166","18194652","18385515"],"confidence":"High","gaps":["Structural basis of Vta1 stimulation not yet known","Quantitative integration of multiple stimulators unresolved"]},{"year":2010,"claim":"Connecting the enzyme to cell division, depletion experiments showed VPS4 and ESCRT-III are required for cytokinetic abscission and that VPS4 localizes to spindle poles and midbodies.","evidence":"siRNA knockdown of VPS4A/B and CHMP proteins with live-cell imaging and division assays","pmids":["20616062"],"confidence":"High","gaps":["Spatial trigger of abscission-associated VPS4 recruitment not defined","Centrosome phenotype mechanism unclear"]},{"year":2010,"claim":"Quantifying autoinhibition and activation, human VPS4A was shown to be intrinsically inactive and stimulated by ESCRT-III via MIM plus adjacent residues with substrate engaging the pore, and a multi-interaction assembly network was mapped.","evidence":"In vitro ATPase assays with purified human proteins and mutagenesis; systematic yeast interaction-network genetics","pmids":["20805225","20110351"],"confidence":"High","gaps":["Structure of the active substrate-engaged hexamer not yet available","Order of cofactor recruitment in mammals not defined"]},{"year":2013,"claim":"Settling the oligomeric state, biophysics and crystallography established that active Vps4 is a hexamer whose assembly interface is essential for ATPase activity and function.","evidence":"Size-exclusion chromatography, analytical ultracentrifugation, crystal structures, yeast functional assays","pmids":["24161953"],"confidence":"High","gaps":["Asymmetry and nucleotide-state coupling not yet resolved","Substrate path through the pore not visualized"]},{"year":2014,"claim":"Linking enzyme action to membrane geometry and division control, work showed Vps4 binding to Vps2/Snf7 drives intraluminal-vesicle scission and that an Aurora-B/ANCHR checkpoint restrains VPS4 at the midbody.","evidence":"Yeast genetics with electron tomography; co-IP, imaging, and Aurora-B inhibition in cells","pmids":["24711499","24814515"],"confidence":"High","gaps":["How the timing signal translates to pore activity not defined","Molecular trigger relocating VPS4 to the abscission zone unclear"]},{"year":2015,"claim":"Defining the disassembly mechanism, biophysics demonstrated VPS4 globally unfolds ESCRT-III by threading it through the pore, and binding studies showed MIM engagement relieves MIT autoinhibition to permit central-pore substrate capture.","evidence":"HDX-MS and cysteine cross-linking; quantitative binding, cross-linking and mutagenesis; LIP5NTD crystal structure with CHMP MIMs","pmids":["25938660","25833946","25637630"],"confidence":"High","gaps":["Step-resolved coupling of hydrolysis to translocation not yet imaged","CHMP5 inhibitory braking not tested in vivo"]},{"year":2017,"claim":"Achieving the mechanistic synthesis, cryo-EM of active hexamers with substrate, Vta1, and nucleotide established a sequential, processive 'conveyor-belt' translocation through an asymmetric spiral ring and the Vta1 bridging mode that promotes the active oligomer.","evidence":"Multiple cryo-EM structures (3.2-4.3 Å) of Vps4-substrate, Vps4-Vta1, and ATP-bound hexamers with biochemical/mutant-doping validation","pmids":["28379137","29165244","28714467","28439563"],"confidence":"High","gaps":["Full-length filament constriction not captured in these structures","In-cell stoichiometry of functional units not established here"]},{"year":2017,"claim":"Bridging structure to cell biology, quantitative live imaging showed productive MVB budding requires multiple Vps4 hexamers with continuous ATPase-dependent component exchange, and acute disruption stalls budding.","evidence":"Lattice light-sheet microscopy and tomographic EM","pmids":["29019322"],"confidence":"High","gaps":["Exact number and lifetime of hexamers per scission event approximate","Generalization to other membrane sites untested"]},{"year":2019,"claim":"Visualizing constriction directly, real-time AFM/EM showed VPS4 progressively narrows and coils CHMP2A-CHMP3 filaments into end caps before disassembly, and a cyclic-peptide structure generalized the pore translocation mechanism to hairpin and crosslinked chains.","evidence":"High-speed AFM and EM of reconstituted filaments; cryo-EM of Vps4-cyclic peptide complexes","pmids":["30989108","31184588"],"confidence":"High","gaps":["Coupling of constriction to membrane fission not directly observed","Physiological substrate topology range not fully mapped"]},{"year":2020,"claim":"Establishing disease causation, de novo ATPase-domain variants were shown to produce enlarged endosomes with aberrant IST1 accumulation, cytokinesis/abscission failures in erythropoiesis, and defects in centrosome, cilium, nuclear membrane and chromosome segregation.","evidence":"Patient-derived fibroblasts, iPSC-derived neurons and erythroid cells, immunofluorescence, flow cytometry, dominant-negative comparison","pmids":["33186545","33186543"],"confidence":"Medium","gaps":["Dominant-negative vs loss-of-function basis of variants not fully dissected","Tissue-specific severity determinants unknown"]},{"year":2022,"claim":"Identifying paralog-specific post-translational control, HCV-induced ROS/JNK signaling was shown to activate Itch, which polyubiquitylates VPS4A at K23/K121, enhancing CHMP1B binding and ATPase activity to promote virus release.","evidence":"siRNA, site-directed mutagenesis, co-IP, ATPase assay, JNK inhibition, HCV titer assay","pmids":["35044214"],"confidence":"High","gaps":["Whether this ubiquitylation operates outside HCV infection unknown","Structural effect of K23/K121 modification on the hexamer undefined"]},{"year":2024,"claim":"Revealing a moonlighting catabolic role, VPS4A was shown to act as a selective lipophagy receptor: fasting-induced Ser95/97 phosphorylation drives lipid-droplet localization and LC3 binding for lysosomal co-degradation.","evidence":"Mouse liver phosphoproteomics, 3D imaging, co-IP, siRNA, phospho-site mutagenesis, lysosomal degradation assays; plus aloperine target-ID and CRISPR KO autophagy-flux work","pmids":["39520981","39166458"],"confidence":"High","gaps":["Relationship between lipophagy receptor role and ESCRT ATPase activity unresolved","Kinase phosphorylating Ser95/97 not identified"]},{"year":null,"claim":"How the well-defined biochemical hexamer mechanism is partitioned among ESCRT-dependent scission and the ESCRT-III-independent roles (centrosome/cilium, endosomal cholesterol, lipophagy), and how VPS4A versus VPS4B specialize, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model for ESCRT-III-independent substrate engagement","Paralog-specific functional division not defined","Upstream kinases/regulators of non-ESCRT functions largely unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,7,20,23]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,16,18]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,3,4,16]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5,6,26,30]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[11,34]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[36]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[26,36]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,13,17]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[11,12,13]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[36,37]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[13,17,26]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[34]}],"complexes":["VPS4 hexamer","ESCRT-III disassembly machinery","VPS4-Vta1/LIP5 complex"],"partners":["CHMP1B","CHMP2A","CHMP3","CHMP6","LIP5","IST1","CHMP4B","CHMP5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UN37","full_name":"Vacuolar protein sorting-associated protein 4A","aliases":["Protein SKD2","VPS4-1","hVPS4"],"length_aa":437,"mass_kda":48.9,"function":"Involved in late steps of the endosomal multivesicular bodies (MVB) pathway. Recognizes membrane-associated ESCRT-III assemblies and catalyzes their disassembly, possibly in combination with membrane fission. Redistributes the ESCRT-III components to the cytoplasm for further rounds of MVB sorting. 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. It is required for proper accomplishment of various processes including the regulation of endosome size, primary cilium organization, mitotic spindle organization, chromosome segregation, and nuclear envelope sealing and spindle disassembly during anaphase (PubMed:33186545). Involved in cytokinesis: retained at the midbody by ZFYVE19/ANCHR and CHMP4C until abscission checkpoint signaling is terminated at late cytokinesis. It is then released following dephosphorylation of CHMP4C, leading to abscission (PubMed:24814515). VPS4A/B are required for the exosomal release of SDCBP, CD63 and syndecan (PubMed:22660413). Critical for normal erythroblast cytokinesis and correct erythropoiesis (PubMed:33186543) (Microbial infection) In conjunction with the ESCRT machinery also appears to function in topologically equivalent membrane fission events, such as the terminal stages of cytokinesis and enveloped virus budding (HIV-1 and other lentiviruses)","subcellular_location":"Late endosome membrane; Midbody; Cytoplasm, cytoskeleton, spindle","url":"https://www.uniprot.org/uniprotkb/Q9UN37/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/VPS4A","classification":"Not Classified","n_dependent_lines":355,"n_total_lines":1208,"dependency_fraction":0.29387417218543044},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000132612","cell_line_id":"CID000260","localizations":[{"compartment":"vesicles","grade":3},{"compartment":"cytoplasmic","grade":2},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"CHMP1A","stoichiometry":0.2},{"gene":"IST1","stoichiometry":0.2},{"gene":"CHMP2A","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000260","total_profiled":1310},"omim":[{"mim_id":"621486","title":"MICROTUBULE-INTERACTING AND TRAFFICKING DOMAIN-CONTAINING PROTEIN 1; MITD1","url":"https://www.omim.org/entry/621486"},{"mim_id":"619768","title":"ARRESTIN DOMAIN-CONTAINING PROTEIN 1; ARRDC1","url":"https://www.omim.org/entry/619768"},{"mim_id":"619635","title":"ZINC FINGER FYVE DOMAIN-CONTAINING PROTEIN 19; ZFYVE19","url":"https://www.omim.org/entry/619635"},{"mim_id":"619273","title":"CIMDAG SYNDROME; CIMDAG","url":"https://www.omim.org/entry/619273"},{"mim_id":"616434","title":"IST1 FACTOR ASSOCIATED WITH ESCRT-III; IST1","url":"https://www.omim.org/entry/616434"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Midbody","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/VPS4A"},"hgnc":{"alias_symbol":["VPS4","VPS4-1","FLJ22197","SKD2","SKD1","SKD1A"],"prev_symbol":[]},"alphafold":{"accession":"Q9UN37","domains":[{"cath_id":"1.20.58.80","chopping":"4-75","consensus_level":"high","plddt":89.2415,"start":4,"end":75},{"cath_id":"3.40.50.300","chopping":"117-289","consensus_level":"high","plddt":87.4673,"start":117,"end":289},{"cath_id":"1.10.8.60","chopping":"295-417","consensus_level":"high","plddt":95.2661,"start":295,"end":417}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UN37","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UN37-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UN37-F1-predicted_aligned_error_v6.png","plddt_mean":85.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VPS4A","jax_strain_url":"https://www.jax.org/strain/search?query=VPS4A"},"sequence":{"accession":"Q9UN37","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UN37.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UN37/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UN37"}},"corpus_meta":[{"pmid":"18687924","id":"PMC_18687924","title":"Helical 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ATPase assay\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with ATP hydrolysis assay and structural imaging, replicated concept across multiple labs\",\n      \"pmids\": [\"18687924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The MIT domain of human VPS4A binds conserved C-terminal MIM1 amphipathic helices of CHMP1-3 class ESCRT-III proteins; crystal structures of VPS4A MIT-CHMP1A complex reveal the CHMP motif binds in a groove between helices α2 and α3 of the TPR-like repeat in a reversed orientation relative to canonical TPR interactions, with three conserved leucines docking into distinct MIT pockets.\",\n      \"method\": \"Crystal structure, NMR, mutagenesis, in vitro binding assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis validated in both yeast and human systems, replicated by two simultaneous Nature papers\",\n      \"pmids\": [\"17928862\", \"17928861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of the yeast Vps4 MIT domain bound to the Vps2 C-terminus reveals that MIT helices α2 and α3 recognize a (D/E)xxLxxRLxxL(K/R) motif; only Vps2 and Did2 among six yeast ESCRT-III-related subunits bind the Vps4 MIT domain, and mutations in the motif cause sorting defects in yeast.\",\n      \"method\": \"Crystal structure, yeast two-hybrid, mutagenesis, vacuolar sorting assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis with functional readout, independently corroborated by Stuchell-Brereton et al. 2007\",\n      \"pmids\": [\"17928861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The N-terminal MIT domain of VPS4A forms an asymmetric three-helix bundle resembling the first three helices of a TPR motif; it binds the C-terminal half of ESCRT-III protein CHMP1B (Kd ~20 µM); a conserved Leu-64 on helix 3 mediates CHMP1B binding by completing the TPR motif.\",\n      \"method\": \"NMR solution structure, mutagenesis, in vitro binding assay\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with mutagenesis and quantitative binding data, single lab\",\n      \"pmids\": [\"16174732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"VPS4 MIT domains bind a second MIM2 motif found in a subset of ESCRT-III subunits (e.g., CHMP6); the NMR solution structure of VPS4 MIT-CHMP6 MIM2 complex shows MIM2 elements bind in extended conformations along the groove between helices 1 and 3 of the MIT domain, distinct from the MIM1 binding site.