{"gene":"PSMD5","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2000,"finding":"PSMD5 (S5b) forms a tetramer with ATPase subunits S4 (PSMC1) and S7 (PSMC2) and the non-ATPase subunit S2 (PSMD1) within the 19S regulatory complex. Using in vitro co-translation, immunoprecipitation, and sedimentation, S5b was shown to bind the C-terminal two-thirds of S4 and the N-terminal region of S7, while S2 binds the N-terminal region of S4 and the C-terminal two-thirds of S7, forming a defined S2-S4-S7-S5b tetramer. S5b did not associate with the S6-S8 or S6'-S10b ATPase dimers, indicating specificity for the S4/S7 pair.","method":"In vitro co-translation in reticulocyte lysate with [35S]methionine, co-immunoprecipitation, sedimentation analysis, chimeric ATPase domain-swap experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (co-translation, co-IP, sedimentation, domain-swap chimeras) in a single rigorous study","pmids":["10625621"],"is_preprint":false},{"year":2009,"finding":"Yeast Hsm3 is a dedicated assembly chaperone for the 19S regulatory particle base subcomplex and is the functional homolog of mammalian S5b/PSMD5. Hsm3 associates with 19S base subcomplexes via the C-terminal domain of the Rpt1 (base) ATPase subunit but is absent from the final mature 26S proteasome, demonstrating that it acts transiently during assembly and is not a stoichiometric subunit of the final complex. Hsm3 is specifically required for base subcomplex assembly.","method":"Co-immunoprecipitation, sucrose gradient sedimentation, yeast genetics (deletion mutants), structural/sequence homology analysis between Hsm3 and human S5b/PSMD5","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, genetic epistasis, multiple orthogonal methods; independently confirmed by commentary (PMID:19250902)","pmids":["19217412","19250902"],"is_preprint":false},{"year":2012,"finding":"PSMD5 (S5b) acts as a negative regulator of 26S proteasome assembly and activity. TNF-α increases PSMD5 expression via NFκB activation, and surplus PSMD5 directly inhibits 26S proteasome assembly and activity. Mechanistically, TNF-α enhances the interaction of PSMD5 with the ATPase subunit S7/PSMC2 in non-proteasome complexes (i.e., free PSMD5-PSMC2 complexes outside the assembled proteasome), and interfering with this interaction rescues TNF-α-induced proteasome inhibition. Downregulation of PSMD5 abolished TNF-α-induced proteasome inhibition. Transgenic mice overexpressing PSMD5 show reduced proteasome activity and premature aging phenotypes, while PSMD5 deficiency in Drosophila ameliorates tauopathy and extends lifespan.","method":"Genome-wide cDNA screen (5,500 cDNAs), co-immunoprecipitation, proteasome activity assays, siRNA knockdown, transgenic mouse overexpression, Drosophila genetic knockout, in vivo proteasome activity measurements in tissues","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — genome-wide screen plus Co-IP, KD, KO, and in vivo transgenic validation across multiple organisms with defined phenotypic readouts","pmids":["22921402"],"is_preprint":false},{"year":2018,"finding":"PSMD5 expression is reduced during intestinal tumor progression, and this loss promotes enhanced 26S proteasome assembly in malignant cells. Re-expression of PSMD5 in tumor cells decreased 26S proteasome assembly and caused accumulation of polyubiquitinated proteins, confirming PSMD5 as an inhibitor of 26S proteasome assembly in cancer cells. Enhanced proteasome assembly was observed specifically in transformed cells but not in other rapidly dividing cells, and occurred without changes in levels of individual proteasome subunits.","method":"26S proteasome native gel activity assays, PSMD5 re-expression (rescue experiments), polyubiquitinated protein accumulation assays, comparison of transformed vs. non-transformed dividing cells","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — functional rescue by re-expression combined with direct biochemical readouts of assembly and ubiquitin accumulation; corroborates mechanistic model from PMID:22921402","pmids":["29716915"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM and PhIX-MS (Photo-induced In situ Crosslinking-Mass Spectrometry) resolved PSMD5 bound to the 19S regulatory particle (RP) in the absence of the 20S catalytic core particle (CP). The C-terminus of PSMD5 inserts into the ATPase pore of the RP, physically blocking CP binding. This provides a structural explanation for how PSMD5 acts as an assembly chaperone/inhibitor that prevents premature or aberrant association of the RP with the CP.","method":"PhIX-MS (UV-activated in-cell crosslinking mass spectrometry), cryo-electron microscopy, AlphaFold structural modeling","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — cryo-EM structure with crosslinking MS validation, but preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.07.31.667872"],"is_preprint":true},{"year":2025,"finding":"BAG3 physically interacts with PSMD5 (along with PSMF1) in human iPSC-derived astrocytes, as identified by co-immunoprecipitation. BAG3 loss reduces proteasome function in astrocytes, placing PSMD5 within the BAG3 