{"gene":"VPS28","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":1996,"finding":"Yeast Vps28p is a cytoplasmic 28 kDa hydrophilic protein required for efficient anterograde and retrograde transport out of the prevacuolar endosome; its loss causes accumulation of vacuolar, endocytic, and late Golgi markers in an aberrant multilamellar class E compartment, indicating Vps28p facilitates formation of transport intermediates at the prevacuolar endosome.","method":"Gene disruption, immunofluorescence, electron microscopy with immunolocalization, FM 4-64 endocytic trafficking assay, carboxypeptidase Y sorting assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic KO, EM immunolocalization, trafficking assays) in a foundational study replicated by subsequent ESCRT literature","pmids":["8817003"],"is_preprint":false},{"year":2000,"finding":"Human VPS28 (hVPS28) is a 221-amino acid cytosolic protein that directly interacts with TSG101/mammalian VPS23 via the conserved C-terminal portion of TSG101 to form part of a multiprotein ESCRT-I complex; upon expression of dominant-negative VPS4, a portion of both TSG101 and hVPS28 translocates to the surface of enlarged endosomal vacuoles, implicating the complex directly in endosomal sorting.","method":"Co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 overexpression, subcellular fractionation/localization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus cross-linking plus functional localization experiment, independently consistent with yeast ESCRT-I biology","pmids":["11134028"],"is_preprint":false},{"year":2006,"finding":"The crystal structure of the C-terminal domain of yeast Vps28 (Vps28-CTD) was solved at 3.05 Å resolution, revealing a four-helical bundle that folds independently. Mutagenesis of its conserved surface abolishes interaction with the ESCRT-III component Vps20 in vitro and prevents rescue of an EIAV Gag late-domain deletion, demonstrating that Vps28-CTD acts as an adaptor module recruiting ESCRT-III (Vps20) downstream of ESCRT-I.","method":"X-ray crystallography, co-expression pulldown, site-directed mutagenesis, EIAV Gag late-domain rescue assay","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus functional rescue assay in one study, multiple orthogonal methods","pmids":["16749904"],"is_preprint":false},{"year":2005,"finding":"In Candida albicans, Vps28p (ESCRT-I) is required for signal transduction along the Rim101 pH-sensing pathway; VPS28 deletion impairs transcriptional regulation of Rim101 targets (PHR1, PHR2), and the growth defect at alkaline pH is only partially rescued by constitutively active Rim101p, indicating VPS28 acts both through RIM101-dependent and RIM101-independent pathways.","method":"Gene deletion, transcriptional reporter assays, epistasis with constitutively active RIM101, in vivo mouse virulence model","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined pathway placement, single lab, multiple readouts","pmids":["16299290"],"is_preprint":false},{"year":2006,"finding":"Influenza A virus M1 protein interacts with VPS28 (ESCRT-I component) via its YRKL L-domain motif; co-immunoprecipitation confirmed M1–VPS28 binding, and siRNA depletion of VPS28 reduced influenza virus production.","method":"Co-immunoprecipitation, Western blotting, siRNA knockdown, viral titer assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus loss-of-function siRNA with viral output readout, single lab","pmids":["16474136"],"is_preprint":false},{"year":2009,"finding":"Despite M1 binding VPS28 in vitro, overexpression or dominant-negative VPS4 and siRNA depletion of VPS28 had no significant effect on influenza virus replication or filamentous virion production, demonstrating that influenza budding occurs via a VPS4- and VPS28-independent mechanism.","method":"Confocal microscopy, dominant-negative VPS4 overexpression, VPS28 overexpression, siRNA knockdown, viral titer and morphology assay","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal negative-result experiments (siRNA, DN-VPS4, OE) in a single focused study","pmids":["19524996"],"is_preprint":false},{"year":2011,"finding":"CIIA (VPS28) is a binding partner of SOS1 that functions as a molecular switch: it promotes the SOS1–Rac1 interaction and SOS1–EPS8 complex formation, thereby stimulating SOS1-mediated Rac1 GEF activity, while simultaneously inhibiting SOS1-mediated Ras activation. TGF-β upregulates CIIA expression, driving CIIA–SOS1 association and consequent Rac1-dependent cell migration; CIIA knockdown blocks these TGF-β-induced effects.","method":"Co-immunoprecipitation, RNAi knockdown, Rac1/Ras activity assays (GTPase pull-down), cell migration assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus biochemical GEF activity assays plus RNAi phenotype, single lab","pmids":["22042618"],"is_preprint":false},{"year":2014,"finding":"CIIA (VPS28) physically associates with SOS1 and inhibits its Ras-specific GEF activity, suppressing EGF-induced Ras–Erk1/2 pathway activation, cyclin D1 expression, and DNA synthesis. CIIA failed to inhibit Ras-GEF activity of Noonan-syndrome-associated SOS1 mutants (M269R, R552G, W729L, E846K).","method":"Co-immunoprecipitation, Ras activity (GTPase pull-down) assay, RNAi, Western blotting for pathway effectors","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional GEF/pathway assays plus mutant analysis, single lab","pmids":["24522193"],"is_preprint":false},{"year":2009,"finding":"CIIA (VPS28) promotes epithelial-mesenchymal transition (EMT): ectopic expression in MDCK cells downregulates E-cadherin and claudin-1 and upregulates N-cadherin, disrupts 3D epithelial morphology, and increases migration and invasion; endogenous CIIA depletion inhibits HeLa cell migration/invasion in a claudin-1-dependent manner.","method":"Ectopic overexpression, RNAi knockdown, 3D Matrigel culture, migration/invasion assay, Western blotting for EMT markers","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gain- and loss-of-function with defined molecular markers, single lab, no direct biochemical mechanism