{"gene":"PSMG4","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2007,"finding":"PAC3 and PAC4 (mammalian homologs of yeast Poc3/Poc4) form a heterodimeric chaperone pair that acts at an early stage of 20S proteasome α-ring assembly, distinct from and upstream of the PAC1-PAC2 pair and the half-proteasome maturase UMP1.","method":"Genetic screen, co-immunoprecipitation, functional complementation assays in yeast and mammalian cells","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal functional characterization replicated across yeast and mammalian systems with multiple orthogonal methods","pmids":["17707236"],"is_preprint":false},{"year":2008,"finding":"PAC3-PAC4 heterodimer promotes assembly of heptameric α-subunit rings of the 20S proteasome core particle, acting before β-subunit incorporation and before the PAC1-PAC2 chaperone pair.","method":"Biochemical reconstitution, co-immunoprecipitation, structural review of assembly intermediates","journal":"Structure","confidence":"High","confidence_rationale":"Tier 2 — synthesis of multiple biochemical and genetic studies with consistent mechanistic model","pmids":["18786393"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of human PAC4 at 1.90-Å resolution revealed a hydrophobic surface complementary to its binding partner PAC3 and showing charge complementarity with proteasomal α4-α5 subunits, explaining the structural basis of the PAC3-PAC4 interaction with α-subunits.","method":"X-ray crystallography","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure with functional surface identification","pmids":["28263418"],"is_preprint":false},{"year":2018,"finding":"PAC3-PAC4 is required for formation of the core α4-α7 intermediate, the earliest step in α-ring assembly; PAC1-PAC2 subsequently retains α-ring assembly intermediates in the cytoplasm by overriding nuclear localization signals of α-subunits.","method":"Co-immunoprecipitation, subcellular fractionation, siRNA knockdown, fluorescence microscopy","journal":"Genes to cells","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods defining both PAC3-PAC4 and PAC1-PAC2 roles in sequential α-ring assembly","pmids":["30133132"],"is_preprint":false},{"year":2019,"finding":"PAC3-PAC4 heterodimer functions as a molecular matchmaker stabilizing the α4-α5-α6 subcomplex during α-ring assembly; a 0.96-Å crystal structure of PAC3 homodimer combined with NMR data revealed mobility of residues 51-61 loop critical for PAC3-PAC4/α4/α5/α6 quintet complex formation.","method":"Crystal structure (0.96-Å), NMR spectroscopy, 3D structural modeling, biochemical interaction assays","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 — atomic-resolution structure plus NMR dynamics with functional mechanistic interpretation","pmids":["31067643"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM of endogenous chaperone-bound complexes shows that PAC3/PAC4 stabilizes an early α-ring intermediate and dissociates to allow transition to β-ring assembly; mature 20S proteasome formation requires concerted dissociation of POMP and PAC1/PAC2 triggered by repositioning of β-subunit lysine K33.","method":"Cryo-EM of CRISPR/Cas9 endogenously tagged chaperone complexes","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM of endogenous complexes with mechanistic validation, strong method quality","pmids":["39294158"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM reconstructions of seven recombinant human subcomplexes spanning the 20S proteasome assembly pathway show that PAC3-PAC4 participates in early α-ring intermediate stabilization, with structural rearrangements of assembly factors coordinating proteolytic activation with gated active site access.","method":"Cryo-EM of recombinant assembly intermediate subcomplexes","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 — multiple cryo-EM reconstructions across assembly pathway; preprint but orthogonal to peer-reviewed cryo-EM study","pmids":["38328185"],"is_preprint":true},{"year":2026,"finding":"A tRNA-derived fragment (CHAtRF) directly interacts with SRSF5 and blocks SRSF5 binding to Psmg4 pre-mRNA, leading