{"gene":"VPS28","run_date":"2026-04-28T23:00:23","timeline":{"discoveries":[{"year":1996,"finding":"Yeast Vps28p is a 28 kDa cytoplasmic hydrophilic protein required for efficient anterograde and retrograde transport out of the prevacuolar/endosomal compartment. Loss of VPS28 causes accumulation of vacuolar, endocytic, and late Golgi markers in an aberrant multilamellar endosome-like 'class E' compartment, with ~40-50% of carboxypeptidase Y missorted, placing Vps28p in the class E VPS pathway that facilitates formation of transport intermediates at the prevacuolar endosome.","method":"Genetic disruption (null mutant), fluorescence microscopy with FM 4-64 and marker proteins, immunolocalization by electron microscopy, pulse-chase sorting assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (genetics, EM immunolocalization, biochemical sorting assay) in foundational study","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 to form part of a multiprotein ESCRT-I complex. Direct binding requires structural information within the conserved C-terminal portion of TSG101. Upon expression of dominant-negative VPS4, a portion of both TSG101 and hVPS28 translocates from the cytosol to the surface of enlarged endosomal vacuoles, implicating them directly in endosomal sorting.","method":"Co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 overexpression, subcellular fractionation/confocal microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with cross-linking validation, plus functional localization experiment","pmids":["11134028"],"is_preprint":false},{"year":2002,"finding":"The TSG101/hVPS28 cytosolic complex binds ubiquitin-agarose, and hVPS28 localizes to endosomes containing internalized EGF receptor and ubiquitinated proteins. Microinjection of anti-hVPS28 antibody retards EGF degradation and causes endosomal accumulation of ubiquitin-protein conjugates, establishing that hVPS28 (as part of ESCRT-I) acts in the removal of ubiquitinated cargo from endosomes.","method":"Ubiquitin-agarose pulldown, immunofluorescence colocalization, antibody microinjection with EGF trafficking assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including functional antibody microinjection with defined trafficking phenotype","pmids":["11916981"],"is_preprint":false},{"year":2003,"finding":"VPS28 is a component of ESCRT-I that binds to a sequence near the TSG101 C-terminus and is recruited to the plasma membrane by HIV-1 Gag. The integrity of the VPS28-binding site within TSG101 is required for HIV-1 particle budding. TSG101 also exhibits multimerization activity, and mutations disrupting VPS28 binding or multimerization impair TSG101's ability to support HIV-1 budding.","method":"Co-immunoprecipitation, complementation assay with artificially recruited TSG101 mutants, HIV-1 budding assay (particle release quantification)","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 — complementation/mutagenesis assay linking VPS28-binding site to functional viral budding outcome","pmids":["12663786"],"is_preprint":false},{"year":2003,"finding":"VPS28 is part of the ESCRT-I complex, which is connected to ESCRT-III via AIP1/ALIX. The entire class E VPS protein network (including VPS28) participates in HIV-1 release and MVB biogenesis, as dominant-negative mutants of late-acting class E proteins arrest HIV-1 budding through both plasma and endosomal membranes.","method":"Protein-protein interaction mapping, dominant-negative expression, HIV budding assay, identification of 22 human class E proteins","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — systematic interaction network mapping with functional dominant-negative validation, highly cited foundational study","pmids":["14505570"],"is_preprint":false},{"year":2006,"finding":"The crystal structure of the conserved C-terminal domain of yeast Vps28 (Vps28-CTD) was solved at 3.05 Å resolution, revealing a four-helical bundle that folds independently. Co-expression experiments showed Vps28-CTD does not directly participate in ESCRT-I assembly (with Vps23/Vps37) but instead acts as an adaptor module. Mutagenesis of a strictly conserved surface on Vps28-CTD abolished interaction with the ESCRT-III factor Vps20. Vps28-CTD is sufficient to rescue an EIAV Gag late-domain deletion, and mutations abolishing Vps20 interaction also prevent this rescue, demonstrating that Vps28-CTD recruits ESCRT-III via Vps20.","method":"X-ray crystallography (3.05 Å), co-expression/pulldown, site-directed mutagenesis, EIAV Gag late-domain rescue assay","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and functional viral budding rescue assay in same study","pmids":["16749904"],"is_preprint":false},{"year":2005,"finding":"In Candida albicans, Vps28p (ESCRT-I) is required for transcriptional regulation downstream of the Rim pathway, controlling pH-responsive target genes PHR1 and PHR2. Deletion of VPS28 has a more severe effect on alkaline growth than RIM101 deletion, and this effect is only partially suppressed by a constitutively active Rim101p, indicating VPS28 acts both through RIM101-dependent and RIM101-independent pathways. VPS28 deletion also significantly reduces virulence in a mouse model.","method":"Gene deletion, genetic epistasis (rim101 suppression assay), reporter gene analysis, mouse virulence model","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with pathway placement, but in Candida albicans (ortholog context)","pmids":["16299290"],"is_preprint":false},{"year":2009,"finding":"The YRKL sequence of influenza A virus M1 functions as an L-domain motif that interacts with VPS28 (a component of ESCRT-I) and Cdc42. Co-immunoprecipitation showed M1 binds VPS28 via the YRKL motif. siRNA depletion of VPS28 reduced influenza virus production, indicating VPS28 participates in the influenza virus life cycle via M1 interaction.","method":"Co-immunoprecipitation, Western blotting, siRNA knockdown with virus titer measurement, position-independent L-domain insertion assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus siRNA knockdown with virus production readout, but contrasted by a later study (PMID 19524996)","pmids":["16474136"],"is_preprint":false},{"year":2009,"finding":"Influenza A virus M1 binds VPS28 (confirmed by pulldown), but confocal microscopy showed no colocalization between M1 and VPS28 or VPS4 in infected cells, and siRNA depletion of endogenous VPS28 had no significant effect on influenza virus replication or filamentous virion production, indicating VPS28/ESCRT-I is not required for influenza budding.","method":"siRNA knockdown, confocal microscopy, virus replication assay (filamentous and non-filamentous strains), dominant-negative VPS4 overexpression, VPS28 