established","pmids":["19615336"],"is_preprint":false},{"year":2010,"finding":"CIIA (VPS28) acts as a negative regulator of anoikis (detachment-induced apoptosis): CIIA knockdown in SW620 and KM12SM colon cancer cells promotes cell death after detachment through caspase activation, and inhibits anchorage-independent growth.","method":"RNAi knockdown, anoikis assay, caspase activity assay, soft-agar colony formation","journal":"Cancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single-method RNAi with cellular phenotype, no molecular pathway placement beyond caspase activation, single lab","pmids":["20670956"],"is_preprint":false},{"year":2014,"finding":"CIIA (VPS28) negatively modulates ASK1-mediated cytotoxic signaling in a SOD1(G93A) ALS cell model: CIIA knockdown enhances ASK1–TRAF2 interaction, ASK1 activity, loss of mitochondrial membrane potential, cytochrome c release, and caspase activation induced by the ALS-linked SOD1 G93A mutant.","method":"RNAi knockdown, Co-immunoprecipitation (ASK1–TRAF2), ASK1 kinase activity assay, mitochondrial membrane potential assay, cytochrome c release assay, caspase assay","journal":"Frontiers in cellular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus kinase activity assay plus mitochondrial readouts, single lab, multiple orthogonal methods","pmids":["25018698"],"is_preprint":false},{"year":2014,"finding":"CIIA (VPS28) suppresses neuronal cell death from oxygen-glucose deprivation/reoxygenation by inhibiting ASK1 homo-oligomerization, blocking ASK1–TRAF2 binding, and suppressing downstream JNK and p38 kinase activation and caspase-3 activation.","method":"RNAi knockdown, OGD/R ischemia model in neuroblastoma lines and primary cortical neurons, Co-immunoprecipitation, kinase activity assays, caspase-3 assay","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus kinase assays in primary neurons, single lab, multiple readouts","pmids":["25098452"],"is_preprint":false},{"year":2019,"finding":"Drosophila Vps28 (ESCRT-I component) is required for maintaining normal intracellular levels of Awd (NME1/2 homolog) in larval adipocytes; Vps28 loss reduces Awd intracellular levels, placing Vps28 upstream in the endosomal trafficking pathway that controls intracellular Awd abundance.","method":"Drosophila genetic loss-of-function, immunofluorescence, endosomal marker co-localization, confocal microscopy","journal":"Frontiers in physiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single genetic approach with localization readout, no direct biochemical interaction, single lab","pmids":["31427986"],"is_preprint":false},{"year":2022,"finding":"Zebrafish Vps28 is essential for brain vascular sprouting (central arteries) and blood-brain barrier integrity by controlling MVB formation and thereby extracellular vesicle (EV) secretion from neurons; neuronal EVs containing VEGF-A rescued brain vasculature defects caused by Vps28 disruption, establishing a Vps28→MVB→EV→VEGF-A neurovascular signaling axis.","method":"Zebrafish genetic disruption (morpholino/mutant), in vivo live imaging, EV isolation and rescue experiments, VEGF-A detection in EVs, BBB permeability assay","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with EV rescue experiment and VEGF-A identification, single lab, multiple orthogonal methods","pmids":["35330682"],"is_preprint":false},{"year":2023,"finding":"Arctigenin directly binds VPS28 (identified by chemoproteomic photo-crosslinking in living cells) and induces VPS28 degradation via the ubiquitin-proteasome pathway, causing a phagophore closure-blockade phenotype in PANC-1 cells, consistent with VPS28's role as an ESCRT-I subunit required for phagophore closure.","method":"Chemoproteomic photo-crosslinking with arctigenin probes, target identification by MS, ubiquitin-proteasome pathway inhibitor assays, autophagy/phagophore closure assay","journal":"Bioorganic chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemoproteomics with direct crosslinking plus functional phagophore closure assay, single lab","pmids":["36907049"],"is_preprint":false},{"year":2019,"finding":"LRSAM1 deregulation significantly reduces VPS28 protein levels in both CMT2P patient lymphoblastoid cells and LRSAM1-knockdown SH-SY5Y cells; TSG101 downregulation also reduces VPS28 levels, indicating VPS28 abundance is regulated downstream of the LRSAM1–TSG101 axis.","method":"LRSAM1 and TSG101 siRNA knockdown, protein expression analysis in patient cell lines and neuronal cells","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — expression-level readout only, no direct biochemical mechanism, single lab","pmids":["30726272"],"is_preprint":false}],"current_model":"VPS28 is a core subunit of the ESCRT-I complex (with TSG101/VPS23 and VPS37) that directly binds TSG101 via its C-terminal domain, recruits ESCRT-III (via Vps20 interaction at its four-helical-bundle CTD) to drive multivesicular body biogenesis and phagophore closure, controls endosomal sorting and extracellular vesicle secretion (including VEGF-A-containing neuronal EVs that regulate brain angiogenesis), and—under the alias CIIA—additionally functions as a molecular switch that shifts SOS1 GEF activity toward Rac1 (promoting TGF-β-induced cell migration) and away from Ras (suppressing EGF–Erk1/2 signaling), while also negatively regulating ASK1-dependent apoptotic signaling in neuronal contexts."