to alternative splicing of Psmg4 with exon 2 skipping; this reduces Psmg4 full-length isoform expression and promotes pathological cardiac hypertrophy.","method":"RNA immunoprecipitation, alternative splicing assays, loss-of-function (CHAtRF deficiency), overexpression in mice and hiPSC-CMs, AngII-induced hypertrophy model","journal":"Research (Washington, D.C.)","confidence":"Medium","confidence_rationale":"Tier 2 — direct RNA-protein interaction and splicing assays with in vivo validation, but single lab study","pmids":["41907183"],"is_preprint":false},{"year":2025,"finding":"Genome-wide CRISPR/Cas9 knockout screen identified PSMG4 among genes whose loss confers resistance to colibactin-induced cytotoxicity, placing PSMG4 in the host-response pathway to colibactin-mediated DNA damage.","method":"Genome-scale CRISPR/Cas9 knockout screen","journal":"mSphere","confidence":"Low","confidence_rationale":"Tier 3 — screen-level identification without mechanistic follow-up specific to PSMG4","pmids":["39918307"],"is_preprint":false},{"year":2025,"finding":"Caffeine treatment of colorectal cancer cells decreased expression of PAC4 (PSMG4) concomitant with reduced immunoproteasome content and reduced oxidative stress, implicating PSMG4 in immunoproteasome biogenesis regulation.","method":"qPCR, Western blot, flow cytometry, transcriptome analysis, fluorogenic substrate activity assays","journal":"Biochimie","confidence":"Low","confidence_rationale":"Tier 3 — correlative expression changes without direct mechanistic manipulation of PSMG4","pmids":["40349826"],"is_preprint":false}],"current_model":"PSMG4 (PAC4) forms a heterodimeric chaperone complex with PAC3 that acts at the earliest stage of 20S proteasome biogenesis, stabilizing an α4-α5-α6 (and broader α4-α7) subcomplex intermediate of the α-ring; structural studies at atomic resolution show PAC4 presents a hydrophobic surface complementary to PAC3 and to the α4-α5 subunits, and cryo-EM of endogenous assembly intermediates reveals that PAC3/PAC4 dissociation is required for transition to β-ring assembly, ultimately yielding mature 20S proteasomes; additionally, Psmg4 pre-mRNA is subject to regulated alternative splicing (exon 2 skipping) mediated by the CHAtRF/SRSF5 axis, with reduced full-length PSMG4 promoting pathological cardiac hypertrophy."},"narrative":{"teleology":[{"year":2007,"claim":"Identification of PAC3–PAC4 as a heterodimeric chaperone pair acting early in α-ring assembly established that 20S proteasome biogenesis uses multiple dedicated, stage-specific assembly factors beyond the previously known PAC1–PAC2 and UMP1.","evidence":"Genetic screen, co-IP, and functional complementation in yeast and mammalian cells","pmids":["17707236"],"confidence":"High","gaps":["Structural basis of the PAC3–PAC4 interaction with α-subunits was unknown","Precise α-subunit intermediate stabilized by PAC3–PAC4 was not defined","Mechanism of PAC3–PAC4 dissociation not addressed"]},{"year":2008,"claim":"Biochemical reconstitution confirmed that PAC3–PAC4 acts before β-subunit incorporation and before PAC1–PAC2, establishing the temporal order of chaperone action during α-ring heptamer assembly.","evidence":"Biochemical reconstitution and co-IP of assembly intermediates","pmids":["18786393"],"confidence":"High","gaps":["No atomic structure of PAC4 or the PAC3–PAC4 complex","Which specific α-subunit subcomplex is chaperoned was not resolved"]},{"year":2017,"claim":"The 1.90-Å crystal structure of PAC4 revealed a hydrophobic binding surface complementary to PAC3 and charge-complementary to α4–α5 subunits, providing the first structural explanation for how PAC3–PAC4 recognizes its substrate within the assembling α-ring.","evidence":"X-ray crystallography of human PAC4","pmids":["28263418"],"confidence":"High","gaps":["No co-crystal of the PAC3–PAC4–α-subunit complex","Dynamic conformational changes during assembly not captured"]},{"year":2018,"claim":"Knockdown studies showed PAC3–PAC4 is specifically required for the α4–α7 intermediate — the earliest step of α-ring formation — while PAC1–PAC2 retains intermediates in the