astrocytic proteostasis network.","method":"Co-immunoprecipitation of BAG3 in human astrocytes followed by mass spectrometry interactome mapping; western blot validation","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP/MS identification; PSMD5 is one of many interactors identified; preprint only; no direct functional follow-up on PSMD5 specifically","pmids":["bio_10.1101_2025.09.20.677505"],"is_preprint":true}],"current_model":"PSMD5 (S5b/Hsm3 in yeast) is an assembly chaperone for the 19S regulatory particle (RP) base subcomplex that transiently binds the ATPase subunits PSMC1 (S4) and PSMC2 (S7) during RP assembly and is excluded from the mature 26S proteasome; when expressed in excess (e.g., induced by TNF-α/NFκB signaling), PSMD5 sequesters PSMC2 in non-proteasome complexes and inserts its C-terminus into the ATPase pore to block 20S core particle docking, thereby acting as a negative regulator of 26S proteasome assembly and activity."},"narrative":{"teleology":[{"year":2000,"claim":"Identifying that PSMD5 associates specifically with the PSMC1/PSMC2 ATPase pair within a defined tetramer established its binding specificity within the 19S base and distinguished it from other RP sub-assemblies.","evidence":"In vitro co-translation, co-immunoprecipitation, sedimentation, and chimeric domain-swap experiments in reticulocyte lysate","pmids":["10625621"],"confidence":"High","gaps":["Whether PSMD5 is a stoichiometric subunit of the mature 26S or a transient factor was unresolved","No in vivo functional consequence of PSMD5 binding was demonstrated","Structural basis of the PSMD5–ATPase interaction was unknown"]},{"year":2009,"claim":"Demonstrating that yeast Hsm3 (PSMD5 ortholog) is absent from the mature 26S proteasome and functions as a transient base-assembly chaperone resolved the long-standing question of whether PSMD5 is a true proteasome subunit or a biogenesis factor.","evidence":"Reciprocal co-immunoprecipitation, sucrose gradient sedimentation, and yeast deletion genetics","pmids":["19217412","19250902"],"confidence":"High","gaps":["Functional consequences of PSMD5 overexpression or depletion in mammalian cells were not yet tested","Signal-regulated control of PSMD5 expression was unknown","Mechanism by which PSMD5 prevents premature RP–CP association was undefined"]},{"year":2012,"claim":"Revealing that TNF-α/NF-κB signaling upregulates PSMD5 to inhibit 26S proteasome assembly—and that overexpression causes aging phenotypes in mice while loss ameliorates tauopathy in Drosophila—established PSMD5 as a physiologically regulated negative regulator of proteasome activity with in vivo consequences.","evidence":"Genome-wide cDNA screen, co-immunoprecipitation, siRNA knockdown, proteasome activity assays, transgenic mouse overexpression, and Drosophila knockout","pmids":["22921402"],"confidence":"High","gaps":["Structural mechanism by which excess PSMD5 blocks 20S CP docking was not resolved","Whether PSMD5 regulation is relevant in human disease contexts beyond aging models was unclear","Quantitative stoichiometry of PSMD5 versus assembled proteasomes in vivo was not determined"]},{"year":2018,"claim":"Finding that PSMD5 expression is lost during intestinal tumor progression and that its re-expression restores assembly inhibition linked the PSMD5 regulatory axis to cancer biology and showed that enhanced proteasome assembly in tumors is not merely a consequence of increased subunit production.","evidence":"Native gel proteasome activity assays, PSMD5 rescue experiments, polyubiquitin accumulation assays in transformed vs. non-transformed cells","pmids":["29716915"],"confidence":"High","gaps":["Causal role of PSMD5 loss in tumor initiation or progression was not genetically tested","Mechanism of PSMD5 downregulation in tumors was not identified","Whether restoring PSMD5 has therapeutic potential was not explored"]},{"year":2025,"claim":"Cryo-EM and crosslinking mass spectrometry showed that the PSMD5 C-terminus inserts into the RP ATPase pore, directly occluding the 20S CP binding interface and providing the first structural explanation for assembly inhibition.","evidence":"Cryo-electron microscopy, PhIX-MS in-cell crosslinking, and AlphaFold modeling (preprint)","pmids":["bio_10.1101_2025.07.31.667872"],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Dynamic steps of PSMD5 insertion and ejection during normal assembly are uncharacterized","Whether specific post-translational modifications regulate C-terminal pore insertion is unknown"]},{"year":null,"claim":"Key unresolved questions include the precise trigger for PSMD5 release during normal RP maturation, the mechanism of PSMD5 downregulation in cancer, and whether pharmacological modulation of PSMD5 levels can tune proteasome activity therapeutically.","evidence":"","pmids":[],"confidence":"High","gaps":["Release mechanism from the maturing RP base is structurally undefined","Transcriptional or post-translational regulation of PSMD5 beyond NF-κB is unexplored","No genetic study has tested whether PSMD5 loss is causally