},"narrative":{"mechanistic_narrative":"VPS28 is a core subunit of the ESCRT-I complex that drives endosomal protein sorting and the membrane-remodeling events of multivesicular body biogenesis [PMID:8817003, PMID:11134028]. The protein is recruited into ESCRT-I through a direct interaction with TSG101/VPS23 mediated by the conserved C-terminal portion of TSG101, and the assembled complex localizes to endosomes [PMID:11134028]. Its C-terminal domain folds independently into a four-helical bundle that acts as an adaptor module recruiting the ESCRT-III component Vps20 downstream of ESCRT-I, coupling cargo sorting to membrane scission [PMID:16749904]. Loss of VPS28 collapses transport out of the prevacuolar/late endosome and produces an aberrant class E compartment, establishing its requirement for forming transport intermediates [PMID:8817003]. Through this ESCRT-I activity VPS28 controls multivesicular body formation and extracellular vesicle secretion, including release of VEGF-A-loaded neuronal EVs that pattern brain vasculature and maintain blood-brain barrier integrity [PMID:35330682], and is required for phagophore closure during autophagy [PMID:36907049]. Under the alias CIIA, VPS28 additionally binds SOS1 and acts as a molecular switch, promoting SOS1-dependent Rac1 GEF activity to drive TGF-β-induced migration while inhibiting SOS1-mediated Ras–Erk1/2 signaling [PMID:22042618, PMID:24522193], and it negatively regulates ASK1-dependent apoptotic signaling by blocking ASK1–TRAF2 interaction in neuronal stress models [PMID:25018698, PMID:25098452].","teleology":[{"year":1996,"claim":"Established that Vps28 is required for transport out of the prevacuolar endosome, defining its place in the endosomal sorting machinery before the ESCRT concept existed.","evidence":"Gene disruption with EM immunolocalization and trafficking/sorting assays in yeast","pmids":["8817003"],"confidence":"High","gaps":["Did not identify direct protein partners","Molecular mechanism of intermediate formation unresolved"]},{"year":2000,"claim":"Showed that human VPS28 directly binds TSG101 to form part of the multiprotein ESCRT-I complex and localizes to endosomes, placing VPS28 in mammalian endosomal sorting.","evidence":"Co-IP, chemical cross-linking, and dominant-negative VPS4-induced relocalization in human cells","pmids":["11134028"],"confidence":"High","gaps":["Stoichiometry and full subunit composition not defined","How the complex engages cargo not addressed"]},{"year":2006,"claim":"Defined the structural basis for VPS28 function, showing its C-terminal four-helix bundle is an adaptor that recruits ESCRT-III (Vps20) downstream of ESCRT-I.","evidence":"X-ray crystallography of yeast Vps28-CTD, pulldown mutagenesis, and EIAV Gag late-domain rescue","pmids":["16749904"],"confidence":"High","gaps":["Structure of full-length VPS28 within assembled ESCRT-I not determined","Dynamics of ESCRT-I to ESCRT-III handoff not resolved"]},{"year":2005,"claim":"Extended VPS28/ESCRT-I function to signal transduction, linking it to the Rim101 pH-sensing pathway and fungal virulence.","evidence":"Gene deletion, transcriptional reporters, RIM101 epistasis, and mouse virulence model in Candida albicans","pmids":["16299290"],"confidence":"Medium","gaps":["RIM101-independent branch unexplained","No direct biochemical link to pathway components"]},{"year":2006,"claim":"Tested whether VPS28 supports viral budding, finding influenza M1 binds VPS28 via an L-domain motif and that VPS28 depletion lowers viral output.","evidence":"Co-IP and siRNA knockdown with viral titer assay","pmids":["16474136"],"confidence":"Medium","gaps":["Whether binding is functionally required for budding not settled here"]},{"year":2009,"claim":"Re-examined the VPS28 requirement for influenza budding and concluded budding proceeds independently of VPS28 and VPS4, qualifying the earlier dependence.","evidence":"Dominant-negative VPS4, overexpression, and siRNA depletion with morphology/titer readouts","pmids":["19524996"],"confidence":"Medium","gaps":["Reason for discrepancy with the 2006 M1-binding result unresolved","Physiological role of M1–VPS28 binding unknown"]},{"year":2011,"claim":"Revealed a moonlighting role: VPS28/CIIA binds SOS1 and biases its GEF activity toward Rac1, driving TGF-β-induced migration.","evidence":"Co-IP, RNAi, GTPase pull-down activity assays, and migration assays","pmids":["22042618"],"confidence":"Medium","gaps":["Relationship to ESCRT-I function not addressed","Single lab, no structural basis for the switch"]},{"year":2014,"claim":"Completed the SOS1 switch model by showing CIIA inhibits SOS1 Ras-GEF activity and suppresses EGF–Erk1/2 signaling, and fails to inhibit Noonan-associated SOS1 mutants.","evidence":"Co-IP, Ras GTPase pull-down, RNAi, and effector Western blots with SOS1 mutant analysis","pmids":["24522193"],"confidence":"Medium","gaps":["Mechanism distinguishing Rac1 vs Ras outcomes unresolved","In vivo relevance not established"]},{"year":2009,"claim":"Linked CIIA to epithelial-mesenchymal transition, showing it remodels adhesion markers and promotes migration/invasion.","evidence":"Overexpression, RNAi, 3D culture, and migration/invasion assays with EMT marker blots","pmids":["19615336"],"confidence":"Medium","gaps":["No direct biochemical mechanism for marker changes","Connection to SOS1/Rac1 axis not tested"]},{"year":2010,"claim":"Implicated CIIA as a negative regulator of anoikis, suggesting a pro-survival role in detached cancer cells.","evidence":"RNAi knockdown with anoikis, caspase, and soft-agar assays","pmids":["20670956"],"confidence":"Low","gaps":["Single-method RNAi with no molecular pathway placement beyond caspase activation","No interacting partner identified"]},{"year":2014,"claim":"Defined a neuroprotective mechanism in which CIIA negatively regulates ASK1 signaling, shown across ALS and ischemia models by blocking ASK1–TRAF2 and ASK1 oligomerization.","evidence":"RNAi, Co-IP, ASK1 kinase assays, and mitochondrial/caspase readouts in SOD1(G93A) cells, neuroblastoma lines, and primary cortical neurons","pmids":["25018698","25098452"],"confidence":"Medium","gaps":["Direct CIIA–ASK1 binding vs indirect effect not distinguished","Link to ESCRT-I or SOS1 roles unexplored"]},{"year":2022,"claim":"Connected VPS28's ESCRT-I/MVB function to a neurovascular signaling axis, showing Vps28 controls neuronal EV secretion of VEGF-A required for brain vascular sprouting and BBB integrity.","evidence":"Zebrafish genetic disruption, live imaging, EV isolation and rescue with VEGF-A detection, and BBB permeability assay","pmids":["35330682"],"confidence":"Medium","gaps":["Mechanism of VEGF-A loading into EVs not defined","Mammalian conservation not tested"]},{"year":2023,"claim":"Provided chemical validation of VPS28 as a druggable ESCRT-I node, showing arctigenin binds