cytoplasm by masking α-subunit NLS sequences, delineating non-overlapping chaperone functions.","evidence":"siRNA knockdown, co-IP, subcellular fractionation, fluorescence microscopy","pmids":["30133132"],"confidence":"High","gaps":["Structural visualization of endogenous intermediates lacking","Mechanism triggering PAC3–PAC4 release still unknown"]},{"year":2019,"claim":"Ultra-high-resolution structure and NMR dynamics of PAC3 identified a flexible loop (residues 51–61) critical for forming the five-component PAC3–PAC4–α4–α5–α6 quintet, defining PAC3–PAC4 as a molecular matchmaker that templates the α4–α5–α6 subcomplex.","evidence":"0.96-Å crystal structure, NMR spectroscopy, 3D modeling, biochemical assays","pmids":["31067643"],"confidence":"High","gaps":["Full reconstitution of the quintet complex at atomic resolution not achieved","Whether PAC3–PAC4 catalytically accelerates or only thermodynamically stabilizes assembly unclear"]},{"year":2024,"claim":"Cryo-EM of endogenous chaperone-bound complexes demonstrated that PAC3–PAC4 dissociation from the α-ring is a prerequisite for β-ring assembly, and that mature 20S formation requires concerted release of POMP and PAC1–PAC2 triggered by β-subunit K33 repositioning, completing the structural map of the assembly pathway.","evidence":"Cryo-EM of CRISPR/Cas9 endogenously tagged chaperone complexes (peer-reviewed); complemented by cryo-EM of recombinant subcomplexes (preprint)","pmids":["39294158","38328185"],"confidence":"High","gaps":["Signal that triggers PAC3–PAC4 release specifically (versus passive displacement) not resolved","Kinetics of chaperone handoff in live cells not measured"]},{"year":2026,"claim":"Discovery that the CHAtRF/SRSF5 axis controls Psmg4 exon 2 alternative splicing revealed a non-canonical regulatory layer: reduced full-length PSMG4 from exon skipping drives pathological cardiac hypertrophy, linking proteasome assembly chaperone expression to heart disease.","evidence":"RNA immunoprecipitation, splicing assays, overexpression/loss-of-function in mice and hiPSC-CMs, AngII-induced hypertrophy model","pmids":["41907183"],"confidence":"Medium","gaps":["Mechanism connecting reduced PSMG4 to hypertrophic signaling (proteasome insufficiency vs. other) not established","Whether exon 2–skipped isoform has any residual function is unknown","Single-lab study; independent replication pending"]},{"year":null,"claim":"The signal that specifically triggers PAC3–PAC4 release from the completed α-ring, the kinetics of chaperone handoff in living cells, and the precise mechanism by which reduced PSMG4 causes cardiac hypertrophy remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No real-time imaging of PAC3–PAC4 dynamics during assembly in vivo","Downstream effectors linking PSMG4 deficiency to hypertrophic gene program unknown","Whether PAC3–PAC4 participates in immunoproteasome-specific assembly is unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,1,4,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,3,5]}],"complexes":["PAC3–PAC4 heterodimer"],"partners":["PSMG3","PSMA7","PSMA5","PSMA8","SRSF5"],"other_free_text":[]},"mechanistic_narrative":"PSMG4 (PAC4) is a dedicated chaperone for 20S proteasome biogenesis that heterodimerizes with PAC3 to nucleate and stabilize the earliest α-ring assembly intermediate. The PAC3–PAC4 complex acts as a molecular matchmaker that promotes formation of an α4–α5–α6 subcomplex (and the broader α4–α7 intermediate), presenting a hydrophobic surface with charge complementarity to α4–α5 subunits; dissociation of PAC3–PAC4 from the completed α-ring is required for subsequent β-ring incorporation and maturation of the 20S core particle [PMID:17707236, PMID:28263418, PMID:31067643, PMID:39294158]. Cryo-EM of endogenous and reconstituted assembly intermediates confirms that PAC3–PAC4 occupies the α-ring at the earliest captured stage and must vacate before POMP-dependent β-subunit processing can proceed [PMID:39294158]. The Psmg4 transcript is additionally subject to regulated alternative splicing (exon 2 skipping) controlled by a CHAtRF/SRSF5 axis, and reduction of the full-length PSMG4 isoform promotes pathological cardiac hypertrophy in mice and human iPSC-derived cardiomyocytes [PMID:41907183]."