\",\n      \"method\": \"NMR solution structure, mutagenesis, HIV-1 budding assay, lysosomal protein targeting assay\",\n      \"journal\": \"Developmental Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus mutagenesis with two orthogonal functional assays (HIV budding and lysosomal sorting), single lab\",\n      \"pmids\": [\"18606141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"ATPase-defective human VPS4 (unable to bind or hydrolyze ATP) localizes to membranes and specifically induced endocytic vacuoles, whereas wild-type hVPS4 is cytosolic; expression of mutant hVPS4 causes a kinetic block in postendosomal cholesterol sorting without affecting recycling to the TGN or recycling compartment.\",\n      \"method\": \"Transient expression of wild-type and ATPase-defective hVPS4 mutants in cultured cells, subcellular fractionation, fluorescence microscopy, cholesterol staining\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with dominant-negative approach and defined phenotypic readouts, multiple orthogonal methods\",\n      \"pmids\": [\"10637304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mammalian cells express two non-allelic VPS4 paralogs, VPS4-A and VPS4-B (80% identity); both associate with endosomal compartments; VPS4-A and VPS4-B form heteromeric complexes as shown by two-hybrid analysis; expression of dominant-negative VPS4-A(E228Q) or VPS4-B(E235Q) induces vacuolar protein sorting defects in yeast.\",\n      \"method\": \"Yeast complementation, two-hybrid interaction, subcellular localization (fluorescence microscopy), dominant-negative expression\",\n      \"journal\": \"Journal of Molecular Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (yeast complementation, two-hybrid) in single lab\",\n      \"pmids\": [\"11563910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Vta1 (human SBP1/LIP5) positively regulates Vps4 by promoting proper assembly and stimulating its ATPase activity through the conserved VSL (Vta1/SBP1/LIP5) region, representing an evolutionarily conserved mechanism for regulating ESCRT disassembly.\",\n      \"method\": \"In vitro ATPase assay, yeast genetic analysis, in vivo ESCRT sorting assay\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ATPase reconstitution plus in vivo genetic assay, single lab with two orthogonal methods\",\n      \"pmids\": [\"16505166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Two distinct mechanisms stimulate Vps4 ATPase activity: Vps2 directly stimulates Vps4 via its MIT domain, whereas Vps60 stimulates Vps4 via Vta1; Did2 can stimulate Vps4 by both mechanisms in distinct contexts.\",\n      \"method\": \"In vitro ATPase assay, protein interaction studies, yeast genetics\",\n      \"journal\": \"Developmental Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro ATPase assay with multiple substrates and genetic validation, single lab\",\n      \"pmids\": [\"18194652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Ist1 has a dual role in Vps4 regulation: it positively regulates Vps4 recruitment to ESCRT-III via Did2, and negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer in which Vps4 cannot bind ESCRT machinery.\",\n      \"method\": \"Yeast genetic analysis, protein interaction assays, localization studies\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (genetics and interaction studies) in single lab\",\n      \"pmids\": [\"18032582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Assembly of active Vps4 on ESCRT-III requires a network of at least 12 distinct interactions; the most critical interactions are with Vta1, and ESCRT-III subunits Vps2 and Snf7; a recruitment complex of Did2-Ist1-Vps4 forms in the cytoplasm and upon binding to ESCRT-III recruits Vta1, which triggers assembly of the active Vps4 oligomer.\",\n      \"method\": \"Systematic in vivo interaction network analysis, yeast genetics, localization studies\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic genetic dissection with multiple mutants, single lab\",\n      \"pmids\": [\"20110351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Depletion of VPS4A or VPS4B, or any of the 11 human ESCRT-III (CHMP) proteins, inhibits cytokinetic abscission; VPS4 proteins concentrate at spindle poles during mitosis and at midbodies during cytokinesis; depletion also alters centrosome and spindle pole numbers, producing multipolar or monopolar spindles.\",\n      \"method\": \"siRNA knockdown, live-cell fluorescence microscopy, immunofluorescence, cell division assays\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic RNAi depletion of multiple ESCRT-III and VPS4 proteins with defined phenotypic readouts and direct localization, replicated across protein family members\",\n      \"pmids\": [\"20616062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ANCHR (ZFYVE19) associates with VPS4 and CHMP4C at the midbody ring in an Aurora-B-dependent manner to delay abscission; this association prevents VPS4 relocalization from the midbody ring to the abscission zone; Aurora B inactivation relieves this constraint to allow abscission.\",\n      \"method\": \"Co-immunoprecipitation, fluorescence microscopy, siRNA knockdown, Aurora-B kinase inhibition, cell division assays\",\n      \"journal\": \"Nature Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional localization studies with Aurora-B manipulation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24814515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Coordinated binding of Vps4 to ESCRT-III subunits Vps2 and Snf7 is coupled to membrane neck constriction during intraluminal vesicle (ILV) formation; Vps4 alone (not ESCRT-III alone) is required to complete ILV biogenesis and membrane scission.\",\n      \"method\": \"Yeast genetics, biochemistry, electron tomography\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — electron tomography combined with genetics and biochemistry showing mechanistic step, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24711499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structure at 4.3 Å of active Vps4 hexamer with cofactor Vta1, ADP·BeFx, and ESCRT-III substrate peptide shows four Vps4 subunits forming a helix that binds substrate; Vta1 stabilizes the helix; the structure supports a 'walking' model in which ATP binding propagates helix growth at one end while hydrolysis promotes disassembly at the other, translocating ESCRT-III through the pore.