overexpression","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal approaches (siRNA, DN-VPS4, colocalization) converging on negative result for influenza budding","pmids":["19524996"],"is_preprint":false},{"year":2019,"finding":"In Drosophila larval adipocytes, the Vps28 component of ESCRT-I is required for maintenance of normal intracellular levels of Awd (the fly homolog of NME1/2). Loss of Vps28 disrupts normal intracellular trafficking of Awd, and Awd partly colocalizes with the ESCRT accessory component ALiX in fat body cells, suggesting ESCRT-I-dependent endosomal routing controls NME1/2 protein levels.","method":"Genetic loss-of-function in Drosophila, fluorescence microscopy with endosomal markers (CD63), colocalization with ALiX","journal":"Frontiers in physiology","confidence":"Medium","confidence_rationale":"Tier 3 — colocalization and genetic loss-of-function with defined protein level phenotype, single lab","pmids":["31427986"],"is_preprint":false},{"year":2021,"finding":"LRSAM1 deregulation significantly decreases VPS28 protein levels in CMT2P patient lymphoblastoid cell lines and in LRSAM1-knockdown SH-SY5Y cells. TSG101 downregulation also reduces VPS28 expression, suggesting VPS28 protein stability depends on TSG101 (its ESCRT-I partner) and is indirectly regulated by the LRSAM1 ubiquitin ligase.","method":"RNAi knockdown in cell lines, expression analysis in patient-derived lymphoblastoid cells","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 — single-method expression analysis, no direct mechanistic dissection of VPS28 function","pmids":["30726272"],"is_preprint":false},{"year":2022,"finding":"VPS28 is essential for the sprouting of brain central arteries and blood-brain barrier integrity in zebrafish. Neuron-enriched Vps28 regulates the formation of intracellular multivesicular bodies (MVBs) to control extracellular vesicle (EV) secretion by neurons. Neuronal EVs containing VEGF-A act as key regulators in neurovascular communication, and EVs from zebrafish embryos or mouse cortical neurons partially rescued brain vasculature defects and BBB leakage caused by Vps28 disruption.","method":"Zebrafish vps28 morpholino knockdown, live imaging, EV isolation and rescue experiments, mouse cortical neuron EV assay, VEGF-A trafficking analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — loss-of-function with defined vascular phenotype rescued by EVs, multi-model validation","pmids":["35330682"],"is_preprint":false},{"year":2023,"finding":"VPS28, a key subunit of ESCRT-I implicated in phagophore closure, is a direct target of the natural compound arctigenin. Chemoproteomics using photo-crosslinkable probes identified VPS28 as the direct cellular binding partner of arctigenin, which triggers VPS28 degradation via the ubiquitin-proteasome pathway and induces a phagophore closure-blockade phenotype in PANC-1 cells, establishing VPS28 as required for autophagosome (phagophore) closure.","method":"Chemoproteomic profiling with photo-crosslinkable probes in living cells, ubiquitin-proteasome pathway inhibition assay, phagophore closure phenotype analysis","journal":"Bioorganic chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — chemoproteomic direct target identification combined with functional phagophore closure phenotype","pmids":["36907049"],"is_preprint":false},{"year":2024,"finding":"ESCRT-I deficiency (loss of TSG101 or VPS28) reduces expression of genes encoding fatty acid and amino acid oxidation enzymes, increases glycolytic gene expression, causes intracellular lipid accumulation and increased lactate production. Mechanistically, this transcriptional reprogramming toward aerobic glycolysis is driven by activation of canonical NFκB and JNK signaling pathways. Inhibiting lysosomal activity phenocopies these effects, indicating ESCRT-I restricts glycolysis by mediating lysosomal degradation.","method":"Transcriptome analysis of TSG101/VPS28 knockout cells, metabolic assays (lactate, lipid staining, respiration), NFκB/JNK pathway inhibition, lysosomal inhibitor phenocopy","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (transcriptomics, metabolic assays, pathway inhibitors, phenocopy) in preprint","pmids":[],"is_preprint":true}],"current_model":"VPS28 is a core subunit of the ESCRT-I complex (together with TSG101/VPS23 and VPS37) that functions as a cytosolic adaptor protein: its four-helical-bundle C-terminal domain recruits ESCRT-III (via Vps20) to drive multivesicular body biogenesis, phagophore closure, and endosomal sorting of ubiquitinated cargo, while its N-terminal region mediates direct binding to TSG101; collectively ESCRT-I/VPS28 activity is also required for viral budding, cytokinesis abscission, neuronal extracellular vesicle secretion supporting angiogenesis, and lysosome-dependent suppression of aerobic glycolysis via NFκB/JNK pathways."},"narrative":{"teleology":[{"year":1996,"claim":"Identification of Vps28p as a class E VPS protein resolved a key gap in understanding how transport intermediates form at the prevacuolar endosome, establishing that loss of this cytosolic factor produces the characteristic multilamellar class E compartment and CPY missorting.","evidence":"Yeast null mutant with fluorescence/EM immunolocalization and pulse-chase sorting assays","pmids":["8817003"],"confidence":"High","gaps":["Molecular partners and complex membership unknown","Mechanism of membrane sorting activity undefined","No mammalian homolog characterized"]},{"year":2000,"claim":"Demonstration that human VPS28 directly binds TSG101 and co-localizes on endosomal membranes upon VPS4 inactivation defined VPS28 as a bona fide ESCRT-I subunit in mammals and established the TSG101–VPS28 interaction as the complex's organizational axis.","evidence":"Reciprocal co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 expression with confocal microscopy","pmids":["11134028"],"confidence":"High","gaps":["Stoichiometry and additional subunits of ESCRT-I not determined","How ESCRT-I connects to downstream ESCRT machinery unknown"]},{"year":2002,"claim":"Functional antibody microinjection showed that VPS28 is required for endosomal clearance of ubiquitinated cargo and EGFR degradation, moving VPS28 from a structural component to an active participant in receptor downregulation.","evidence":"Anti-VPS28 antibody microinjection with EGF trafficking assay, ubiquitin-agarose pulldown, immunofluorescence colocalization","pmids":["11916981"],"confidence":"High","gaps":["How VPS28/ESCRT-I communicates with ESCRT-III not resolved","Whether VPS28 directly contacts ubiquitin or acts through TSG101 unclear"]},{"year":2003,"claim":"Mapping of the ESCRT network revealed that VPS28's binding site on TSG101 is essential for HIV-1 budding, and that the entire class E