tumorigenic"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,3]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[1,2]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,2]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2]}],"complexes":["19S regulatory particle base assembly intermediate"],"partners":["PSMC1","PSMC2","PSMD1"],"other_free_text":[]},"mechanistic_narrative":"PSMD5 (S5b) is a dedicated assembly chaperone and negative regulator of the 26S proteasome that transiently associates with the 19S regulatory particle (RP) base subcomplex during its biogenesis but is excluded from the mature 26S holoenzyme. It forms a specific tetramer with the ATPase subunits PSMC1 (S4) and PSMC2 (S7) and the non-ATPase subunit PSMD1 (S2), and its C-terminus inserts into the RP ATPase pore to physically block docking of the 20S core particle [PMID:10625621, PMID:19217412]. When PSMD5 expression is elevated—for example by TNF-α/NF-κB signaling—it sequesters PSMC2 in non-proteasome complexes, inhibiting 26S assembly and overall proteasome activity; conversely, PSMD5 loss in intestinal tumors enhances 26S proteasome assembly, and re-expression restores assembly inhibition with concomitant accumulation of polyubiquitinated substrates [PMID:22921402, PMID:29716915]. Transgenic mice overexpressing PSMD5 exhibit reduced proteasome activity and premature aging, while PSMD5 deficiency in Drosophila ameliorates tauopathy and extends lifespan [PMID:22921402]."},"prefetch_data":{"uniprot":{"accession":"Q16401","full_name":"26S proteasome non-ATPase regulatory subunit 5","aliases":["26S protease subunit S5 basic","26S proteasome subunit S5B"],"length_aa":504,"mass_kda":56.2,"function":"Acts as a chaperone during the assembly of the 26S proteasome, specifically of the base subcomplex of the PA700/19S regulatory complex (RC). In the initial step of the base subcomplex assembly is part of an intermediate PSMD5:PSMC2:PSMC1:PSMD2 module which probably assembles with a PSMD10:PSMC4:PSMC5:PAAF1 module followed by dissociation of PSMD5","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q16401/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PSMD5","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PSMD5","total_profiled":1310},"omim":[{"mim_id":"617218","title":"TRANSMEMBRANE AND TETRATRICOPEPTIDE REPEAT DOMAINS-CONTAINING PROTEIN 3; TMTC3","url":"https://www.omim.org/entry/617218"},{"mim_id":"605490","title":"LON PEPTIDASE 1, MITOCHONDRIAL; LONP1","url":"https://www.omim.org/entry/605490"},{"mim_id":"604452","title":"PROTEASOME 26S SUBUNIT, NON-ATPase, 5; PSMD5","url":"https://www.omim.org/entry/604452"},{"mim_id":"601648","title":"PROTEASOME 26S SUBUNIT, NON-ATPase, 4; PSMD4","url":"https://www.omim.org/entry/601648"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Centrosome","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"},{"location":"Connecting piece","reliability":"Additional"},{"location":"Mid piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PSMD5"},"hgnc":{"alias_symbol":["S5B","KIAA0072"],"prev_symbol":[]},"alphafold":{"accession":"Q16401","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16401","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q16401-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q16401-F1-predicted_aligned_error_v6.png","plddt_mean":93.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PSMD5","jax_strain_url":"https://www.jax.org/strain/search?query=PSMD5"},"sequence":{"accession":"Q16401","fasta_url":"https://rest.uniprot.org/uniprotkb/Q16401.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q16401/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16401"}},"corpus_meta":[{"pmid":"19217412","id":"PMC_19217412","title":"Hsm3/S5b participates in the assembly pathway of the 19S regulatory particle of the proteasome.","date":"2009","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/19217412","citation_count":93,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11011914","id":"PMC_11011914","title":"Differential expression of leptin receptor in high- and low-fat-fed Osborne-Mendel and S5B/Pl rats.","date":"2000","source":"Obesity research","url":"https://pubmed.ncbi.nlm.nih.gov/11011914","citation_count":54,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"10625621","id":"PMC_10625621","title":"Mapping subunit contacts in the regulatory complex of the 26 S proteasome. 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ligase.","date":"2007","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/17392787","citation_count":464,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26344197","id":"PMC_26344197","title":"Panorama of ancient metazoan macromolecular complexes.