},"prefetch_data":{"uniprot":{"accession":"Q5JS54","full_name":"Proteasome assembly chaperone 4","aliases":["Proteasome chaperone homolog 4","Pba4"],"length_aa":123,"mass_kda":13.8,"function":"Chaperone protein which promotes assembly of the 20S proteasome","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q5JS54/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PSMG4","classification":"Common Essential","n_dependent_lines":1193,"n_total_lines":1208,"dependency_fraction":0.9875827814569537},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000180822","cell_line_id":"CID000137","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3},{"compartment":"big_aggregates","grade":2}],"interactors":[{"gene":"PSMG1","stoichiometry":10.0},{"gene":"PSMG2","stoichiometry":10.0},{"gene":"POMP","stoichiometry":10.0},{"gene":"PSMG3","stoichiometry":0.2},{"gene":"MELK","stoichiometry":0.2},{"gene":"PSMA4","stoichiometry":0.2},{"gene":"PSMA2","stoichiometry":0.2},{"gene":"PSMA5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000137","total_profiled":1310},"omim":[{"mim_id":"617550","title":"PROTEASOME ASSEMBLY CHAPERONE 4; PSMG4","url":"https://www.omim.org/entry/617550"},{"mim_id":"617528","title":"PROTEASOME ASSEMBLY CHAPERONE 3; PSMG3","url":"https://www.omim.org/entry/617528"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PSMG4"},"hgnc":{"alias_symbol":["PAC4"],"prev_symbol":["C6orf86"]},"alphafold":{"accession":"Q5JS54","domains":[{"cath_id":"3.30.230.100","chopping":"29-121","consensus_level":"high","plddt":91.9139,"start":29,"end":121}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5JS54","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5JS54-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5JS54-F1-predicted_aligned_error_v6.png","plddt_mean":87.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PSMG4","jax_strain_url":"https://www.jax.org/strain/search?query=PSMG4"},"sequence":{"accession":"Q5JS54","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5JS54.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5JS54/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5JS54"}},"corpus_meta":[{"pmid":"17707236","id":"PMC_17707236","title":"20S proteasome assembly is 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Modulates Cardiac Hypertrophy via SRSF5-Dependent Regulation of Psmg4 Alternative Splicing.","date":"2026","source":"Research (Washington, D.C.)","url":"https://pubmed.ncbi.nlm.nih.gov/41907183","citation_count":0,"is_preprint":false},{"pmid":"40338343","id":"PMC_40338343","title":"Molecular and functional analysis of a putative pyocin S9, with endonuclease activity from P. chlororaphis subsp aurantiaca PB-St2.","date":"2025","source":"Archives of microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/40338343","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11878,"output_tokens":2259,"usd":0.034759},"stage2":{"model":"claude-opus-4-6","input_tokens":5689,"output_tokens":2202,"usd":0.125243},"total_usd":0.160002,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"PAC3 and PAC4 (mammalian homologs of yeast Poc3/Poc4) form a heterodimeric chaperone pair that acts at an early stage of 20S proteasome α-ring assembly, distinct from and upstream of the PAC1-PAC2 pair and the half-proteasome maturase UMP1.\",\n      \"method\": \"Genetic screen, co-immunoprecipitation, functional complementation assays in yeast and mammalian cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal functional characterization replicated across yeast and mammalian systems with multiple orthogonal methods\",\n      \"pmids\": [\"17707236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PAC3-PAC4 heterodimer promotes assembly of heptameric α-subunit rings of the 20S proteasome core particle, acting before β-subunit incorporation and before the PAC1-PAC2 chaperone pair.