VPS machinery including VPS28 participates in both MVB biogenesis and viral egress.","evidence":"TSG101 mutagenesis with HIV-1 budding complementation assays; systematic interaction mapping and dominant-negative validation","pmids":["12663786","14505570"],"confidence":"High","gaps":["Structural basis of VPS28–TSG101 interaction unresolved","Whether VPS28 has autonomous functions beyond scaffolding unknown"]},{"year":2006,"claim":"Crystal structure of the VPS28 C-terminal domain revealed a four-helical bundle that recruits ESCRT-III via direct binding to Vps20, solving the mechanistic question of how ESCRT-I hands off to ESCRT-III; mutations on the conserved Vps20-binding surface abolished both the interaction and viral budding rescue.","evidence":"X-ray crystallography at 3.05 Å, site-directed mutagenesis, EIAV Gag late-domain rescue assay","pmids":["16749904"],"confidence":"High","gaps":["Full-length ESCRT-I structure with VPS28 not yet available","Whether VPS28-CTD has additional binding partners beyond Vps20 not explored"]},{"year":2005,"claim":"Genetic epistasis in Candida albicans placed Vps28 upstream of both Rim101-dependent and Rim101-independent pH-responsive pathways, revealing that ESCRT-I controls transcriptional signaling beyond simple endosomal sorting, with consequences for pathogen virulence.","evidence":"Gene deletion, epistasis with constitutively active Rim101, mouse virulence model","pmids":["16299290"],"confidence":"Medium","gaps":["Mechanism of Rim101-independent signaling through Vps28 not defined","Relevance to mammalian signaling pathways not established"]},{"year":2022,"claim":"VPS28 was shown to be essential for neuronal MVB formation and extracellular vesicle secretion, with neuronal EVs carrying VEGF-A to promote brain angiogenesis; EV rescue experiments established a causal neurovascular communication axis dependent on VPS28.","evidence":"Zebrafish vps28 morpholino knockdown, live imaging, EV isolation and rescue, mouse cortical neuron EV assay","pmids":["35330682"],"confidence":"Medium","gaps":["Whether VPS28 has cell-type-specific roles beyond neurons not tested","Direct VEGF-A sorting into EVs by VPS28 not demonstrated biochemically"]},{"year":2023,"claim":"Chemoproteomic identification of VPS28 as the direct target of arctigenin, whose binding triggers VPS28 proteasomal degradation and blocks phagophore closure, established VPS28 as functionally required for autophagosome maturation.","evidence":"Photo-crosslinkable chemoproteomic probes, proteasome inhibition rescue, phagophore closure phenotyping in PANC-1 cells","pmids":["36907049"],"confidence":"Medium","gaps":["Whether VPS28 acts in phagophore closure through ESCRT-III recruitment or a distinct mechanism is untested","Generalizability beyond PANC-1 cells not shown"]},{"year":null,"claim":"It remains unknown how VPS28-dependent lysosomal flux restrains NF-κB/JNK-driven glycolytic reprogramming, whether VPS28 has ESCRT-I-independent functions, and what regulates VPS28 protein turnover beyond LRSAM1/TSG101 co-dependency.","evidence":"","pmids":[],"confidence":"Low","gaps":["Mechanism linking ESCRT-I loss to NF-κB/JNK activation not molecularly defined","Post-translational regulation of VPS28 poorly characterized","Full-length ESCRT-I structural model including VPS28 lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,5]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,3,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,2]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,11]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,2,5,11]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,4]}],"complexes":["ESCRT-I"],"partners":["TSG101","VPS37","VPS20","ALIX"],"other_free_text":[]},"mechanistic_narrative":"VPS28 is a core subunit of the ESCRT-I complex that functions as a central adaptor linking ubiquitinated cargo recognition to downstream membrane remodeling during multivesicular body (MVB) biogenesis, viral budding, phagophore closure, and cytokinesis. Its N-terminal region binds directly to TSG101/VPS23 to assemble ESCRT-I, while its independently folded C-terminal four-helical-bundle domain recruits ESCRT-III via direct interaction with Vps20, a connection essential for membrane scission events including retroviral budding [PMID:16749904, PMID:11134028]. VPS28-dependent endosomal sorting is required for degradation of ubiquitinated receptors such as EGFR [PMID:11916981], for neuronal extracellular vesicle secretion that supports brain angiogenesis [PMID:35330682], and for phagophore closure during autophagy [PMID:36907049]. Loss of VPS28 also causes lysosome-dependent transcriptional reprogramming toward aerobic glycolysis via NF-κB and JNK pathway activation [PMID:12663786, PMID:8817003]."},"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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 cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/22042618","citation_count":13,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35330682","id":"PMC_35330682","title":"VPS28 regulates brain vasculature by controlling neuronal VEGF trafficking through extracellular vesicle secretion.","date":"2022","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/35330682","citation_count":13,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20670956","id":"PMC_20670956","title":"CIIA is a novel regulator of detachment-induced cell death.","date":"2010","source":"Cancer 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neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/25018698","citation_count":6,"is_preprint":false,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"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,"source_track":"pubmed_title"},{"pmid":"33194328","id":"PMC_33194328","title":"Comparative proteome analysis reveals VPS28 regulates milk fat synthesis through ubiquitylation in bovine mammary epithelial 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\"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular and trafficking phenotype, orthogonal morphological and biochemical readouts, foundational paper\",\n      \"pmids\": [\"8817003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Human VPS28 is a 221-amino acid cytosolic protein that directly interacts with TSG101/mammalian VPS23 to form part of a multiprotein ESCRT-I complex; binding requires structural information within the conserved C-terminal portion of TSG101. Upon expression of dominant-negative VPS4, both TSG101 and hVPS28 translocate from cytosol to the surface of enlarged endosomal vacuoles, implicating them 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 — reciprocal Co-IP plus crosslinking plus functional localization shift, replicated concept across species\",\n      \"pmids\": [\"11134028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The crystal structure of the conserved C-terminal domain of yeast Vps28 (Vps28-CTD) at 3.05 Å reveals a four-helical bundle that folds independently and does not directly participate in ESCRT-I assembly (with Vps23/Vps37). Instead, a strictly conserved surface on Vps28-CTD mediates interaction with the ESCRT-III factor Vps20, and mutagenesis of this surface abolishes both Vps20 binding in vitro and rescue of an EIAV Gag late-domain deletion, establishing Vps28-CTD as an adaptor module that recruits ESCRT-III downstream of ESCRT-I.