\",\n      \"method\": \"Cryo-EM structure (4.3 Å resolution), biochemical assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — near-atomic resolution cryo-EM structure of the active complex with substrate, single lab\",\n      \"pmids\": [\"28379137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structure at 3.2 Å of Vps4 bound to ESCRT-III peptide substrate reveals the peptide adopts a β-strand conformation with helical symmetry matching the five Vps4 subunits it contacts; adjacent Vps4 subunits make equivalent interactions with successive substrate dipeptides through two classes of pore loop 1 side-chain binding pockets, supporting a 'conveyor belt' translocation model.\",\n      \"method\": \"Cryo-EM structure (3.2 Å resolution)\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with detailed mechanistic interpretation, builds on prior structure from same lab\",\n      \"pmids\": [\"29165244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Vps4 completely unfolds ESCRT-III substrates during disassembly (EX1 hydrogen/deuterium exchange behavior); Vps4 hexamers containing single pore-loop cysteines cross-link to cysteines in the folded core domain of ESCRT-III subunits, supporting a mechanism in which Vps4 disassembles ESCRT-III by globally unfolding substrates and threading them through the central pore.\",\n      \"method\": \"Hydrogen/deuterium exchange mass spectrometry, cysteine cross-linking, in vitro disassembly assay\",\n      \"journal\": \"Nature Structural & Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — two orthogonal biophysical methods (HDX and cross-linking) establishing the unfolding mechanism, single lab\",\n      \"pmids\": [\"25938660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Quantitative lattice light-sheet microscopy shows that productive MVB budding events require at least two Vps4 hexamers during ESCRT-III assembly, and that membrane budding involves continuous stochastic exchange of Vps4 and ESCRT-III components dependent on Vps4 ATPase activity; acute disruption of Vps4 recruitment by tomographic EM stalled membrane budding.\",\n      \"method\": \"Quantitative fluorescence lattice light-sheet microscopy, tomographic electron microscopy\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative live imaging with orthogonal EM validation, single lab with multiple methods\",\n      \"pmids\": [\"29019322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"VPS4 constricts and cleaves CHMP2A-CHMP3 helical filaments in vitro as observed by high-speed atomic force microscopy and EM; constriction starts asymmetrically and progressively decreases filament diameter, coiling filaments into dome-like end caps before complete disassembly.\",\n      \"method\": \"High-speed atomic force microscopy, electron microscopy, in vitro reconstitution with purified proteins\",\n      \"journal\": \"Science Advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct real-time visualization of VPS4-driven constriction with two orthogonal structural methods, single lab\",\n      \"pmids\": [\"30989108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structures of the ATP-bound Vps4E233Q hexamer (3.9 Å) and its complex with Vta1 (4.2 Å) reveal six subunits in a spiral-shaped ring; Vta1 dimer bridges two adjacent Vps4 subunits via two different interaction modes at the ring periphery to promote active Vps4 hexamer formation.\",\n      \"method\": \"Cryo-EM structure determination, single-molecule analysis, biochemical assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — near-atomic cryo-EM structures with biochemical validation, single lab\",\n      \"pmids\": [\"28714467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Active wild-type yeast and archaeal Vps4 enzymes form hexamers (not dodecamers) in the presence of ATP/ADP; Vta1 binds the hexameric Vps4 without changing its oligomeric state; hexamerization interface mutations abolish ATPase activity and block Vps4p function in yeast.\",\n      \"method\": \"Size-exclusion chromatography, equilibrium analytical ultracentrifugation, crystal structures, mutagenesis, yeast functional assay\",\n      \"journal\": \"Journal of Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures combined with solution biophysics and functional mutagenesis, multiple orthogonal approaches\",\n      \"pmids\": [\"24161953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM of hydrolysis-deficient Vps4 with ATP shows an asymmetric hexameric ring with a mobile subunit whose substrate-binding loop moves 33 Å from the top to the bottom of the central pore upon a conformational transition; mutant-doping experiments support a sequential and processive ATP hydrolysis mechanism.\",\n      \"method\": \"Cryo-EM, mutant-doping biochemical assay, ATPase activity assay\",\n      \"journal\": \"Science Advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM with functional biochemical validation, single lab\",\n      \"pmids\": [\"28439563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Substrate engagement to the central pore of Vps4 is autoinhibited by the MIT domain and relieved by binding of either Type 1 (MIM1) or Type 2 (MIM2) MIT-interacting motifs; residues from helix 5 of Vps2 bind the central pore of an asymmetric Vps4 hexamer in a nucleotide-state-dependent manner.\",\n      \"method\": \"Quantitative binding assays, cross-linking, mutagenesis\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative binding studies with mutagenesis revealing autoinhibitory mechanism, single lab with two orthogonal methods\",\n      \"pmids\": [\"25833946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Purified human VPS4A is essentially inactive but is stimulated to hydrolyze ATP by ESCRT-III proteins (CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, CHMP5) in a reaction requiring their MIT-interacting motifs plus ~50 adjacent residues; the MIT domain and adjacent linker autoinhibit VPS4A; pore loop mutations alter the response to ESCRT-III, consistent with substrate engagement inside the pore.\",\n      \"method\": \"In vitro ATPase assay with purified proteins, mutagenesis, liposome-based oligomerization assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ATPase reconstitution with multiple substrates and mutagenesis, single lab\",\n      \"pmids\": [\"20805225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The N-terminal domain of LIP5 (LIP5NTD) stimulates VPS4 ATPase activity; CHMP5 binding to LIP5NTD strongly inhibits this stimulation via insertion of a conserved Tyr182 residue into the core of LIP5NTD; crystal structure at 1 Å of LIP5NTD bound to CHMP5 and CHMP1B MIMs reveals ESCRT-III binding-induced conformational change in LIP5NTD.\",\n      \"method\": \"Crystal structure (1 Å resolution), ATPase assay, mutagenesis\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — atomic-resolution crystal structure with ATPase assay and mutagenesis validating the mechanism, single lab\",\n      \"pmids\": [\"25637630\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"LIP5 binds to CHMP5, CHMP1B, CHMP2A, and CHMP3 but not CHMP4A or CHMP6; LIP5 binds a different region in CHMP5 than in other ESCRT-III proteins; a second VPS4 binding site exists in CHMP2A and CHMP1B allowing simultaneous binding of LIP5 and VPS4; LIP5 preferentially binds soluble CHMP5 but polymerized CHMP2A.