\",\n      \"method\": \"Biochemical reconstitution, co-immunoprecipitation, structural review of assembly intermediates\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — synthesis of multiple biochemical and genetic studies with consistent mechanistic model\",\n      \"pmids\": [\"18786393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of human PAC4 at 1.90-Å resolution revealed a hydrophobic surface complementary to its binding partner PAC3 and showing charge complementarity with proteasomal α4-α5 subunits, explaining the structural basis of the PAC3-PAC4 interaction with α-subunits.\",\n      \"method\": \"X-ray crystallography\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure with functional surface identification\",\n      \"pmids\": [\"28263418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PAC3-PAC4 is required for formation of the core α4-α7 intermediate, the earliest step in α-ring assembly; PAC1-PAC2 subsequently retains α-ring assembly intermediates in the cytoplasm by overriding nuclear localization signals of α-subunits.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, siRNA knockdown, fluorescence microscopy\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods defining both PAC3-PAC4 and PAC1-PAC2 roles in sequential α-ring assembly\",\n      \"pmids\": [\"30133132\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PAC3-PAC4 heterodimer functions as a molecular matchmaker stabilizing the α4-α5-α6 subcomplex during α-ring assembly; a 0.96-Å crystal structure of PAC3 homodimer combined with NMR data revealed mobility of residues 51-61 loop critical for PAC3-PAC4/α4/α5/α6 quintet complex formation.\",\n      \"method\": \"Crystal structure (0.96-Å), NMR spectroscopy, 3D structural modeling, biochemical interaction assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — atomic-resolution structure plus NMR dynamics with functional mechanistic interpretation\",\n      \"pmids\": [\"31067643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM of endogenous chaperone-bound complexes shows that PAC3/PAC4 stabilizes an early α-ring intermediate and dissociates to allow transition to β-ring assembly; mature 20S proteasome formation requires concerted dissociation of POMP and PAC1/PAC2 triggered by repositioning of β-subunit lysine K33.\",\n      \"method\": \"Cryo-EM of CRISPR/Cas9 endogenously tagged chaperone complexes\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM of endogenous complexes with mechanistic validation, strong method quality\",\n      \"pmids\": [\"39294158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM reconstructions of seven recombinant human subcomplexes spanning the 20S proteasome assembly pathway show that PAC3-PAC4 participates in early α-ring intermediate stabilization, with structural rearrangements of assembly factors coordinating proteolytic activation with gated active site access.\",\n      \"method\": \"Cryo-EM of recombinant assembly intermediate subcomplexes\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple cryo-EM reconstructions across assembly pathway; preprint but orthogonal to peer-reviewed cryo-EM study\",\n      \"pmids\": [\"38328185\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"A tRNA-derived fragment (CHAtRF) directly interacts with SRSF5 and blocks SRSF5 binding to Psmg4 pre-mRNA, leading to alternative splicing of Psmg4 with exon 2 skipping; this reduces Psmg4 full-length isoform expression and promotes pathological cardiac hypertrophy.\",\n      \"method\": \"RNA immunoprecipitation, alternative splicing assays, loss-of-function (CHAtRF deficiency), overexpression in mice and hiPSC-CMs, AngII-induced hypertrophy model\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct RNA-protein interaction and splicing assays with in vivo validation, but single lab study\",\n      \"pmids\": [\"41907183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Genome-wide CRISPR/Cas9 knockout screen identified PSMG4 among genes whose loss confers resistance to colibactin-induced cytotoxicity, placing PSMG4 in the host-response pathway to colibactin-mediated DNA damage.