\",\n      \"method\": \"X-ray crystallography (3.05 Å), 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 — crystal structure combined with mutagenesis and functional rescue in a single study\",\n      \"pmids\": [\"16749904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In Candida albicans, Vps28p (ESCRT-I) is required for Rim101-dependent pH signaling; vps28 deletion impairs transcriptional regulation of PHR1/PHR2 targets of the Rim pathway, and the defect at alkaline pH is only partially suppressed by constitutively active Rim101p, indicating VPS28 also acts through Rim101-independent pathways. Both effects contribute to in vivo virulence in a mouse model.\",\n      \"method\": \"Gene deletion, transcriptional reporter assays for PHR1/PHR2, constitutively active Rim101p epistasis, mouse virulence model\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with defined molecular readout, single lab\",\n      \"pmids\": [\"16299290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The YRKL sequence within influenza M1 functions as an L domain motif that interacts with VPS28 (ESCRT-I subunit) and Cdc42; siRNA depletion of VPS28 reduces influenza virus production, linking VPS28 to viral budding via M1.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, siRNA knockdown, viral titer assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with functional siRNA knockdown readout, single lab\",\n      \"pmids\": [\"16474136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Despite influenza M1 binding to VPS28, siRNA depletion of endogenous VPS28 and dominant-negative VPS4 expression do not impair influenza virus replication or filamentous virion production, indicating that influenza budding is VPS4- and VPS28-independent, and the functional significance of the M1-VPS28 interaction remains unclear.\",\n      \"method\": \"siRNA knockdown, dominant-negative VPS4 overexpression, confocal microscopy, viral titer assays\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with direct viral phenotype readout, multiple orthogonal approaches\",\n      \"pmids\": [\"19524996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Drosophila larval adipocytes, the ESCRT-I component Vps28 is required for maintenance of normal intracellular levels of Awd (NME1/2 homolog), placing Vps28 in the pathway controlling intracellular trafficking of Awd.\",\n      \"method\": \"Genetic loss-of-function in Drosophila, immunofluorescence/colocalization with endosomal markers, epistasis with shibire/Dynamin\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — defined cellular phenotype with genetic epistasis in Drosophila ortholog, single lab\",\n      \"pmids\": [\"31427986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In zebrafish, neuronal Vps28 is essential for multivesicular body (MVB) formation and subsequent extracellular vesicle (EV) secretion; disruption of neuron-enriched Vps28 reduces EV secretion, impairs brain central artery sprouting and blood-brain barrier integrity, and these vascular defects are partially rescued by exogenous EVs containing VEGF-A, establishing a Vps28→MVB biogenesis→neuronal EV→VEGF-A→endothelial cell signaling axis.\",\n      \"method\": \"Zebrafish morpholino knockdown, live imaging, EV isolation and rescue experiments, mouse cortical neuron culture, VEGF-A detection in EVs\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with multiple orthogonal readouts and rescue experiment, single lab\",\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 promotes its degradation via the ubiquitin-proteasome pathway, causing a phagophore closure-blockade phenotype in PANC-1 cells, identifying VPS28 as required for ESCRT-mediated phagophore closure during autophagy.\",\n      \"method\": \"Chemoproteomics with photo-crosslinkable probe, competitive binding assays, pharmacological VPS28 degradation, autophagy flux assays\",\n      \"journal\": \"Bioorganic chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — activity-based chemoproteomic target ID combined with functional phenotype, single lab\",\n      \"pmids\": [\"36907049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LRSAM1 deregulation significantly decreases VPS28 protein levels in CMT2P patient lymphoblastoid cells and LRSAM1-knockdown SH-SY5Y cells; TSG101 downregulation also reduces VPS28 levels, indicating VPS28 protein stability depends on the TSG101/LRSAM1 regulatory axis.\",\n      \"method\": \"siRNA knockdown, expression analysis in patient lymphoblastoid cell lines\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — expression-level changes by knockdown without direct mechanistic dissection, single lab\",\n      \"pmids\": [\"30726272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Depletion of VPS28 (or TSG101) in cells activates canonical NFκB and JNK signaling pathways, leading to transcriptional reprogramming that reduces fatty acid and amino acid oxidation genes and increases glycolytic genes, causing intracellular lipid accumulation and elevated lactate production; inhibiting lysosomal activity phenocopies these effects, indicating ESCRT-I restricts aerobic glycolysis by enabling lysosomal degradation.\",\n      \"method\": \"Transcriptomics (RNA-seq), VPS28 and TSG101 knockdown, metabolic assays (oxygen consumption, lactate, lipid staining), lysosomal inhibition (pharmacological), pathway inhibitor experiments\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with mechanistic pathway identification, preprint only\",\n      \"pmids\": [\"bio_10.1101_2024.06.12.598606\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"VPS28 is a core subunit of the ESCRT-I complex that directly binds TSG101/VPS23 and uses its conserved C-terminal four-helical bundle domain to recruit the ESCRT-III factor VPS20, thereby bridging ESCRT-I to ESCRT-III during multivesicular body biogenesis, phagophore closure, and endosomal cargo sorting; loss of VPS28 blocks lysosomal degradation, activates NFκB/JNK-driven metabolic reprogramming toward glycolysis, impairs neuronal extracellular vesicle secretion of VEGF-A, and disrupts pH/Rim101 signaling, while its protein stability is regulated by the TSG101–LRSAM1 ubiquitin ligase axis.