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays, competition binding studies\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple binding assays but no structural or functional validation, single lab\",\n      \"pmids\": [\"18385515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"VPS4/SKD1 regulates endosomal cholesterol transport independently of ESCRT-III: VPS4 knockdown in HeLa cells causes LDL-cholesterol accumulation in late endosomes/lysosomes, while depletion of any ESCRT-III component has no significant effect on endosomal cholesterol transport.\",\n      \"method\": \"siRNA knockdown, fluorescence cholesterol staining, NPC1/NPC2 localization assays\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional separation of VPS4 from ESCRT-III by systematic knockdown with defined phenotypic readout, single lab\",\n      \"pmids\": [\"23009658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Dominant-negative VPS4 impairs MVB biogenesis and blocks lysosomal targeting of α-synuclein while facilitating its extracellular secretion; hypersecretion of α-synuclein in VPS4-defective cells is restored by functional disruption of recycling endosome regulator Rab11a.\",\n      \"method\": \"Dominant-negative VPS4 expression, secretion assays, Rab11a co-manipulation, immunostaining\",\n      \"journal\": \"PLoS ONE\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative approach with epistasis (Rab11a rescue) and multiple assay readouts, single lab\",\n      \"pmids\": [\"22216284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Vps4A facilitates the secretion of oncogenic miRNAs into exosomes and the accumulation of tumor suppressor miRNAs within HCC cells; overexpression of Vps4A inactivates the PI3K/Akt signaling pathway in hepatoma cells.\",\n      \"method\": \"Small RNA sequencing, Vps4A overexpression/knockdown, exosome isolation, PI3K/Akt pathway analysis\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional studies with gain/loss of function and defined pathway readout, single lab\",\n      \"pmids\": [\"25503676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Vps4A interacts with β-catenin and CHMP4B (identified by mass spectrometry of immunoprecipitated Vps4A complex); through this interaction, Vps4A promotes plasma membrane localization and exosome release of β-catenin; Vps4A overexpression decreases β-catenin signaling and inhibits EMT in HCC cells.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, siRNA knockdown, cell fractionation, EMT marker analysis\",\n      \"journal\": \"Cancer Letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with MS identification plus functional assays, single lab\",\n      \"pmids\": [\"31059752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"VPS4-A directly binds the Rho family GTPase Rnd2 regardless of its nucleotide-bound state (GTP- or GDP-bound); co-expression of Rnd2 with the ATPase-defective VPS4-A(E228Q) recruits Rnd2 to VPS4-A-bound early endosomes.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, co-immunoprecipitation, fluorescence microscopy\",\n      \"journal\": \"Biochemical Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — three orthogonal binding assays plus localization, but no downstream functional mechanism defined, single lab\",\n      \"pmids\": [\"11931639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VPS4A and CHMP6/VPS20 interact directly with H-Ras (but not K-Ras) in endosomes in a GTP-bound and ubiquitylated state-dependent manner; repressing CHMP6 and VPS4A blocks Ras-induced transformation and impairs Ras recycling to the plasma membrane and EGFR recycling.\",\n      \"method\": \"Co-immunoprecipitation, cell fractionation, FRAP, siRNA knockdown, transformation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus multiple functional assays establishing pathway position, single lab\",\n      \"pmids\": [\"22231449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"De novo missense variants in the ATPase domain of VPS4A cause enlarged endosomal vacuoles; proband-derived fibroblasts show enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane; VPS4A is also required for normal centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, and mitotic spindle formation.\",\n      \"method\": \"Patient-derived fibroblasts, iPSC-derived neurons, immunofluorescence, dominant-negative VPS4A overexpression as comparison\",\n      \"journal\": \"American Journal of Human Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with multiple orthogonal cellular phenotype readouts, single study\",\n      \"pmids\": [\"33186545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"VPS4A mutations cause cytokinesis and abscission defects in erythropoiesis (binucleated erythroblasts, cytoplasmic bridges); VPS4A is required for reticulocyte maturation including removal of transferrin receptor (CD71) from the red blood cell membrane; iPSC-derived erythroid cells from probands recapitulate the dyserythropoietic phenotype.\",\n      \"method\": \"Bone marrow analysis, iPSC differentiation, flow cytometry (CD71), patient-derived fibroblast studies\",\n      \"journal\": \"American Journal of Human Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived iPSC model with defined mechanistic readouts (cytokinesis, transferrin receptor trafficking), single study\",\n      \"pmids\": [\"33186543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GFP-VPS4 is a dynamic component of both mother and daughter centrioles; ATPase-defective VPS4EQ accumulates at centrosomes and reduces γ-tubulin levels, decreases microtubule growth, eliminates centriolar satellites, and pauses ciliogenesis after ciliary vesicle formation; ESCRT-III proteins rarely localize to centrosomes and their depletion does not phenocopy VPS4EQ, indicating an ESCRT-III-independent centrosomal function.\",\n      \"method\": \"Live-cell fluorescence microscopy, FRAP, siRNA knockdown, zebrafish embryo injection, immunofluorescence\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct live-cell localization with functional consequence, ESCRT-III independence established by parallel knockdowns, single lab\",\n      \"pmids\": [\"29463826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HCV infection activates ROS/JNK signaling to phosphorylate and activate the E3 ubiquitin ligase Itch, which specifically polyubiquitylates VPS4A (at K23 and K121) but not VPS4B; this polyubiquitylation enhances the VPS4A-CHMP1B interaction, increases VPS4A ATPase activity, and promotes HCV particle release.