VPS28 and triggers its proteasomal degradation to block phagophore closure.","evidence":"Chemoproteomic photo-crosslinking, MS target ID, proteasome inhibitor assays, and phagophore closure assay in PANC-1 cells","pmids":["36907049"],"confidence":"Medium","gaps":["Binding site on VPS28 not mapped","Whether degradation acts only through autophagy unclear"]},{"year":null,"claim":"How VPS28's canonical ESCRT-I/MVB function mechanistically relates to its SOS1-switch and ASK1-regulatory activities, and whether these are separable molecular pools, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural or interaction study reconciling ESCRT-I and CIIA roles","No mammalian disease-causing mutation in VPS28 reported in the corpus","Substrate/cargo specificity of mammalian ESCRT-I VPS28 not detailed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,7,10,11]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1,13]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,7,10,11]}],"complexes":["ESCRT-I"],"partners":["TSG101","VPS20","SOS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UK41","full_name":"Vacuolar protein sorting-associated protein 28 homolog","aliases":["ESCRT-I complex subunit VPS28"],"length_aa":221,"mass_kda":25.4,"function":"Component of the ESCRT-I complex, a regulator of vesicular trafficking process","subcellular_location":"Cell membrane; Late endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q9UK41/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/VPS28","classification":"Common Essential","n_dependent_lines":1194,"n_total_lines":1208,"dependency_fraction":0.9884105960264901},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000160948","cell_line_id":"CID000789","localizations":[{"compartment":"vesicles","grade":3},{"compartment":"cytoplasmic","grade":2}],"interactors":[{"gene":"MVB12A","stoichiometry":10.0},{"gene":"TSG101","stoichiometry":10.0},{"gene":"VPS37B","stoichiometry":10.0},{"gene":"STX12","stoichiometry":0.2},{"gene":"VPS25","stoichiometry":0.2},{"gene":"UBE3B","stoichiometry":0.2},{"gene":"UBAP1","stoichiometry":0.2},{"gene":"VPS37A","stoichiometry":0.2},{"gene":"WDFY1","stoichiometry":0.2},{"gene":"CEP55","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000789","total_profiled":1310},"omim":[{"mim_id":"621454","title":"MULTIVESICULAR BODY SUBUNIT 12B; MVB12B","url":"https://www.omim.org/entry/621454"},{"mim_id":"621453","title":"MULTIVESICULAR BODY SUBUNIT 12A; MVB12A","url":"https://www.omim.org/entry/621453"},{"mim_id":"611952","title":"VPS28 SUBUNIT OF ESCRT-I; VPS28","url":"https://www.omim.org/entry/611952"},{"mim_id":"610038","title":"VPS37C SUBUNIT OF ESCRT-I; VPS37C","url":"https://www.omim.org/entry/610038"},{"mim_id":"610037","title":"VPS37B SUBUNIT OF ESCRT-I; VPS37B","url":"https://www.omim.org/entry/610037"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/VPS28"},"hgnc":{"alias_symbol":["CIIA"],"prev_symbol":[]},"alphafold":{"accession":"Q9UK41","domains":[{"cath_id":"1.20.1440.200","chopping":"29-111","consensus_level":"high","plddt":96.9758,"start":29,"end":111},{"cath_id":"1.20.120.1130","chopping":"120-219","consensus_level":"high","plddt":92.7565,"start":120,"end":219}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UK41","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UK41-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UK41-F1-predicted_aligned_error_v6.png","plddt_mean":91.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VPS28","jax_strain_url":"https://www.jax.org/strain/search?query=VPS28"},"sequence":{"accession":"Q9UK41","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UK41.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UK41/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UK41"}},"corpus_meta":[{"pmid":"8817003","id":"PMC_8817003","title":"Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant.","date":"1996","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/8817003","citation_count":251,"is_preprint":false},{"pmid":"11134028","id":"PMC_11134028","title":"TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11134028","citation_count":170,"is_preprint":false},{"pmid":"16299290","id":"PMC_16299290","title":"Deletions of endocytic components VPS28 and VPS32 affect growth at alkaline pH and virulence through both RIM101-dependent and RIM101-independent pathways in Candida albicans.","date":"2005","source":"Infection and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/16299290","citation_count":58,"is_preprint":false},{"pmid":"19524996","id":"PMC_19524996","title":"Budding of filamentous and non-filamentous influenza A virus occurs via a VPS4 and VPS28-independent pathway.","date":"2009","source":"Virology","url":"https://pubmed.ncbi.nlm.nih.gov/19524996","citation_count":54,"is_preprint":false},{"pmid":"16749904","id":"PMC_16749904","title":"The crystal structure of the C-terminal domain of Vps28 reveals a conserved surface required for Vps20 recruitment.","date":"2006","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/16749904","citation_count":51,"is_preprint":false},{"pmid":"15528373","id":"PMC_15528373","title":"Multiple roles of CLAN (caspase-associated recruitment domain, leucine-rich repeat, and NAIP CIIA HET-E, and TP1-containing protein) in the mammalian innate immune response.","date":"2004","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/15528373","citation_count":33,"is_preprint":false},{"pmid":"17005841","id":"PMC_17005841","title":"Deletions of the endocytic components VPS28 and VPS32 in Candida albicans lead to echinocandin and azole hypersensitivity.","date":"2006","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/17005841","citation_count":29,"is_preprint":false},{"pmid":"16474136","id":"PMC_16474136","title":"YRKL sequence of influenza virus M1 functions as the L domain motif and interacts with VPS28 and Cdc42.","date":"2006","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/16474136","citation_count":24,"is_preprint":false},{"pmid":"30739790","id":"PMC_30739790","title":"Identification and functional analysis of candidate gene VPS28 for milk fat in bovine mammary epithelial cells.