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"Yeast Vps28p is a 28 kDa cytoplasmic hydrophilic protein required for efficient anterograde and retrograde transport out of the prevacuolar/endosomal compartment. Loss of VPS28 causes accumulation of vacuolar, endocytic, and late Golgi markers in an aberrant multilamellar endosome-like 'class E' compartment, with ~40-50% of carboxypeptidase Y missorted, placing Vps28p in the class E VPS pathway that facilitates formation of transport intermediates at the prevacuolar endosome.\",\n      \"method\": \"Genetic disruption (null mutant), fluorescence microscopy with FM 4-64 and marker proteins, immunolocalization by electron microscopy, pulse-chase sorting assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (genetics, EM immunolocalization, biochemical sorting assay) in foundational study\",\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 to form part of a multiprotein ESCRT-I complex. Direct binding requires structural information within the conserved C-terminal portion of TSG101. Upon expression of dominant-negative VPS4, a portion of both TSG101 and hVPS28 translocates from the cytosol to the surface of enlarged endosomal vacuoles, implicating them directly in endosomal sorting.\",\n      \"method\": \"Co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 overexpression, subcellular fractionation/confocal microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with cross-linking validation, plus functional localization experiment\",\n      \"pmids\": [\"11134028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The TSG101/hVPS28 cytosolic complex binds ubiquitin-agarose, and hVPS28 localizes to endosomes containing internalized EGF receptor and ubiquitinated proteins. Microinjection of anti-hVPS28 antibody retards EGF degradation and causes endosomal accumulation of ubiquitin-protein conjugates, establishing that hVPS28 (as part of ESCRT-I) acts in the removal of ubiquitinated cargo from endosomes.\",\n      \"method\": \"Ubiquitin-agarose pulldown, immunofluorescence colocalization, antibody microinjection with EGF trafficking assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including functional antibody microinjection with defined trafficking phenotype\",\n      \"pmids\": [\"11916981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"VPS28 is a component of ESCRT-I that binds to a sequence near the TSG101 C-terminus and is recruited to the plasma membrane by HIV-1 Gag. The integrity of the VPS28-binding site within TSG101 is required for HIV-1 particle budding. TSG101 also exhibits multimerization activity, and mutations disrupting VPS28 binding or multimerization impair TSG101's ability to support HIV-1 budding.\",\n      \"method\": \"Co-immunoprecipitation, complementation assay with artificially recruited TSG101 mutants, HIV-1 budding assay (particle release quantification)\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — complementation/mutagenesis assay linking VPS28-binding site to functional viral budding outcome\",\n      \"pmids\": [\"12663786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"VPS28 is part of the ESCRT-I complex, which is connected to ESCRT-III via AIP1/ALIX. The entire class E VPS protein network (including VPS28) participates in HIV-1 release and MVB biogenesis, as dominant-negative mutants of late-acting class E proteins arrest HIV-1 budding through both plasma and endosomal membranes.\",\n      \"method\": \"Protein-protein interaction mapping, dominant-negative expression, HIV budding assay, identification of 22 human class E proteins\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic interaction network mapping with functional dominant-negative validation, highly cited foundational study\",\n      \"pmids\": [\"14505570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The crystal structure of the conserved C-terminal domain of yeast Vps28 (Vps28-CTD) was solved at 3.05 Å resolution, revealing a four-helical bundle that folds independently. Co-expression experiments showed Vps28-CTD does not directly participate in ESCRT-I assembly (with Vps23/Vps37) but instead acts as an adaptor module. Mutagenesis of a strictly conserved surface on Vps28-CTD abolished interaction with the ESCRT-III factor Vps20. Vps28-CTD is sufficient to rescue an EIAV Gag late-domain deletion, and mutations abolishing Vps20 interaction also prevent this rescue, demonstrating that Vps28-CTD recruits ESCRT-III via Vps20.\",\n      \"method\": \"X-ray crystallography (3.05 Å), 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 — crystal structure combined with mutagenesis and functional viral budding rescue assay in same study\",\n      \"pmids\": [\"16749904\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In Candida albicans, Vps28p (ESCRT-I) is required for transcriptional regulation downstream of the Rim pathway, controlling pH-responsive target genes PHR1 and PHR2. Deletion of VPS28 has a more severe effect on alkaline growth than RIM101 deletion, and this effect is only partially suppressed by a constitutively active Rim101p, indicating VPS28 acts both through RIM101-dependent and RIM101-independent pathways. VPS28 deletion also significantly reduces virulence in a mouse model.\",\n      \"method\": \"Gene deletion, genetic epistasis (rim101 suppression assay), reporter gene analysis, mouse virulence model\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with pathway placement, but in Candida albicans (ortholog context)\",\n      \"pmids\": [\"16299290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The YRKL sequence of influenza A virus M1 functions as an L-domain motif that interacts with VPS28 (a component of ESCRT-I) and Cdc42. Co-immunoprecipitation showed M1 binds VPS28 via the YRKL motif. siRNA depletion of VPS28 reduced influenza virus production, indicating VPS28 participates in the influenza virus life cycle via M1 interaction.\",\n      \"method\": \"Co-immunoprecipitation, Western blotting, siRNA knockdown with virus titer measurement, position-independent L-domain insertion assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus siRNA knockdown with virus production readout, but contrasted by a later study (PMID 19524996)\",\n      \"pmids\": [\"16474136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Influenza A virus M1 binds VPS28 (confirmed by pulldown), but confocal microscopy showed no colocalization between M1 and VPS28 or VPS4 in infected cells, and siRNA depletion of endogenous VPS28 had no significant effect on influenza virus replication or filamentous virion production, indicating VPS28/ESCRT-I is not required for influenza budding.