\",\n      \"method\": \"siRNA knockdown, site-directed mutagenesis, co-immunoprecipitation, ATPase activity assay, JNK inhibition, HCV infectivity titer assay\",\n      \"journal\": \"Journal of Virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — identifies writer (Itch), modification sites (K23/K121), demonstrates functional consequence on ATPase activity and CHMP1B interaction with orthogonal methods, single lab\",\n      \"pmids\": [\"35044214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"VPS4A functions as a selective receptor for lipophagy: phosphorylation of VPS4A at Ser95 and Ser97 drives its localization to lipid droplets in response to fasting; VPS4A and lipid droplets are concomitantly degraded in lysosomes in an ATG7-sensitive manner; silencing VPS4A or blocking its LC3 binding specifically inhibits lipophagy without affecting other selective autophagy pathways.\",\n      \"method\": \"Mouse liver phosphoproteomics, 3D imaging reconstruction, co-immunoprecipitation, siRNA knockdown, phosphorylation site mutagenesis, lysosomal degradation assay\",\n      \"journal\": \"Molecular Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — identifies phosphorylation events, localization to lipid droplets, LC3 interaction, and lysosomal co-degradation with multiple orthogonal methods including in vivo and in vitro evidence\",\n      \"pmids\": [\"39520981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"VPS4A is a direct target of the compound aloperine; amino acids F153 and D263 of VPS4A are confirmed as aloperine binding sites; VPS4A knockout mimics aloperine treatment, inhibiting autophagosome-lysosome fusion and blocking autophagic flux.\",\n      \"method\": \"Target identification by drug-protein interaction assays, CRISPR knockout, autophagy flux assays\",\n      \"journal\": \"Advanced Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding site identification with CRISPR KO phenocopying drug effect, single lab\",\n      \"pmids\": [\"39166458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The conserved V domain of Bro1 (yeast homologue of ALIX/HD-PTP) directly stimulates Vps4; this stimulatory activity is required for MVB cargo sorting; ubiquitin binding enhances V domain stimulation of Vps4 to promote intraluminal vesicle formation.\",\n      \"method\": \"In vitro ATPase assay, yeast genetics, in vivo MVB sorting assay\",\n      \"journal\": \"Journal of Cell Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro stimulation assay combined with yeast functional genetics, single lab\",\n      \"pmids\": [\"34160559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ist1 can both inhibit and stimulate Vps4 ATPase activity depending on its conformation: inhibition requires both the MIM and a conserved ELYC surface; binding of the ESCRT-III partner Did2 converts Ist1 from an inhibitor to a stimulator of Vps4, coordinating Vps4 activity with the timing of ESCRT-III disassembly.\",\n      \"method\": \"In vitro ATPase assay, ESCRT-III disassembly assay, mutagenesis\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ATPase and disassembly assays with mutagenesis, single lab\",\n      \"pmids\": [\"26515066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The Vps4 stimulatory element (VSE) of Vta1 contacts Vps4 α-helices 7 and 9 of the small AAA domain to stimulate ATP hydrolysis; intergenic compensatory mutations between VSE and these Vps4 helices validate the interaction surface.\",\n      \"method\": \"Mutagenesis, ATPase assay, yeast complementation, structural modeling\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with compensatory mutations and functional ATPase assay, single lab\",\n      \"pmids\": [\"25164817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Vfa1 binds to the MIT domain of Vps4 via its C-terminal 17 residues adopting a canonical MIM2 conformation (crystal structure determined); this interaction is high affinity and greatly stimulates Vps4 ATPase activity.\",\n      \"method\": \"Crystal structure of Vps4-MIT/Vfa1 complex, ATPase assay, SPR binding measurement\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with quantitative binding and ATPase stimulation assay, single lab\",\n      \"pmids\": [\"24567329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Vps4 can bind and translocate cyclic peptides in a hairpin conformation through its central pore, with one strand making primary contacts and the second returning through the pore without intimate contacts; this reveals a general mechanism for translocation of extended chains, hairpins, and crosslinked polypeptides.\",\n      \"method\": \"Cryo-EM structure of cyclic peptide complexes, binding affinity measurements\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with binding assays revealing translocation mechanism, single lab\",\n      \"pmids\": [\"31184588\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"VPS4A is a hexameric AAA+ ATPase that functions as the primary energy-providing enzyme of the ESCRT pathway: its N-terminal MIT domain binds C-terminal MIM1 and MIM2 motifs on ESCRT-III subunits (CHMP proteins) to recruit the enzyme to ESCRT-III filaments, whereupon ESCRT-III binding relieves MIT-domain autoinhibition and activates the central pore, which translocates and globally unfolds ESCRT-III subunits by a processive 'conveyor belt' mechanism driven by sequential ATP hydrolysis around an asymmetric hexameric ring, thereby constricting and disassembling ESCRT-III filaments to drive membrane neck constriction and fission during MVB biogenesis, cytokinesis, nuclear envelope/NPC surveillance, and enveloped virus budding; VPS4A activity is positively regulated by the cofactor LIP5/Vta1 (which bridges adjacent subunits and contains a VSE element that contacts Vps4 α7/α9) and by direct stimulation from ESCRT-III subunits Vps2 and Snf7/CHMP4, and is negatively regulated by Ist1 unless it is partnered with Did2; additionally, VPS4A performs ESCRT-III-independent functions at centrosomes (regulating γ-tubulin, centriolar satellites, and ciliogenesis), in lipophagy (acting as a selective receptor for lipid droplet autophagy upon phosphorylation at Ser95/97 enabling LC3 binding), and in endosomal cholesterol trafficking, and its activity is modulated post-translationally by polyubiquitylation at K23/K121 by the E3 ligase Itch downstream of HCV-induced ROS/JNK signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"VPS4A is the energy-providing AAA+ ATPase of the ESCRT pathway, a hexameric enzyme that disassembles ESCRT-III filaments to drive membrane constriction and fission [#0, #20]. Its N-terminal MIT domain forms an asymmetric three-helix bundle that recognizes ESCRT-III subunits through two distinct interaction modes: it binds C-terminal MIM1 amphipathic helices of CHMP1-3-class subunits in a groove between MIT helices α2 and α3 [#1, #3], and binds a second MIM2 motif of subunits such as CHMP6 in an extended conformation along the groove between helices 1 and 3 [#4]. Purified VPS4A is essentially inactive and autoinhibited by its MIT domain and adjacent linker; ESCRT-III binding relieves this autoinhibition and engages substrate in the central pore to stimulate ATP hydrolysis [#22, #23]. Active VPS4 assembles into