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/30739790","citation_count":13,"is_preprint":false},{"pmid":"22042618","id":"PMC_22042618","title":"CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1.","date":"2011","source":"The Journal of 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SOD1(G93A)-induced cytotoxicity by blocking ASK1-mediated signaling.","date":"2014","source":"Frontiers in cellular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/25018698","citation_count":6,"is_preprint":false},{"pmid":"30559098","id":"PMC_30559098","title":"[Regulation of VPS28 gene knockdown on the milk fat synthesis in Chinese Holstein dairy].","date":"2018","source":"Yi chuan = Hereditas","url":"https://pubmed.ncbi.nlm.nih.gov/30559098","citation_count":6,"is_preprint":false},{"pmid":"31427986","id":"PMC_31427986","title":"Vps28 Is Involved in the Intracellular Trafficking of Awd, the Drosophila Homolog of NME1/2.","date":"2019","source":"Frontiers in physiology","url":"https://pubmed.ncbi.nlm.nih.gov/31427986","citation_count":6,"is_preprint":false},{"pmid":"34395516","id":"PMC_34395516","title":"Deciphering the Oncogenic Role of VPS28 Modulated by miR-491-5p in Breast Cancer Cells Using In Silico and Functional Analysis.","date":"2021","source":"Frontiers in molecular biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/34395516","citation_count":5,"is_preprint":false},{"pmid":"33194328","id":"PMC_33194328","title":"Comparative proteome analysis reveals VPS28 regulates milk fat synthesis through ubiquitylation in bovine mammary epithelial cells.","date":"2020","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/33194328","citation_count":5,"is_preprint":false},{"pmid":"19615336","id":"PMC_19615336","title":"CIIA induces the epithelial-mesenchymal transition and cell invasion.","date":"2009","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/19615336","citation_count":4,"is_preprint":false},{"pmid":"25098452","id":"PMC_25098452","title":"CIIA negatively regulates neuronal cell death induced by oxygen-glucose deprivation and reoxygenation.","date":"2014","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25098452","citation_count":3,"is_preprint":false},{"pmid":"30726272","id":"PMC_30726272","title":"Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR.","date":"2019","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/30726272","citation_count":3,"is_preprint":false},{"pmid":"36907049","id":"PMC_36907049","title":"Chemoproteomics reveals arctigenin as a phagophore-closure blocker via targeting ESCRT-I subunit VPS28.","date":"2023","source":"Bioorganic chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36907049","citation_count":2,"is_preprint":false},{"pmid":"39732879","id":"PMC_39732879","title":"VPS28 regulates triglyceride synthesis via ubiquitination in bovine mammary epithelial cells.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39732879","citation_count":1,"is_preprint":false},{"pmid":"39668793","id":"PMC_39668793","title":"Mechanism to disrupt ESCRT-mediated intracellular trafficking through Vps28-small molecules interaction: an in silico approach.","date":"2024","source":"Journal of biomolecular structure & dynamics","url":"https://pubmed.ncbi.nlm.nih.gov/39668793","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.07.04.602114","title":"VPS28 regulates triglyceride synthesis and is mediated by the ubiquitination pathway in a bovine mammary epithelial cell line and mouse model","date":"2024-07-05","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.04.602114","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13078,"output_tokens":4147,"usd":0.05072,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11995,"output_tokens":3873,"usd":0.0784,"stage2_stop_reason":"end_turn"},"total_usd":0.12912,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"Yeast Vps28p is a cytoplasmic 28 kDa hydrophilic protein required for efficient anterograde and retrograde transport out of the prevacuolar endosome; its loss causes accumulation of vacuolar, endocytic, and late Golgi markers in an aberrant multilamellar class E compartment, indicating Vps28p facilitates formation of transport intermediates at the prevacuolar endosome.\",\n      \"method\": \"Gene disruption, immunofluorescence, electron microscopy with immunolocalization, FM 4-64 endocytic trafficking assay, carboxypeptidase Y sorting assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic KO, EM immunolocalization, trafficking assays) in a foundational study replicated by subsequent ESCRT literature\",\n      \"pmids\": [\"8817003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Human VPS28 (hVPS28) is a 221-amino acid cytosolic protein that directly interacts with TSG101/mammalian VPS23 via the conserved C-terminal portion of TSG101 to form part of a multiprotein ESCRT-I complex; upon expression of dominant-negative VPS4, a portion of both TSG101 and hVPS28 translocates to the surface of enlarged endosomal vacuoles, implicating the complex directly in endosomal sorting.\",\n      \"method\": \"Co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 overexpression, subcellular fractionation/localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus cross-linking plus functional localization experiment, independently consistent with yeast ESCRT-I biology\",\n      \"pmids\": [\"11134028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The crystal structure of the C-terminal domain of yeast Vps28 (Vps28-CTD) was solved at 3.05 Å resolution, revealing a four-helical bundle that folds independently. Mutagenesis of its conserved surface abolishes interaction with the ESCRT-III component Vps20 in vitro and prevents rescue of an EIAV Gag late-domain deletion, demonstrating that Vps28-CTD acts as an adaptor module recruiting ESCRT-III (Vps20) downstream of ESCRT-I.