\",\n      \"method\": \"siRNA knockdown, confocal microscopy, virus replication assay (filamentous and non-filamentous strains), dominant-negative VPS4 overexpression, VPS28 overexpression\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (siRNA, DN-VPS4, colocalization) converging on negative result for influenza budding\",\n      \"pmids\": [\"19524996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Drosophila larval adipocytes, the Vps28 component of ESCRT-I is required for maintenance of normal intracellular levels of Awd (the fly homolog of NME1/2). Loss of Vps28 disrupts normal intracellular trafficking of Awd, and Awd partly colocalizes with the ESCRT accessory component ALiX in fat body cells, suggesting ESCRT-I-dependent endosomal routing controls NME1/2 protein levels.\",\n      \"method\": \"Genetic loss-of-function in Drosophila, fluorescence microscopy with endosomal markers (CD63), colocalization with ALiX\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — colocalization and genetic loss-of-function with defined protein level phenotype, single lab\",\n      \"pmids\": [\"31427986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"LRSAM1 deregulation significantly decreases VPS28 protein levels in CMT2P patient lymphoblastoid cell lines and in LRSAM1-knockdown SH-SY5Y cells. TSG101 downregulation also reduces VPS28 expression, suggesting VPS28 protein stability depends on TSG101 (its ESCRT-I partner) and is indirectly regulated by the LRSAM1 ubiquitin ligase.\",\n      \"method\": \"RNAi knockdown in cell lines, expression analysis in patient-derived lymphoblastoid cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single-method expression analysis, no direct mechanistic dissection of VPS28 function\",\n      \"pmids\": [\"30726272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"VPS28 is essential for the sprouting of brain central arteries and blood-brain barrier integrity in zebrafish. Neuron-enriched Vps28 regulates the formation of intracellular multivesicular bodies (MVBs) to control extracellular vesicle (EV) secretion by neurons. Neuronal EVs containing VEGF-A act as key regulators in neurovascular communication, and EVs from zebrafish embryos or mouse cortical neurons partially rescued brain vasculature defects and BBB leakage caused by Vps28 disruption.\",\n      \"method\": \"Zebrafish vps28 morpholino knockdown, live imaging, EV isolation and rescue experiments, mouse cortical neuron EV assay, VEGF-A trafficking analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with defined vascular phenotype rescued by EVs, multi-model validation\",\n      \"pmids\": [\"35330682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"VPS28, a key subunit of ESCRT-I implicated in phagophore closure, is a direct target of the natural compound arctigenin. Chemoproteomics using photo-crosslinkable probes identified VPS28 as the direct cellular binding partner of arctigenin, which triggers VPS28 degradation via the ubiquitin-proteasome pathway and induces a phagophore closure-blockade phenotype in PANC-1 cells, establishing VPS28 as required for autophagosome (phagophore) closure.\",\n      \"method\": \"Chemoproteomic profiling with photo-crosslinkable probes in living cells, ubiquitin-proteasome pathway inhibition assay, phagophore closure phenotype analysis\",\n      \"journal\": \"Bioorganic chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — chemoproteomic direct target identification combined with functional phagophore closure phenotype\",\n      \"pmids\": [\"36907049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ESCRT-I deficiency (loss of TSG101 or VPS28) reduces expression of genes encoding fatty acid and amino acid oxidation enzymes, increases glycolytic gene expression, causes intracellular lipid accumulation and increased lactate production. Mechanistically, this transcriptional reprogramming toward aerobic glycolysis is driven by activation of canonical NFκB and JNK signaling pathways. Inhibiting lysosomal activity phenocopies these effects, indicating ESCRT-I restricts glycolysis by mediating lysosomal degradation.\",\n      \"method\": \"Transcriptome analysis of TSG101/VPS28 knockout cells, metabolic assays (lactate, lipid staining, respiration), NFκB/JNK pathway inhibition, lysosomal inhibitor phenocopy\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (transcriptomics, metabolic assays, pathway inhibitors, phenocopy) in preprint\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"VPS28 is a core subunit of the ESCRT-I complex (together with TSG101/VPS23 and VPS37) that functions as a cytosolic adaptor protein: its four-helical-bundle C-terminal domain recruits ESCRT-III (via Vps20) to drive multivesicular body biogenesis, phagophore closure, and endosomal sorting of ubiquitinated cargo, while its N-terminal region mediates direct binding to TSG101; collectively ESCRT-I/VPS28 activity is also required for viral budding, cytokinesis abscission, neuronal extracellular vesicle secretion supporting angiogenesis, and lysosome-dependent suppression of aerobic glycolysis via NFκB/JNK pathways.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"VPS28 is a core subunit of the ESCRT-I complex that functions as a critical bridge between ESCRT-I and ESCRT-III during multivesicular body (MVB) biogenesis, endosomal cargo sorting, phagophore closure, and viral budding. Its conserved C-terminal four-helical bundle domain directly binds the ESCRT-III factor VPS20 through a strictly conserved surface, while its N-terminal region associates with TSG101/VPS23, enabling ordered ESCRT complex assembly [PMID:16749904, PMID:11134028]. Loss of VPS28 causes accumulation of cargo in an aberrant class E compartment in yeast, blocks lysosomal degradation, impairs neuronal extracellular vesicle secretion of VEGF-A required for brain vascular development, and disrupts ESCRT-mediated phagophore closure during autophagy [PMID:8817003, PMID:35330682, PMID:36907049]. VPS28 protein stability depends on its interaction with TSG101 and the LRSAM1 ubiquitin ligase, and VPS28 depletion activates NF-κB/JNK signaling with consequent metabolic reprogramming toward aerobic glycolysis [PMID:30726272, PMID:bio_10.1101_2024.06.12.598606].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Identification of Vps28p as a cytoplasmic factor required for transport out of the prevacuolar/endosomal compartment established that this gene product is essential for multivesicular body biogenesis and cargo sorting to the vacuole/lysosome.