an asymmetric spiral hexamer that processively translocates and globally unfolds ESCRT-III substrates through its central pore by a sequential, 'conveyor-belt' hydrolysis mechanism, thereby constricting and severing ESCRT-III filaments [#14, #15, #16, #18, #21]. This activity is tuned by an extensive cofactor network: LIP5/Vta1 stabilizes the active hexamer and stimulates ATPase activity via its conserved VSE/VSL elements that bridge adjacent subunits and contact VPS4 helices α7/α9 [#7, #19, #24, #40], ESCRT-III subunits Vps2 and Snf7 and the Bro1 V domain directly stimulate the enzyme [#8, #13, #38], and Ist1 acts as a context-dependent inhibitor or activator depending on its partnership with Did2 [#9, #39]. Through this machinery VPS4A executes membrane scission in MVB/intraluminal-vesicle biogenesis and cytokinetic abscission, where it concentrates at spindle poles and midbodies and is held in check by an Aurora-B/ANCHR checkpoint at the midbody ring [#11, #12, #13, #17]. VPS4A additionally performs ESCRT-III-independent roles: regulation of endosomal cholesterol transport [#5, #26], centrosome/centriole homeostasis and ciliogenesis [#34], and lipophagy, where phosphorylation at Ser95/97 directs it to lipid droplets and enables LC3 binding for selective autophagic degradation [#36]. De novo missense variants in the VPS4A ATPase domain cause a multisystem disorder featuring enlarged endosomal vacuoles, dyserythropoiesis with cytokinesis defects, and neurodevelopmental phenotypes [#32, #33].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing that VPS4 ATPase activity gates a discrete membrane-trafficking step, the first cell-based work showed catalytically dead enzyme dominantly blocks a defined sorting process rather than acting cytosolically.\",\n      \"evidence\": \"Expression of ATPase-defective hVPS4 mutants in cultured cells with fractionation and cholesterol staining\",\n      \"pmids\": [\"10637304\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the molecular substrate of the ATPase\", \"Mechanism of membrane recruitment unknown at this stage\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolving the gene architecture in mammals, two paralogs VPS4A and VPS4B were shown to associate with endosomes and form heteromeric complexes, framing later paralog-specific questions.\",\n      \"evidence\": \"Yeast complementation, two-hybrid, dominant-negative expression and localization\",\n      \"pmids\": [\"11563910\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional division of labor between VPS4A and VPS4B not resolved\", \"Oligomeric state misassigned (later corrected to hexamer)\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"To explain how VPS4 finds its targets, the MIT domain was solved and shown to recognize the C-terminal half of an ESCRT-III subunit, identifying the substrate-recruitment module.\",\n      \"evidence\": \"NMR solution structure of VPS4A MIT with CHMP1B binding and mutagenesis\",\n      \"pmids\": [\"16174732\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Only one ESCRT-III partner tested\", \"Did not address how binding couples to ATPase activation\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defining the MIM1 recognition code, crystal structures established the conserved amphipathic-helix motif and the reversed TPR-like binding mode, explaining ESCRT-III selectivity of the MIT domain across yeast and human.\",\n      \"evidence\": \"Crystal structures of VPS4 MIT-CHMP1A and yeast Vps4 MIT-Vps2 with mutagenesis and sorting assays\",\n      \"pmids\": [\"17928862\", \"17928861\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not explain subunits lacking MIM1\", \"Did not capture the assembled hexamer-substrate complex\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Extending the recognition repertoire, a second motif class (MIM2) bound at a distinct MIT site was identified, showing VPS4 reads multiple ESCRT-III tags relevant to HIV budding and lysosomal sorting.\",\n      \"evidence\": \"NMR structure of VPS4 MIT-CHMP6 MIM2 with HIV-1 budding and lysosomal targeting assays\",\n      \"pmids\": [\"18606141\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of MIM1 vs MIM2 in vivo unresolved\", \"How dual motifs coordinate on filaments not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating direct disassembly, reconstitution showed VPS4 binds inside ESCRT-III tubes and severs them upon ATP hydrolysis, providing the mechanistic basis for filament turnover.\",\n      \"evidence\": \"In vitro reconstitution with purified CHMP2A/CHMP3, EM, ATPase assay\",\n      \"pmids\": [\"18687924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic mechanism of substrate engagement still unknown\", \"Cofactor requirements for full activity not yet defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Beginning to map cofactor control, Vta1/LIP5 was shown to stimulate ATPase activity and promote assembly, and distinct stimulation routes (Vps2 direct vs Vps60/Did2 via Vta1) were distinguished.\",\n      \"evidence\": \"In vitro ATPase assays, yeast genetics, binding studies; LIP5-CHMP co-IP/competition assays\",\n      \"pmids\": [\"16505166\", \"18194652\", \"18385515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Vta1 stimulation not yet known\", \"Quantitative integration of multiple stimulators unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Connecting the enzyme to cell division, depletion experiments showed VPS4 and ESCRT-III are required for cytokinetic abscission and that VPS4 localizes to spindle poles and midbodies.\",\n      \"evidence\": \"siRNA knockdown of VPS4A/B and CHMP proteins with live-cell imaging and division assays\",\n      \"pmids\": [\"20616062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial trigger of abscission-associated VPS4 recruitment not defined\", \"Centrosome phenotype mechanism unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Quantifying autoinhibition and activation, human VPS4A was shown to be intrinsically inactive and stimulated by ESCRT-III via MIM plus adjacent residues with substrate engaging the pore, and a multi-interaction assembly network was mapped.\",\n      \"evidence\": \"In vitro ATPase assays with purified human proteins and mutagenesis; systematic yeast interaction-network genetics\",\n      \"pmids\": [\"20805225\", \"20110351\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the active substrate-engaged hexamer not yet available\", \"Order of cofactor recruitment in mammals not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Settling the oligomeric state, biophysics and crystallography established that active Vps4 is a hexamer whose assembly interface is essential for ATPase activity and function.