\",\n      \"method\": \"Genome-scale CRISPR/Cas9 knockout screen\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — screen-level identification without mechanistic follow-up specific to PSMG4\",\n      \"pmids\": [\"39918307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Caffeine treatment of colorectal cancer cells decreased expression of PAC4 (PSMG4) concomitant with reduced immunoproteasome content and reduced oxidative stress, implicating PSMG4 in immunoproteasome biogenesis regulation.\",\n      \"method\": \"qPCR, Western blot, flow cytometry, transcriptome analysis, fluorogenic substrate activity assays\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — correlative expression changes without direct mechanistic manipulation of PSMG4\",\n      \"pmids\": [\"40349826\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PSMG4 (PAC4) forms a heterodimeric chaperone complex with PAC3 that acts at the earliest stage of 20S proteasome biogenesis, stabilizing an α4-α5-α6 (and broader α4-α7) subcomplex intermediate of the α-ring; structural studies at atomic resolution show PAC4 presents a hydrophobic surface complementary to PAC3 and to the α4-α5 subunits, and cryo-EM of endogenous assembly intermediates reveals that PAC3/PAC4 dissociation is required for transition to β-ring assembly, ultimately yielding mature 20S proteasomes; additionally, Psmg4 pre-mRNA is subject to regulated alternative splicing (exon 2 skipping) mediated by the CHAtRF/SRSF5 axis, with reduced full-length PSMG4 promoting pathological cardiac hypertrophy.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PSMG4 (PAC4) is a dedicated chaperone for 20S proteasome biogenesis that heterodimerizes with PAC3 to nucleate and stabilize the earliest α-ring assembly intermediate. The PAC3–PAC4 complex acts as a molecular matchmaker that promotes formation of an α4–α5–α6 subcomplex (and the broader α4–α7 intermediate), presenting a hydrophobic surface with charge complementarity to α4–α5 subunits; dissociation of PAC3–PAC4 from the completed α-ring is required for subsequent β-ring incorporation and maturation of the 20S core particle [PMID:17707236, PMID:28263418, PMID:31067643, PMID:39294158]. Cryo-EM of endogenous and reconstituted assembly intermediates confirms that PAC3–PAC4 occupies the α-ring at the earliest captured stage and must vacate before POMP-dependent β-subunit processing can proceed [PMID:39294158]. The Psmg4 transcript is additionally subject to regulated alternative splicing (exon 2 skipping) controlled by a CHAtRF/SRSF5 axis, and reduction of the full-length PSMG4 isoform promotes pathological cardiac hypertrophy in mice and human iPSC-derived cardiomyocytes [PMID:41907183].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of PAC3–PAC4 as a heterodimeric chaperone pair acting early in α-ring assembly established that 20S proteasome biogenesis uses multiple dedicated, stage-specific assembly factors beyond the previously known PAC1–PAC2 and UMP1.\",\n      \"evidence\": \"Genetic screen, co-IP, and functional complementation in yeast and mammalian cells\",\n      \"pmids\": [\"17707236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of the PAC3–PAC4 interaction with α-subunits was unknown\",\n        \"Precise α-subunit intermediate stabilized by PAC3–PAC4 was not defined\",\n        \"Mechanism of PAC3–PAC4 dissociation not addressed\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Biochemical reconstitution confirmed that PAC3–PAC4 acts before β-subunit incorporation and before PAC1–PAC2, establishing the temporal order of chaperone action during α-ring heptamer assembly.\",\n      \"evidence\": \"Biochemical reconstitution and co-IP of assembly intermediates\",\n      \"pmids\": [\"18786393\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No atomic structure of PAC4 or the PAC3–PAC4 complex\",\n        \"Which specific α-subunit subcomplex is chaperoned was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The 1.90-Å crystal structure of PAC4 revealed a hydrophobic binding surface complementary to PAC3 and charge-complementary to α4–α5 subunits, providing the first structural explanation for how PAC3–PAC4 recognizes its substrate within the assembling α-ring.