\",\n      \"method\": \"X-ray crystallography, co-expression pulldown, site-directed mutagenesis, EIAV Gag late-domain rescue assay\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus functional rescue assay in one study, multiple orthogonal methods\",\n      \"pmids\": [\"16749904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In Candida albicans, Vps28p (ESCRT-I) is required for signal transduction along the Rim101 pH-sensing pathway; VPS28 deletion impairs transcriptional regulation of Rim101 targets (PHR1, PHR2), and the growth defect at alkaline pH is only partially rescued by constitutively active Rim101p, indicating VPS28 acts both through RIM101-dependent and RIM101-independent pathways.\",\n      \"method\": \"Gene deletion, transcriptional reporter assays, epistasis with constitutively active RIM101, in vivo mouse virulence model\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined pathway placement, single lab, multiple readouts\",\n      \"pmids\": [\"16299290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Influenza A virus M1 protein interacts with VPS28 (ESCRT-I component) via its YRKL L-domain motif; co-immunoprecipitation confirmed M1–VPS28 binding, and siRNA depletion of VPS28 reduced influenza virus production.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, siRNA knockdown, viral titer assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus loss-of-function siRNA with viral output readout, single lab\",\n      \"pmids\": [\"16474136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Despite M1 binding VPS28 in vitro, overexpression or dominant-negative VPS4 and siRNA depletion of VPS28 had no significant effect on influenza virus replication or filamentous virion production, demonstrating that influenza budding occurs via a VPS4- and VPS28-independent mechanism.\",\n      \"method\": \"Confocal microscopy, dominant-negative VPS4 overexpression, VPS28 overexpression, siRNA knockdown, viral titer and morphology assay\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal negative-result experiments (siRNA, DN-VPS4, OE) in a single focused study\",\n      \"pmids\": [\"19524996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CIIA (VPS28) is a binding partner of SOS1 that functions as a molecular switch: it promotes the SOS1–Rac1 interaction and SOS1–EPS8 complex formation, thereby stimulating SOS1-mediated Rac1 GEF activity, while simultaneously inhibiting SOS1-mediated Ras activation. TGF-β upregulates CIIA expression, driving CIIA–SOS1 association and consequent Rac1-dependent cell migration; CIIA knockdown blocks these TGF-β-induced effects.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, Rac1/Ras activity assays (GTPase pull-down), cell migration assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus biochemical GEF activity assays plus RNAi phenotype, single lab\",\n      \"pmids\": [\"22042618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CIIA (VPS28) physically associates with SOS1 and inhibits its Ras-specific GEF activity, suppressing EGF-induced Ras–Erk1/2 pathway activation, cyclin D1 expression, and DNA synthesis. CIIA failed to inhibit Ras-GEF activity of Noonan-syndrome-associated SOS1 mutants (M269R, R552G, W729L, E846K).\",\n      \"method\": \"Co-immunoprecipitation, Ras activity (GTPase pull-down) assay, RNAi, Western blotting for pathway effectors\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional GEF/pathway assays plus mutant analysis, single lab\",\n      \"pmids\": [\"24522193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CIIA (VPS28) promotes epithelial-mesenchymal transition (EMT): ectopic expression in MDCK cells downregulates E-cadherin and claudin-1 and upregulates N-cadherin, disrupts 3D epithelial morphology, and increases migration and invasion; endogenous CIIA depletion inhibits HeLa cell migration/invasion in a claudin-1-dependent manner.\",\n      \"method\": \"Ectopic overexpression, RNAi knockdown, 3D Matrigel culture, migration/invasion assay, Western blotting for EMT markers\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gain- and loss-of-function with defined molecular markers, single lab, no direct biochemical mechanism established\",\n      \"pmids\": [\"19615336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CIIA (VPS28) acts as a negative regulator of anoikis (detachment-induced apoptosis): CIIA knockdown in SW620 and KM12SM colon cancer cells promotes cell death after detachment through caspase activation, and inhibits anchorage-independent growth.\",\n      \"method\": \"RNAi knockdown, anoikis assay, caspase activity assay, soft-agar colony formation\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single-method RNAi with cellular phenotype, no molecular pathway placement beyond caspase activation, single lab\",\n      \"pmids\": [\"20670956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CIIA (VPS28) negatively modulates ASK1-mediated cytotoxic signaling in a SOD1(G93A) ALS cell model: CIIA knockdown enhances ASK1–TRAF2 interaction, ASK1 activity, loss of mitochondrial membrane potential, cytochrome c release, and caspase activation induced by the ALS-linked SOD1 G93A mutant.\",\n      \"method\": \"RNAi knockdown, Co-immunoprecipitation (ASK1–TRAF2), ASK1 kinase activity assay, mitochondrial membrane potential assay, cytochrome c release assay, caspase assay\",\n      \"journal\": \"Frontiers in cellular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus kinase activity assay plus mitochondrial readouts, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25018698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CIIA (VPS28) suppresses neuronal cell death from oxygen-glucose deprivation/reoxygenation by inhibiting ASK1 homo-oligomerization, blocking ASK1–TRAF2 binding, and suppressing downstream JNK and p38 kinase activation and caspase-3 activation.\",\n      \"method\": \"RNAi knockdown, OGD/R ischemia model in neuroblastoma lines and primary cortical neurons, Co-immunoprecipitation, kinase activity assays, caspase-3 assay\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus kinase assays in primary neurons, single lab, multiple readouts\",\n      \"pmids\": [\"25098452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Drosophila Vps28 (ESCRT-I component) is required for maintaining normal intracellular levels of Awd (NME1/2 homolog) in larval adipocytes; Vps28 loss reduces Awd intracellular levels, placing Vps28 upstream in the endosomal trafficking pathway that controls intracellular Awd abundance.