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26344197","citation_count":407,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14614829","id":"PMC_14614829","title":"The Vif protein of HIV triggers degradation of the human antiretroviral DNA deaminase APOBEC3G.","date":"2003","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/14614829","citation_count":405,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11285280","id":"PMC_11285280","title":"Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint.","date":"2001","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11285280","citation_count":372,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9640,"output_tokens":1647,"usd":0.026812},"stage2":{"model":"claude-opus-4-6","input_tokens":4921,"output_tokens":1983,"usd":0.11127},"total_usd":0.306051,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":20944,"output_tokens":2945,"usd":0.053504},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":5587,"output_tokens":1935,"usd":0.114465}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"S5b/PSMD5 forms a tetramer with ATPase subunits S4 and S7 and the non-ATPase subunit S2 within the 19S regulatory complex. S5b binds the C-terminal two-thirds of S4 and the N-terminal region of S7, as demonstrated by chimeric ATPase experiments and co-translation/sedimentation assays.\",\n      \"method\": \"In vitro translation, co-translation, sedimentation, immunoprecipitation, and chimeric ATPase domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with domain-mapping mutagenesis, rigorous controls\",\n      \"pmids\": [\"10625621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Yeast Hsm3, the functional homolog of mammalian S5b/PSMD5, is a dedicated chaperone required for assembly of the 19S base subcomplex. Hsm3 associates with 19S subcomplexes via the C-terminal domain of the Rpt1 (S7) ATPase subunit but is absent from the final 26S proteasome, establishing S5b/PSMD5 as an assembly chaperone rather than a stoichiometric subunit.\",\n      \"method\": \"Genetic deletion, co-immunoprecipitation, yeast two-hybrid, sedimentation, identification of mammalian ortholog by functional complementation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in yeast ortholog, functional complementation with mammalian S5b confirms conservation; replicated in commentary PMID:19250902\",\n      \"pmids\": [\"19217412\", \"19250902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TNF-α upregulates S5b/PSMD5 expression via NFκB, and excess S5b/PSMD5 directly inhibits 26S proteasome assembly and activity. S5b/PSMD5 sequesters S7/PSMC2 in non-proteasome complexes, and interference with this S5b/PSMD5–S7 interaction rescues TNF-α-induced proteasome inhibition. In vivo, transgenic S5b/PSMD5-overexpressing mice show reduced proteasome activity and premature aging, while S5b/PSMD5 deficiency in Drosophila enhances proteasome activity.\",\n      \"method\": \"Genome-wide cDNA screen, RNAi knockdown, co-immunoprecipitation, proteasome activity assay, transgenic mouse model, Drosophila genetics\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods across cell, mouse, and Drosophila models; mechanism placed in TNF-α/NFκB pathway with functional rescue\",\n      \"pmids\": [\"22921402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PSMD5 acts as an inhibitor of 26S proteasome assembly in colorectal cancer cells. Re-expression of PSMD5 in tumor cells decreases 26S proteasome assembly and causes accumulation of polyubiquitinated proteins, while PSMD5 expression is progressively silenced during tumor progression to facilitate enhanced proteasome assembly.\",\n      \"method\": \"Re-expression (rescue experiment), native gel proteasome assembly assay, polyubiquitin immunoblot, tumor progression model in intestinal epithelium\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean gain-of-function with defined molecular phenotype, single lab\",\n      \"pmids\": [\"29716915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM combined with PhIX-MS (photo-induced in situ crosslinking–mass spectrometry) resolved PSMD5 bound to the 19S regulatory particle (RP) in the absence of the 20S catalytic core particle (CP), with its C-terminus inserting into the ATPase pore to block CP binding, providing a structural mechanism for its chaperone/assembly-inhibitor function.\",\n      \"method\": \"Cryo-EM, PhIX-MS (in-cell UV crosslinking coupled to mass spectrometry), AlphaFold structural modeling\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 method (cryo-EM + structural proteomics), but preprint with no independent replication yet\",\n      \"pmids\": [\"bio_10.1101_2025.07.31.667872\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"BAG3 co-immunoprecipitates with PSMD5 in human astrocytes, placing PSMD5 within the BAG3 interactome that coordinates proteasome activity and proteostasis in astrocytes.\",\n      \"method\": \"Co-immunoprecipitation in human iPSC-derived astrocytes, validated by western blot and proximity ligation assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP interaction identified in the context of a broader interactome study; no direct functional dissection of PSMD5 specifically\",\n      \"pmids\": [\"bio_10.1101_2025.09.20.677505\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PSMD5 (S5b) functions as an assembly chaperone of the 19S regulatory particle: it transiently binds the ATPase subunits S4 (PSMC1) and S7 (PSMC2) during base subcomplex assembly, and its C-terminus inserts into the ATPase pore to block association with the 20S catalytic core, such that surplus PSMD5—induced by TNF-α/NFκB signaling—sequesters S7 in non-proteasome complexes to directly inhibit 26S proteasome assembly and activity.