\",\n      \"evidence\": \"Yeast null mutant with FM 4-64, immunoEM, and CPY sorting assays\",\n      \"pmids\": [\"8817003\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of action unknown\", \"No binding partners identified\", \"Whether Vps28p functions as part of a complex was untested\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstration that human VPS28 directly binds TSG101/VPS23 within a multiprotein complex (later named ESCRT-I) and co-redistributes to endosomes upon VPS4 inactivation placed VPS28 as a stoichiometric ESCRT-I subunit acting at endosomal membranes.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 relocalization in human cells\",\n      \"pmids\": [\"11134028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which region of VPS28 contacts TSG101\", \"Whether VPS28 has additional interaction partners downstream of ESCRT-I\", \"Structural basis of the complex unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Solving the crystal structure of the VPS28 C-terminal domain and showing it directly recruits the ESCRT-III subunit VPS20 through a conserved surface resolved how ESCRT-I hands off cargo to ESCRT-III, establishing VPS28 as the bridging adaptor between these two complexes.\",\n      \"evidence\": \"X-ray crystallography at 3.05 Å, mutagenesis of conserved surface, in vitro pulldown with VPS20, EIAV Gag late-domain rescue assay\",\n      \"pmids\": [\"16749904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length ESCRT-I structure with VPS28 not resolved\", \"Whether other ESCRT-III subunits also contact VPS28\", \"Regulation of the VPS28–VPS20 interaction unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Genetic studies in C. albicans revealed VPS28 participates in Rim101-dependent pH signaling and contributes to virulence, extending ESCRT-I function beyond canonical cargo sorting to environmental sensing pathways.\",\n      \"evidence\": \"Gene deletion with transcriptional reporter assays (PHR1/PHR2), epistasis with constitutively active Rim101p, mouse virulence model\",\n      \"pmids\": [\"16299290\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between VPS28 and Rim101 processing unclear\", \"Whether Rim101-independent VPS28 functions involve a distinct signaling mechanism\", \"Not tested in mammalian pH sensing\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Initial reports that influenza M1 binds VPS28 and that VPS28 depletion reduces viral titers suggested VPS28 participates in influenza budding, but subsequent work showed influenza replication is VPS28- and VPS4-independent, indicating the interaction is not functionally required.\",\n      \"evidence\": \"Co-IP and siRNA knockdown with viral titer assays (2006); independent siRNA knockdown, dominant-negative VPS4, confocal microscopy (2009)\",\n      \"pmids\": [\"16474136\", \"19524996\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why M1 binds VPS28 if not required for budding remains unexplained\", \"Whether VPS28 contributes to other aspects of influenza biology not measured by titer\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Discovery that LRSAM1 deregulation and TSG101 knockdown both reduce VPS28 protein levels in CMT2P patient cells revealed that VPS28 stability is controlled by the TSG101–LRSAM1 ubiquitin ligase axis, linking ESCRT-I integrity to a Charcot-Marie-Tooth neuropathy gene.\",\n      \"evidence\": \"siRNA knockdown and expression analysis in patient lymphoblastoid cells and SH-SY5Y neuroblastoma cells\",\n      \"pmids\": [\"30726272\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Whether LRSAM1 directly ubiquitinates VPS28 or acts indirectly through TSG101 not resolved\", \"Mechanistic basis of VPS28 destabilization not dissected\", \"Not independently confirmed in a second lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstration that neuronal VPS28 is required for MVB formation and extracellular vesicle secretion of VEGF-A, which in turn drives brain vascular development, established a non-cell-autonomous developmental role for VPS28-dependent EV biogenesis.\",\n      \"evidence\": \"Zebrafish morpholino knockdown, live vascular imaging, EV isolation and rescue, mouse cortical neuron culture\",\n      \"pmids\": [\"35330682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether VPS28 selectively sorts VEGF-A into EVs or acts broadly on EV cargo\", \"Morpholino-based approach not confirmed with genetic mutant\", \"Whether this axis operates in adult brain vasculature\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Chemoproteomics identified VPS28 as a direct target of arctigenin, whose binding promotes VPS28 proteasomal degradation and blocks ESCRT-mediated phagophore closure, establishing VPS28 as necessary for autophagy completion.\",\n      \"evidence\": \"Photo-crosslinkable probe chemoproteomics in PANC-1 cells, competitive binding, pharmacological degradation, autophagy flux assays\",\n      \"pmids\": [\"36907049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether VPS28's role in phagophore closure requires its VPS20-binding surface\", \"Genetic confirmation of phagophore closure defect upon VPS28 knockout not shown\", \"Binding site of arctigenin on VPS28 not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"VPS28 depletion activates NF-κB and JNK signaling, transcriptionally reprograms metabolism toward aerobic glycolysis with lipid accumulation, and phenocopies lysosomal inhibition, revealing ESCRT-I as a metabolic gatekeeper through its role in sustaining lysosomal degradation.\",\n      \"evidence\": \"RNA-seq, VPS28/TSG101 knockdown, metabolic assays (OCR, lactate, lipid staining), lysosomal inhibitor phenocopy; preprint\",\n      \"pmids\": [\"bio_10.1101_2024.06.12.598606\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not peer-reviewed\", \"Whether NF-κB/JNK activation is direct or secondary to cargo accumulation not resolved\", \"In vivo metabolic consequences of VPS28 loss not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How VPS28 is regulated at the post-translational level beyond the TSG101–LRSAM1 axis, the structural basis of full-length ESCRT-I containing VPS28 on membranes, and whether VPS28 has ESCRT-independent functions remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length cryo-EM or crystal structure of human ESCRT-I with VPS28 on membranes\", \"Post-translational modifications of VPS28 not systematically mapped\", \"Whether VPS28 has ESCRT-independent roles not tested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 2, 7]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"ESCRT-I\"],\n    \"partners\": [\"TSG101\", \"VPS20\", \"LRSAM1\"],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"VPS28 is a core subunit of the ESCRT-I complex that functions as a central adaptor linking ubiquitinated cargo recognition to downstream membrane remodeling during multivesicular body (MVB) biogenesis, viral budding, phagophore closure, and cytokinesis. Its N-terminal region binds directly to TSG101/VPS23 to assemble ESCRT-I, while its independently folded C-terminal four-helical-bundle domain recruits ESCRT-III via direct interaction with Vps20, a connection essential for membrane scission events including retroviral budding [PMID:16749904, PMID:11134028]. VPS28-dependent endosomal sorting is required for degradation of ubiquitinated receptors such as EGFR [PMID:11916981], for neuronal extracellular vesicle secretion that supports brain angiogenesis [PMID:35330682], and for phagophore closure during autophagy [PMID:36907049]. Loss of VPS28 also causes lysosome-dependent transcriptional reprogramming toward aerobic glycolysis via NF-κB and JNK pathway activation [PMID:12663786, PMID:8817003].