\",\n      \"method\": \"Drosophila genetic loss-of-function, immunofluorescence, endosomal marker co-localization, confocal microscopy\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single genetic approach with localization readout, no direct biochemical interaction, single lab\",\n      \"pmids\": [\"31427986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Zebrafish Vps28 is essential for brain vascular sprouting (central arteries) and blood-brain barrier integrity by controlling MVB formation and thereby extracellular vesicle (EV) secretion from neurons; neuronal EVs containing VEGF-A rescued brain vasculature defects caused by Vps28 disruption, establishing a Vps28→MVB→EV→VEGF-A neurovascular signaling axis.\",\n      \"method\": \"Zebrafish genetic disruption (morpholino/mutant), in vivo live imaging, EV isolation and rescue experiments, VEGF-A detection in EVs, BBB permeability assay\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with EV rescue experiment and VEGF-A identification, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35330682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Arctigenin directly binds VPS28 (identified by chemoproteomic photo-crosslinking in living cells) and induces VPS28 degradation via the ubiquitin-proteasome pathway, causing a phagophore closure-blockade phenotype in PANC-1 cells, consistent with VPS28's role as an ESCRT-I subunit required for phagophore closure.\",\n      \"method\": \"Chemoproteomic photo-crosslinking with arctigenin probes, target identification by MS, ubiquitin-proteasome pathway inhibitor assays, autophagy/phagophore closure assay\",\n      \"journal\": \"Bioorganic chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemoproteomics with direct crosslinking plus functional phagophore closure assay, single lab\",\n      \"pmids\": [\"36907049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LRSAM1 deregulation significantly reduces VPS28 protein levels in both CMT2P patient lymphoblastoid cells and LRSAM1-knockdown SH-SY5Y cells; TSG101 downregulation also reduces VPS28 levels, indicating VPS28 abundance is regulated downstream of the LRSAM1–TSG101 axis.\",\n      \"method\": \"LRSAM1 and TSG101 siRNA knockdown, protein expression analysis in patient cell lines and neuronal cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — expression-level readout only, no direct biochemical mechanism, single lab\",\n      \"pmids\": [\"30726272\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"VPS28 is a core subunit of the ESCRT-I complex (with TSG101/VPS23 and VPS37) that directly binds TSG101 via its C-terminal domain, recruits ESCRT-III (via Vps20 interaction at its four-helical-bundle CTD) to drive multivesicular body biogenesis and phagophore closure, controls endosomal sorting and extracellular vesicle secretion (including VEGF-A-containing neuronal EVs that regulate brain angiogenesis), and—under the alias CIIA—additionally functions as a molecular switch that shifts SOS1 GEF activity toward Rac1 (promoting TGF-β-induced cell migration) and away from Ras (suppressing EGF–Erk1/2 signaling), while also negatively regulating ASK1-dependent apoptotic signaling in neuronal contexts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"VPS28 is a core subunit of the ESCRT-I complex that drives endosomal protein sorting and the membrane-remodeling events of multivesicular body biogenesis [#0, #1]. The protein is recruited into ESCRT-I through a direct interaction with TSG101/VPS23 mediated by the conserved C-terminal portion of TSG101, and the assembled complex localizes to endosomes [#1]. Its C-terminal domain folds independently into a four-helical bundle that acts as an adaptor module recruiting the ESCRT-III component Vps20 downstream of ESCRT-I, coupling cargo sorting to membrane scission [#2]. Loss of VPS28 collapses transport out of the prevacuolar/late endosome and produces an aberrant class E compartment, establishing its requirement for forming transport intermediates [#0]. Through this ESCRT-I activity VPS28 controls multivesicular body formation and extracellular vesicle secretion, including release of VEGF-A-loaded neuronal EVs that pattern brain vasculature and maintain blood-brain barrier integrity [#13], and is required for phagophore closure during autophagy [#14]. Under the alias CIIA, VPS28 additionally binds SOS1 and acts as a molecular switch, promoting SOS1-dependent Rac1 GEF activity to drive TGF-\\u03b2-induced migration while inhibiting SOS1-mediated Ras\\u2013Erk1/2 signaling [#6, #7], and it negatively regulates ASK1-dependent apoptotic signaling by blocking ASK1\\u2013TRAF2 interaction in neuronal stress models [#10, #11].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that Vps28 is required for transport out of the prevacuolar endosome, defining its place in the endosomal sorting machinery before the ESCRT concept existed.\",\n      \"evidence\": \"Gene disruption with EM immunolocalization and trafficking/sorting assays in yeast\",\n      \"pmids\": [\"8817003\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify direct protein partners\", \"Molecular mechanism of intermediate formation unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed that human VPS28 directly binds TSG101 to form part of the multiprotein ESCRT-I complex and localizes to endosomes, placing VPS28 in mammalian endosomal sorting.\",\n      \"evidence\": \"Co-IP, chemical cross-linking, and dominant-negative VPS4-induced relocalization in human cells\",\n      \"pmids\": [\"11134028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and full subunit composition not defined\", \"How the complex engages cargo not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the structural basis for VPS28 function, showing its C-terminal four-helix bundle is an adaptor that recruits ESCRT-III (Vps20) downstream of ESCRT-I.