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEPT papers.\n\n**Classification:**\n\nPapers about PSMD5 (the proteasome 19S regulatory particle non-ATPase subunit 5, also called S5b):\n- PMID:19217412 — KEEP (Hsm3/S5b, yeast ortholog of PSMD5, proteasome assembly chaperone)\n- PMID:10625621 — KEEP (S5b/PSMD5 subunit contacts in 26S proteasome)\n- PMID:22921402 — KEEP (S5b/PSMD5 role in TNF-α/NFκB proteasome inhibition)\n- PMID:29716915 — KEEP (PSMD5 in colorectal tumor proteasome assembly)\n- PMID:19250902 — KEEP (commentary on Hsm3/S5b as assembly chaperone)\n- bio_10.1101_2025.07.31.667872 — KEEP (PSMD5 structure at proteasome RP by PhIX-MS/cryo-EM)\n- bio_10.1101_2025.09.20.677505 — KEEP (BAG3 interacts with PSMD5 in astrocytes)\n- PMID:8125911 — KEEP (26S proteasome subunit 5 binds ubiquitin — this is S5/PSMD4, not S5b/PSMD5; EXCLUDE)\n\nPapers about S5B/Pl rat strain (obesity model) — ALIAS COLLISION:\n- PMID:11011914, PMID:20801137, PMID:20404826, PMID:9449144, PMID:15677528, PMID:33662430, PMID:10764902, PMID:26641537, PMID:30872180 — all EXCLUDE\n\nPapers about APOBEC3G/CEM15 — ALIAS COLLISION:\n- PMID:12167863, PMID:12808466, PMID:12809610, PMID:14564014, PMID:14528300, PMID:12859895, PMID:14702039, PMID:14527406, PMID:12750511, PMID:14528301, PMID:14614829 — all EXCLUDE\n\nGeneral interactome/proteasome review papers with no specific PSMD5 mechanistic finding:\n- PMID:8811196 — EXCLUDE (general review)\n- PMID:16189514, PMID:28514442, PMID:26496610, PMID:25416956, PMID:32296183, PMID:33961781, PMID:22939629, PMID:26344197 — EXCLUDE (general interactome maps)\n- PMID:12477932, PMID:15489334 — EXCLUDE (cDNA collections)\n- PMID:21873635 — EXCLUDE (GO annotation method)\n- PMID:23503661 — EXCLUDE (PARKIN ubiquitylome)\n- PMID:17392787 — EXCLUDE (AhR ubiquitin ligase)\n- PMID:12665801 — EXCLUDE (proteomics method)\n- PMID:11285280 — EXCLUDE (cyclin A/APC/C)\n- PMID:8125911 — EXCLUDE (subunit 5 = S5/PSMD4, binds ubiquitin chains — different subunit)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"PSMD5 (S5b) forms a tetramer with ATPase subunits S4 (PSMC1) and S7 (PSMC2) and the non-ATPase subunit S2 (PSMD1) within the 19S regulatory complex. Using in vitro co-translation, immunoprecipitation, and sedimentation, S5b was shown to bind the C-terminal two-thirds of S4 and the N-terminal region of S7, while S2 binds the N-terminal region of S4 and the C-terminal two-thirds of S7, forming a defined S2-S4-S7-S5b tetramer. S5b did not associate with the S6-S8 or S6'-S10b ATPase dimers, indicating specificity for the S4/S7 pair.\",\n      \"method\": \"In vitro co-translation in reticulocyte lysate with [35S]methionine, co-immunoprecipitation, sedimentation analysis, chimeric ATPase domain-swap experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (co-translation, co-IP, sedimentation, domain-swap chimeras) in a single rigorous study\",\n      \"pmids\": [\"10625621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Yeast Hsm3 is a dedicated assembly chaperone for the 19S regulatory particle base subcomplex and is the functional homolog of mammalian S5b/PSMD5. Hsm3 associates with 19S base subcomplexes via the C-terminal domain of the Rpt1 (base) ATPase subunit but is absent from the final mature 26S proteasome, demonstrating that it acts transiently during assembly and is not a stoichiometric subunit of the final complex. Hsm3 is specifically required for base subcomplex assembly.\",\n      \"method\": \"Co-immunoprecipitation, sucrose gradient sedimentation, yeast genetics (deletion mutants), structural/sequence homology analysis between Hsm3 and human S5b/PSMD5\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, genetic epistasis, multiple orthogonal methods; independently confirmed by commentary (PMID:19250902)\",\n      \"pmids\": [\"19217412\", \"19250902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PSMD5 (S5b) acts as a negative regulator of 26S proteasome assembly and activity. TNF-α increases PSMD5 expression via NFκB activation, and surplus PSMD5 directly inhibits 26S proteasome assembly and activity. Mechanistically, TNF-α enhances the interaction of PSMD5 with the ATPase subunit S7/PSMC2 in non-proteasome complexes (i.e., free PSMD5-PSMC2 complexes outside the assembled proteasome), and interfering with this interaction rescues TNF-α-induced proteasome inhibition. Downregulation of PSMD5 abolished TNF-α-induced proteasome inhibition. Transgenic mice overexpressing PSMD5 show reduced proteasome activity and premature aging phenotypes, while PSMD5 deficiency in Drosophila ameliorates tauopathy and extends lifespan.