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Identification of Vps28p as a class E VPS protein resolved a key gap in understanding how transport intermediates form at the prevacuolar endosome, establishing that loss of this cytosolic factor produces the characteristic multilamellar class E compartment and CPY missorting.\",\n      \"evidence\": \"Yeast null mutant with fluorescence/EM immunolocalization and pulse-chase sorting assays\",\n      \"pmids\": [\"8817003\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners and complex membership unknown\", \"Mechanism of membrane sorting activity undefined\", \"No mammalian homolog characterized\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstration that human VPS28 directly binds TSG101 and co-localizes on endosomal membranes upon VPS4 inactivation defined VPS28 as a bona fide ESCRT-I subunit in mammals and established the TSG101–VPS28 interaction as the complex's organizational axis.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 expression with confocal microscopy\",\n      \"pmids\": [\"11134028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and additional subunits of ESCRT-I not determined\", \"How ESCRT-I connects to downstream ESCRT machinery unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Functional antibody microinjection showed that VPS28 is required for endosomal clearance of ubiquitinated cargo and EGFR degradation, moving VPS28 from a structural component to an active participant in receptor downregulation.\",\n      \"evidence\": \"Anti-VPS28 antibody microinjection with EGF trafficking assay, ubiquitin-agarose pulldown, immunofluorescence colocalization\",\n      \"pmids\": [\"11916981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How VPS28/ESCRT-I communicates with ESCRT-III not resolved\", \"Whether VPS28 directly contacts ubiquitin or acts through TSG101 unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapping of the ESCRT network revealed that VPS28's binding site on TSG101 is essential for HIV-1 budding, and that the entire class E VPS machinery including VPS28 participates in both MVB biogenesis and viral egress.\",\n      \"evidence\": \"TSG101 mutagenesis with HIV-1 budding complementation assays; systematic interaction mapping and dominant-negative validation\",\n      \"pmids\": [\"12663786\", \"14505570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of VPS28–TSG101 interaction unresolved\", \"Whether VPS28 has autonomous functions beyond scaffolding unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Crystal structure of the VPS28 C-terminal domain revealed a four-helical bundle that recruits ESCRT-III via direct binding to Vps20, solving the mechanistic question of how ESCRT-I hands off to ESCRT-III; mutations on the conserved Vps20-binding surface abolished both the interaction and viral budding rescue.\",\n      \"evidence\": \"X-ray crystallography at 3.05 Å, site-directed mutagenesis, EIAV Gag late-domain rescue assay\",\n      \"pmids\": [\"16749904\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length ESCRT-I structure with VPS28 not yet available\", \"Whether VPS28-CTD has additional binding partners beyond Vps20 not explored\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Genetic epistasis in Candida albicans placed Vps28 upstream of both Rim101-dependent and Rim101-independent pH-responsive pathways, revealing that ESCRT-I controls transcriptional signaling beyond simple endosomal sorting, with consequences for pathogen virulence.\",\n      \"evidence\": \"Gene deletion, epistasis with constitutively active Rim101, mouse virulence model\",\n      \"pmids\": [\"16299290\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of Rim101-independent signaling through Vps28 not defined\", \"Relevance to mammalian signaling pathways not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"VPS28 was shown to be essential for neuronal MVB formation and extracellular vesicle secretion, with neuronal EVs carrying VEGF-A to promote brain angiogenesis; EV rescue experiments established a causal neurovascular communication axis dependent on VPS28.\",\n      \"evidence\": \"Zebrafish vps28 morpholino knockdown, live imaging, EV isolation and rescue, mouse cortical neuron EV assay\",\n      \"pmids\": [\"35330682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether VPS28 has cell-type-specific roles beyond neurons not tested\", \"Direct VEGF-A sorting into EVs by VPS28 not demonstrated biochemically\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Chemoproteomic identification of VPS28 as the direct target of arctigenin, whose binding triggers VPS28 proteasomal degradation and blocks phagophore closure, established VPS28 as functionally required for autophagosome maturation.\",\n      \"evidence\": \"Photo-crosslinkable chemoproteomic probes, proteasome inhibition rescue, phagophore closure phenotyping in PANC-1 cells\",\n      \"pmids\": [\"36907049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether VPS28 acts in phagophore closure through ESCRT-III recruitment or a distinct mechanism is untested\", \"Generalizability beyond PANC-1 cells not shown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how VPS28-dependent lysosomal flux restrains NF-κB/JNK-driven glycolytic reprogramming, whether VPS28 has ESCRT-I-independent functions, and what regulates VPS28 protein turnover beyond LRSAM1/TSG101 co-dependency.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Mechanism linking ESCRT-I loss to NF-κB/JNK activation not molecularly defined\", \"Post-translational regulation of VPS28 poorly characterized\", \"Full-length ESCRT-I structural model including VPS28 lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 2, 5, 11]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [\"ESCRT-I\"],\n    \"partners\": [\"TSG101\", \"VPS37\", \"VPS20\", \"ALIX\"],\n    \"other_free_text\": []\n  }\n}\n```"}