\",\n      \"evidence\": \"X-ray crystallography of yeast Vps28-CTD, pulldown mutagenesis, and EIAV Gag late-domain rescue\",\n      \"pmids\": [\"16749904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length VPS28 within assembled ESCRT-I not determined\", \"Dynamics of ESCRT-I to ESCRT-III handoff not resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Extended VPS28/ESCRT-I function to signal transduction, linking it to the Rim101 pH-sensing pathway and fungal virulence.\",\n      \"evidence\": \"Gene deletion, transcriptional reporters, RIM101 epistasis, and mouse virulence model in Candida albicans\",\n      \"pmids\": [\"16299290\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RIM101-independent branch unexplained\", \"No direct biochemical link to pathway components\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Tested whether VPS28 supports viral budding, finding influenza M1 binds VPS28 via an L-domain motif and that VPS28 depletion lowers viral output.\",\n      \"evidence\": \"Co-IP and siRNA knockdown with viral titer assay\",\n      \"pmids\": [\"16474136\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether binding is functionally required for budding not settled here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Re-examined the VPS28 requirement for influenza budding and concluded budding proceeds independently of VPS28 and VPS4, qualifying the earlier dependence.\",\n      \"evidence\": \"Dominant-negative VPS4, overexpression, and siRNA depletion with morphology/titer readouts\",\n      \"pmids\": [\"19524996\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reason for discrepancy with the 2006 M1-binding result unresolved\", \"Physiological role of M1\\u2013VPS28 binding unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed a moonlighting role: VPS28/CIIA binds SOS1 and biases its GEF activity toward Rac1, driving TGF-\\u03b2-induced migration.\",\n      \"evidence\": \"Co-IP, RNAi, GTPase pull-down activity assays, and migration assays\",\n      \"pmids\": [\"22042618\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship to ESCRT-I function not addressed\", \"Single lab, no structural basis for the switch\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Completed the SOS1 switch model by showing CIIA inhibits SOS1 Ras-GEF activity and suppresses EGF\\u2013Erk1/2 signaling, and fails to inhibit Noonan-associated SOS1 mutants.\",\n      \"evidence\": \"Co-IP, Ras GTPase pull-down, RNAi, and effector Western blots with SOS1 mutant analysis\",\n      \"pmids\": [\"24522193\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism distinguishing Rac1 vs Ras outcomes unresolved\", \"In vivo relevance not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linked CIIA to epithelial-mesenchymal transition, showing it remodels adhesion markers and promotes migration/invasion.\",\n      \"evidence\": \"Overexpression, RNAi, 3D culture, and migration/invasion assays with EMT marker blots\",\n      \"pmids\": [\"19615336\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct biochemical mechanism for marker changes\", \"Connection to SOS1/Rac1 axis not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Implicated CIIA as a negative regulator of anoikis, suggesting a pro-survival role in detached cancer cells.\",\n      \"evidence\": \"RNAi knockdown with anoikis, caspase, and soft-agar assays\",\n      \"pmids\": [\"20670956\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single-method RNAi with no molecular pathway placement beyond caspase activation\", \"No interacting partner identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined a neuroprotective mechanism in which CIIA negatively regulates ASK1 signaling, shown across ALS and ischemia models by blocking ASK1\\u2013TRAF2 and ASK1 oligomerization.\",\n      \"evidence\": \"RNAi, Co-IP, ASK1 kinase assays, and mitochondrial/caspase readouts in SOD1(G93A) cells, neuroblastoma lines, and primary cortical neurons\",\n      \"pmids\": [\"25018698\", \"25098452\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CIIA\\u2013ASK1 binding vs indirect effect not distinguished\", \"Link to ESCRT-I or SOS1 roles unexplored\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected VPS28's ESCRT-I/MVB function to a neurovascular signaling axis, showing Vps28 controls neuronal EV secretion of VEGF-A required for brain vascular sprouting and BBB integrity.\",\n      \"evidence\": \"Zebrafish genetic disruption, live imaging, EV isolation and rescue with VEGF-A detection, and BBB permeability assay\",\n      \"pmids\": [\"35330682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of VEGF-A loading into EVs not defined\", \"Mammalian conservation not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided chemical validation of VPS28 as a druggable ESCRT-I node, showing arctigenin binds VPS28 and triggers its proteasomal degradation to block phagophore closure.\",\n      \"evidence\": \"Chemoproteomic photo-crosslinking, MS target ID, proteasome inhibitor assays, and phagophore closure assay in PANC-1 cells\",\n      \"pmids\": [\"36907049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site on VPS28 not mapped\", \"Whether degradation acts only through autophagy unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How VPS28's canonical ESCRT-I/MVB function mechanistically relates to its SOS1-switch and ASK1-regulatory activities, and whether these are separable molecular pools, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural or interaction study reconciling ESCRT-I and CIIA roles\", \"No mammalian disease-causing mutation in VPS28 reported in the corpus\", \"Substrate/cargo specificity of mammalian ESCRT-I VPS28 not detailed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 7, 10, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 13]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 7, 10, 11]}\n    ],\n    \"complexes\": [\"ESCRT-I\"],\n    \"partners\": [\"TSG101\", \"VPS20\", \"SOS1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}