\",\n      \"method\": \"Genome-wide cDNA screen (5,500 cDNAs), co-immunoprecipitation, proteasome activity assays, siRNA knockdown, transgenic mouse overexpression, Drosophila genetic knockout, in vivo proteasome activity measurements in tissues\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen plus Co-IP, KD, KO, and in vivo transgenic validation across multiple organisms with defined phenotypic readouts\",\n      \"pmids\": [\"22921402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PSMD5 expression is reduced during intestinal tumor progression, and this loss promotes enhanced 26S proteasome assembly in malignant cells. Re-expression of PSMD5 in tumor cells decreased 26S proteasome assembly and caused accumulation of polyubiquitinated proteins, confirming PSMD5 as an inhibitor of 26S proteasome assembly in cancer cells. Enhanced proteasome assembly was observed specifically in transformed cells but not in other rapidly dividing cells, and occurred without changes in levels of individual proteasome subunits.\",\n      \"method\": \"26S proteasome native gel activity assays, PSMD5 re-expression (rescue experiments), polyubiquitinated protein accumulation assays, comparison of transformed vs. non-transformed dividing cells\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional rescue by re-expression combined with direct biochemical readouts of assembly and ubiquitin accumulation; corroborates mechanistic model from PMID:22921402\",\n      \"pmids\": [\"29716915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM and PhIX-MS (Photo-induced In situ Crosslinking-Mass Spectrometry) resolved PSMD5 bound to the 19S regulatory particle (RP) in the absence of the 20S catalytic core particle (CP). The C-terminus of PSMD5 inserts into the ATPase pore of the RP, physically blocking CP binding. This provides a structural explanation for how PSMD5 acts as an assembly chaperone/inhibitor that prevents premature or aberrant association of the RP with the CP.\",\n      \"method\": \"PhIX-MS (UV-activated in-cell crosslinking mass spectrometry), cryo-electron microscopy, AlphaFold structural modeling\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with crosslinking MS validation, but preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.07.31.667872\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"BAG3 physically interacts with PSMD5 (along with PSMF1) in human iPSC-derived astrocytes, as identified by co-immunoprecipitation. BAG3 loss reduces proteasome function in astrocytes, placing PSMD5 within the BAG3 astrocytic proteostasis network.\",\n      \"method\": \"Co-immunoprecipitation of BAG3 in human astrocytes followed by mass spectrometry interactome mapping; western blot validation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP/MS identification; PSMD5 is one of many interactors identified; preprint only; no direct functional follow-up on PSMD5 specifically\",\n      \"pmids\": [\"bio_10.1101_2025.09.20.677505\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PSMD5 (S5b/Hsm3 in yeast) is an assembly chaperone for the 19S regulatory particle (RP) base subcomplex that transiently binds the ATPase subunits PSMC1 (S4) and PSMC2 (S7) during RP assembly and is excluded from the mature 26S proteasome; when expressed in excess (e.g., induced by TNF-α/NFκB signaling), PSMD5 sequesters PSMC2 in non-proteasome complexes and inserts its C-terminus into the ATPase pore to block 20S core particle docking, thereby acting as a negative regulator of 26S proteasome assembly and activity.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PSMD5 (S5b) is a dedicated assembly chaperone and negative regulator of the 26S proteasome that transiently associates with the 19S regulatory particle during base subcomplex biogenesis. It binds the ATPase subunits PSMC1 (S4) and PSMC2 (S7), forming a tetramer with PSMD2 (S2), and its C-terminus inserts into the ATPase pore to sterically block association of the 19S regulatory particle with the 20S catalytic core [PMID:10625621, PMID:19217412]. TNF-α/NF-κB signaling upregulates PSMD5, and excess PSMD5 sequesters PSMC2 in non-proteasome complexes, directly inhibiting 26S proteasome assembly and activity; accordingly, transgenic overexpression reduces proteasome function and accelerates aging, whereas loss of the ortholog in Drosophila enhances proteasome activity [PMID:22921402]. PSMD5 expression is progressively silenced during colorectal tumor progression, and its re-expression restores accumulation of polyubiquitinated proteins by impeding 26S assembly [PMID:29716915].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"The first question was whether PSMD5/S5b is a stoichiometric 19S subunit or an accessory factor; biochemical reconstitution showed it forms a tetramer with ATPase subunits S4 and S7 and non-ATPase S2, mapping its binding determinants to the C-terminal two-thirds of S4 and the N-terminal region of S7, establishing it as a component of a discrete 19S subassembly.\",\n      \"evidence\": \"In vitro translation, co-sedimentation, immunoprecipitation, and chimeric ATPase domain mapping\",\n      \"pmids\": [\"10625621\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether PSMD5 is a permanent stoichiometric subunit or transiently associated was not resolved\",\n        \"No structural information on the PSMD5–ATPase interface\",\n        \"Functional consequence of PSMD5 binding for proteasome assembly was untested\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The key question of whether PSMD5 is a permanent subunit or a transient chaperone was answered by studies on the yeast ortholog Hsm3: it is required for 19S base assembly but absent from the mature 26S proteasome, establishing PSMD5 as a dedicated, evolutionarily conserved assembly chaperone.