\",\n      \"evidence\": \"Size-exclusion chromatography, analytical ultracentrifugation, crystal structures, yeast functional assays\",\n      \"pmids\": [\"24161953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Asymmetry and nucleotide-state coupling not yet resolved\", \"Substrate path through the pore not visualized\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Linking enzyme action to membrane geometry and division control, work showed Vps4 binding to Vps2/Snf7 drives intraluminal-vesicle scission and that an Aurora-B/ANCHR checkpoint restrains VPS4 at the midbody.\",\n      \"evidence\": \"Yeast genetics with electron tomography; co-IP, imaging, and Aurora-B inhibition in cells\",\n      \"pmids\": [\"24711499\", \"24814515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the timing signal translates to pore activity not defined\", \"Molecular trigger relocating VPS4 to the abscission zone unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defining the disassembly mechanism, biophysics demonstrated VPS4 globally unfolds ESCRT-III by threading it through the pore, and binding studies showed MIM engagement relieves MIT autoinhibition to permit central-pore substrate capture.\",\n      \"evidence\": \"HDX-MS and cysteine cross-linking; quantitative binding, cross-linking and mutagenesis; LIP5NTD crystal structure with CHMP MIMs\",\n      \"pmids\": [\"25938660\", \"25833946\", \"25637630\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Step-resolved coupling of hydrolysis to translocation not yet imaged\", \"CHMP5 inhibitory braking not tested in vivo\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Achieving the mechanistic synthesis, cryo-EM of active hexamers with substrate, Vta1, and nucleotide established a sequential, processive 'conveyor-belt' translocation through an asymmetric spiral ring and the Vta1 bridging mode that promotes the active oligomer.\",\n      \"evidence\": \"Multiple cryo-EM structures (3.2-4.3 Å) of Vps4-substrate, Vps4-Vta1, and ATP-bound hexamers with biochemical/mutant-doping validation\",\n      \"pmids\": [\"28379137\", \"29165244\", \"28714467\", \"28439563\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length filament constriction not captured in these structures\", \"In-cell stoichiometry of functional units not established here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Bridging structure to cell biology, quantitative live imaging showed productive MVB budding requires multiple Vps4 hexamers with continuous ATPase-dependent component exchange, and acute disruption stalls budding.\",\n      \"evidence\": \"Lattice light-sheet microscopy and tomographic EM\",\n      \"pmids\": [\"29019322\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact number and lifetime of hexamers per scission event approximate\", \"Generalization to other membrane sites untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Visualizing constriction directly, real-time AFM/EM showed VPS4 progressively narrows and coils CHMP2A-CHMP3 filaments into end caps before disassembly, and a cyclic-peptide structure generalized the pore translocation mechanism to hairpin and crosslinked chains.\",\n      \"evidence\": \"High-speed AFM and EM of reconstituted filaments; cryo-EM of Vps4-cyclic peptide complexes\",\n      \"pmids\": [\"30989108\", \"31184588\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coupling of constriction to membrane fission not directly observed\", \"Physiological substrate topology range not fully mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Establishing disease causation, de novo ATPase-domain variants were shown to produce enlarged endosomes with aberrant IST1 accumulation, cytokinesis/abscission failures in erythropoiesis, and defects in centrosome, cilium, nuclear membrane and chromosome segregation.\",\n      \"evidence\": \"Patient-derived fibroblasts, iPSC-derived neurons and erythroid cells, immunofluorescence, flow cytometry, dominant-negative comparison\",\n      \"pmids\": [\"33186545\", \"33186543\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dominant-negative vs loss-of-function basis of variants not fully dissected\", \"Tissue-specific severity determinants unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying paralog-specific post-translational control, HCV-induced ROS/JNK signaling was shown to activate Itch, which polyubiquitylates VPS4A at K23/K121, enhancing CHMP1B binding and ATPase activity to promote virus release.\",\n      \"evidence\": \"siRNA, site-directed mutagenesis, co-IP, ATPase assay, JNK inhibition, HCV titer assay\",\n      \"pmids\": [\"35044214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this ubiquitylation operates outside HCV infection unknown\", \"Structural effect of K23/K121 modification on the hexamer undefined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealing a moonlighting catabolic role, VPS4A was shown to act as a selective lipophagy receptor: fasting-induced Ser95/97 phosphorylation drives lipid-droplet localization and LC3 binding for lysosomal co-degradation.\",\n      \"evidence\": \"Mouse liver phosphoproteomics, 3D imaging, co-IP, siRNA, phospho-site mutagenesis, lysosomal degradation assays; plus aloperine target-ID and CRISPR KO autophagy-flux work\",\n      \"pmids\": [\"39520981\", \"39166458\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between lipophagy receptor role and ESCRT ATPase activity unresolved\", \"Kinase phosphorylating Ser95/97 not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the well-defined biochemical hexamer mechanism is partitioned among ESCRT-dependent scission and the ESCRT-III-independent roles (centrosome/cilium, endosomal cholesterol, lipophagy), and how VPS4A versus VPS4B specialize, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model for ESCRT-III-independent substrate engagement\", \"Paralog-specific functional division not defined\", \"Upstream kinases/regulators of non-ESCRT functions largely unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 7, 20, 23]},\n      {\"term_id\": \"GO:0016887\", \"supporting_discovery_ids\": [0, 20, 23]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 16, 18]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 3, 4, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5, 6, 26, 30]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [11, 34]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [36]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [26, 36]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 13, 17]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [11, 12, 13]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [36, 37]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [13, 17, 26]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [34]}\n    ],\n    \"complexes\": [\n      \"VPS4 hexamer\",\n      \"ESCRT-III disassembly machinery\",\n      \"VPS4-Vta1/LIP5 complex\"\n    ],\n    \"partners\": [\n      \"CHMP1B\",\n      \"CHMP2A\",\n      \"CHMP3\",\n      \"CHMP6\",\n      \"LIP5\",\n      \"IST1\",\n      \"CHMP4B\",\n      \"CHMP5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}