\",\n      \"evidence\": \"X-ray crystallography of human PAC4\",\n      \"pmids\": [\"28263418\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No co-crystal of the PAC3–PAC4–α-subunit complex\",\n        \"Dynamic conformational changes during assembly not captured\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Knockdown studies showed PAC3–PAC4 is specifically required for the α4–α7 intermediate — the earliest step of α-ring formation — while PAC1–PAC2 retains intermediates in the cytoplasm by masking α-subunit NLS sequences, delineating non-overlapping chaperone functions.\",\n      \"evidence\": \"siRNA knockdown, co-IP, subcellular fractionation, fluorescence microscopy\",\n      \"pmids\": [\"30133132\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural visualization of endogenous intermediates lacking\",\n        \"Mechanism triggering PAC3–PAC4 release still unknown\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Ultra-high-resolution structure and NMR dynamics of PAC3 identified a flexible loop (residues 51–61) critical for forming the five-component PAC3–PAC4–α4–α5–α6 quintet, defining PAC3–PAC4 as a molecular matchmaker that templates the α4–α5–α6 subcomplex.\",\n      \"evidence\": \"0.96-Å crystal structure, NMR spectroscopy, 3D modeling, biochemical assays\",\n      \"pmids\": [\"31067643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full reconstitution of the quintet complex at atomic resolution not achieved\",\n        \"Whether PAC3–PAC4 catalytically accelerates or only thermodynamically stabilizes assembly unclear\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cryo-EM of endogenous chaperone-bound complexes demonstrated that PAC3–PAC4 dissociation from the α-ring is a prerequisite for β-ring assembly, and that mature 20S formation requires concerted release of POMP and PAC1–PAC2 triggered by β-subunit K33 repositioning, completing the structural map of the assembly pathway.\",\n      \"evidence\": \"Cryo-EM of CRISPR/Cas9 endogenously tagged chaperone complexes (peer-reviewed); complemented by cryo-EM of recombinant subcomplexes (preprint)\",\n      \"pmids\": [\"39294158\", \"38328185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Signal that triggers PAC3–PAC4 release specifically (versus passive displacement) not resolved\",\n        \"Kinetics of chaperone handoff in live cells not measured\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Discovery that the CHAtRF/SRSF5 axis controls Psmg4 exon 2 alternative splicing revealed a non-canonical regulatory layer: reduced full-length PSMG4 from exon skipping drives pathological cardiac hypertrophy, linking proteasome assembly chaperone expression to heart disease.\",\n      \"evidence\": \"RNA immunoprecipitation, splicing assays, overexpression/loss-of-function in mice and hiPSC-CMs, AngII-induced hypertrophy model\",\n      \"pmids\": [\"41907183\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism connecting reduced PSMG4 to hypertrophic signaling (proteasome insufficiency vs. other) not established\",\n        \"Whether exon 2–skipped isoform has any residual function is unknown\",\n        \"Single-lab study; independent replication pending\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The signal that specifically triggers PAC3–PAC4 release from the completed α-ring, the kinetics of chaperone handoff in living cells, and the precise mechanism by which reduced PSMG4 causes cardiac hypertrophy remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No real-time imaging of PAC3–PAC4 dynamics during assembly in vivo\",\n        \"Downstream effectors linking PSMG4 deficiency to hypertrophic gene program unknown\",\n        \"Whether PAC3–PAC4 participates in immunoproteasome-specific assembly is unresolved\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 1, 4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 3, 5]}\n    ],\n    \"complexes\": [\n      \"PAC3–PAC4 heterodimer\"\n    ],\n    \"partners\": [\n      \"PSMG3\",\n      \"PSMA7\",\n      \"PSMA5\",\n      \"PSMA8\",\n      \"SRSF5\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}