\",\n      \"evidence\": \"Genetic deletion, co-immunoprecipitation, yeast two-hybrid, sedimentation in S. cerevisiae; functional complementation with mammalian S5b\",\n      \"pmids\": [\"19217412\", \"19250902\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether mammalian PSMD5 can also actively inhibit (not just assist) proteasome assembly was unknown\",\n        \"The physiological signals regulating PSMD5 expression were unidentified\",\n        \"Structural basis for how PSMD5 prevents 20S–19S joining was unresolved\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"PSMD5 was shown to function not only as a chaperone but as a signal-regulated inhibitor of 26S proteasome assembly: TNF-α/NF-κB upregulates PSMD5, and excess PSMD5 sequesters PSMC2 in non-proteasome complexes, directly reducing 26S activity—with in vivo consequences including premature aging in overexpressing mice and enhanced proteasome activity upon loss in Drosophila.\",\n      \"evidence\": \"Genome-wide cDNA screen, RNAi, co-immunoprecipitation, proteasome activity assays, transgenic mouse and Drosophila genetic models\",\n      \"pmids\": [\"22921402\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural mechanism by which PSMD5 blocks 19S–20S association was still unknown\",\n        \"Whether PSMD5-mediated proteasome inhibition is relevant in disease contexts such as cancer was untested\",\n        \"Quantitative relationship between PSMD5 levels and proteasome pool composition was not determined\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The disease relevance of PSMD5's inhibitory role was demonstrated in colorectal cancer, where PSMD5 silencing during tumor progression permits enhanced 26S assembly, and re-expression restores accumulation of polyubiquitinated substrates by blocking assembly.\",\n      \"evidence\": \"Gain-of-function re-expression, native gel proteasome assembly assay, ubiquitin immunoblot in intestinal tumor models\",\n      \"pmids\": [\"29716915\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Findings from a single lab; independent replication in additional cancer types is lacking\",\n        \"Whether PSMD5 silencing is a driver or passenger event in tumorigenesis was not resolved\",\n        \"Mechanism of transcriptional silencing of PSMD5 in tumors was not identified\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The structural basis for PSMD5's dual chaperone/inhibitor function was resolved: cryo-EM and in situ crosslinking mass spectrometry showed that the PSMD5 C-terminus inserts into the ATPase pore of the 19S base, physically occluding 20S core particle docking.\",\n      \"evidence\": \"Cryo-EM, PhIX-MS (photo-induced in situ crosslinking–mass spectrometry), AlphaFold modeling (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.07.31.667872\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Preprint not yet peer-reviewed or independently replicated\",\n        \"Conformational dynamics of PSMD5 release during the transition to mature 26S proteasome are unresolved\",\n        \"Whether post-translational modifications regulate C-terminal pore insertion is unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Outstanding questions include how PSMD5 is released from the assembling base to allow 20S docking, what post-translational modifications regulate its chaperone-to-inhibitor switch, and whether its tumor-suppressive silencing can be therapeutically reversed.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Mechanism of PSMD5 release during productive 26S maturation is unknown\",\n        \"No post-translational regulatory modifications of PSMD5 have been mapped\",\n        \"Therapeutic relevance of restoring PSMD5 expression in cancer has not been tested\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\n      \"19S regulatory particle (transient assembly intermediate)\"\n    ],\n    \"partners\": [\n      \"PSMC1\",\n      \"PSMC2\",\n      \"PSMD2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"PSMD5 (S5b) is a dedicated assembly chaperone and negative regulator of the 26S proteasome that transiently associates with the 19S regulatory particle (RP) base subcomplex during its biogenesis but is excluded from the mature 26S holoenzyme. It forms a specific tetramer with the ATPase subunits PSMC1 (S4) and PSMC2 (S7) and the non-ATPase subunit PSMD1 (S2), and its C-terminus inserts into the RP ATPase pore to physically block docking of the 20S core particle [PMID:10625621, PMID:19217412]. When PSMD5 expression is elevated—for example by TNF-α/NF-κB signaling—it sequesters PSMC2 in non-proteasome complexes, inhibiting 26S assembly and overall proteasome activity; conversely, PSMD5 loss in intestinal tumors enhances 26S proteasome assembly, and re-expression restores assembly inhibition with concomitant accumulation of polyubiquitinated substrates [PMID:22921402, PMID:29716915]. Transgenic mice overexpressing PSMD5 exhibit reduced proteasome activity and premature aging, while PSMD5 deficiency in Drosophila ameliorates tauopathy and extends lifespan [PMID:22921402].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying that PSMD5 associates specifically with the PSMC1/PSMC2 ATPase pair within a defined tetramer established its binding specificity within the 19S base and distinguished it from other RP sub-assemblies.\",\n      \"evidence\": \"In vitro co-translation, co-immunoprecipitation, sedimentation, and chimeric domain-swap experiments in reticulocyte lysate\",\n      \"pmids\": [\"10625621\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether PSMD5 is a stoichiometric subunit of the mature 26S or a transient factor was unresolved\",\n        \"No in vivo functional consequence of PSMD5 binding was demonstrated\",\n        \"Structural basis of the PSMD5–ATPase interaction was unknown\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrating that yeast Hsm3 (PSMD5 ortholog) is absent from the mature 26S proteasome and functions as a transient base-assembly chaperone resolved the long-standing question of whether PSMD5 is a true proteasome subunit or a biogenesis factor.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, sucrose gradient sedimentation, and yeast deletion genetics\",\n      \"pmids\": [\"19217412\", \"19250902\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Functional consequences of PSMD5 overexpression or depletion in mammalian cells were not yet tested\",\n        \"Signal-regulated control of PSMD5 expression was unknown\",\n        \"Mechanism by which PSMD5 prevents premature RP–CP association was undefined\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Revealing that TNF-α/NF-κB signaling upregulates PSMD5 to inhibit 26S proteasome assembly—and that overexpression causes aging phenotypes in mice while loss ameliorates tauopathy in Drosophila—established PSMD5 as a physiologically regulated negative regulator of proteasome activity with in vivo consequences.\",\n      \"evidence\": \"Genome-wide cDNA screen, co-immunoprecipitation, siRNA knockdown, proteasome activity assays, transgenic mouse overexpression, and Drosophila knockout\",\n      \"pmids\": [\"22921402\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural mechanism by which excess PSMD5 blocks 20S CP docking was not resolved\",\n        \"Whether PSMD5 regulation is relevant in human disease contexts beyond aging models was unclear\",\n        \"Quantitative stoichiometry of PSMD5 versus assembled proteasomes in vivo was not determined\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Finding that PSMD5 expression is lost during intestinal tumor progression and that its re-expression restores assembly inhibition linked the PSMD5 regulatory axis to cancer biology and showed that enhanced proteasome assembly in tumors is not merely a consequence of increased subunit production.\",\n      \"evidence\": \"Native gel proteasome activity assays, PSMD5 rescue experiments, polyubiquitin accumulation assays in transformed vs. non-transformed cells\",\n      \"pmids\": [\"29716915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Causal role of PSMD5 loss in tumor initiation or progression was not genetically tested\",\n        \"Mechanism of PSMD5 downregulation in tumors was not identified\",\n        \"Whether restoring PSMD5 has therapeutic potential was not explored\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM and crosslinking mass spectrometry showed that the PSMD5 C-terminus inserts into the RP ATPase pore, directly occluding the 20S CP binding interface and providing the first structural explanation for assembly inhibition.\",\n      \"evidence\": \"Cryo-electron microscopy, PhIX-MS in-cell crosslinking, and AlphaFold modeling (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.07.31.667872\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Preprint not yet peer-reviewed\",\n        \"Dynamic steps of PSMD5 insertion and ejection during normal assembly are uncharacterized\",\n        \"Whether specific post-translational modifications regulate C-terminal pore insertion is unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the precise trigger for PSMD5 release during normal RP maturation, the mechanism of PSMD5 downregulation in cancer, and whether pharmacological modulation of PSMD5 levels can tune proteasome activity therapeutically.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Release mechanism from the maturing RP base is structurally undefined\",\n        \"Transcriptional or post-translational regulation of PSMD5 beyond NF-κB is unexplored\",\n        \"No genetic study has tested whether PSMD5 loss is causally tumorigenic\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 3]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\n      \"19S regulatory particle base assembly intermediate\"\n    ],\n    \"partners\": [\n      \"PSMC1\",\n      \"PSMC2\",\n      \"PSMD1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}