{"gene":"CCT4","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2013,"finding":"Human CCT4 expressed alone in E. coli forms homo-oligomeric double-ring complexes (two back-to-back rings of eight subunits, ~20S) that hydrolyze ATP at rates similar to the hetero-oligomeric TRiC complex and are active as chaperonins, capable of refolding luciferase and suppressing aggregation/refolding human γD-crystallin.","method":"Recombinant expression in E. coli, sucrose gradient sedimentation, negative stain and cryo-electron microscopy, ATPase assay, luciferase refolding assay, γD-crystallin aggregation suppression assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with multiple orthogonal functional assays (ATPase, luciferase refolding, crystallin aggregation suppression) and structural characterization by cryo-EM in a single rigorous study","pmids":["23612981"],"is_preprint":false},{"year":2008,"finding":"The G345D mutation in CCT4 (anc2-1 allele in S. cerevisiae) abolishes both intra-ring and inter-ring cooperativity in ATP binding by the CCT chaperonin complex, demonstrating that allostery in CCT depends critically on CCT4 and is required for normal actin organization, budding, and cell shape.","method":"Yeast temperature-sensitive mutant analysis, kinetic ATP-binding cooperativity assays (Hill coefficient measurements), actin structure and morphology phenotyping of anc2-1 mutant cells","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical reconstitution of allosteric kinetics combined with in vivo genetic phenotyping in a single focused study; single lab but multiple orthogonal methods","pmids":["18272176"],"is_preprint":false},{"year":2003,"finding":"A C450Y missense mutation in rat Cct4 causes hereditary sensory neuropathy (mutilated foot phenotype) with severe loss of sensory ganglia and fibers, establishing that a functional CCT4 subunit is required for normal sensory neuron maintenance, consistent with its role in folding tubulin, actin, and other cytosolic proteins.","method":"Genetic mapping in Sprague-Dawley rat mutant strain, candidate gene sequencing, identification of 1349G>A (C450Y) mutation in Cct4","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mapping and sequencing established causation in a well-characterized animal model; no in vitro reconstitution but strong in vivo genetic evidence","pmids":["12874111"],"is_preprint":false},{"year":2014,"finding":"The C450Y CCT4 mutation reduces recovery of soluble homo-oligomeric complexes and abolishes formation of ring-shaped structures as seen by electron microscopy, indicating that Cys450 is critical for proper CCT4 oligomeric assembly; wild-type CCT4 homo-oligomers support β-actin refolding in vitro.","method":"Recombinant expression of C450Y CCT4 in E. coli, sucrose gradient centrifugation, negative stain electron microscopy, β-actin refolding assay, huntingtin aggregation suppression assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with structural (EM) and functional (refolding) assays characterizing the mechanistic consequence of the neuropathy-associated mutation; single lab, multiple orthogonal methods","pmids":["25124038"],"is_preprint":false},{"year":2022,"finding":"YB-1 binds the 5'UTR of CCT4 mRNA to promote CCT4 translation; CCT4 in turn acts as a component of the CCT chaperone complex to promote folding of mLST8, an essential component of both mTORC1 and mTORC2, thereby activating mTOR signaling in glioblastoma.","method":"RNA immunoprecipitation, polysome profiling, co-immunoprecipitation, CCT4 knockdown/overexpression with mTOR pathway readouts (p-S6K, p-4EBP1, p-AKT), mLST8 folding assay, mouse xenograft model","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, polysome profiling, functional rescue experiments, and in vivo xenograft validation across multiple orthogonal methods establishing the pathway position of CCT4","pmids":["35239512"],"is_preprint":false},{"year":2022,"finding":"Anticarin-β specifically binds CCT4 and inhibits CCT4-mediated STAT3 maturation/folding, thereby impairing proteostasis and suppressing osteosarcoma cell survival.","method":"Drug-target binding assays, CCT4 knockdown, STAT3 folding/maturation assays, orthotopic and patient-derived xenograft tumor models","journal":"Acta pharmaceutica Sinica. B","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct drug-target binding combined with functional substrate (STAT3) folding assays and in vivo models; single lab","pmids":["35646538"],"is_preprint":false},{"year":2021,"finding":"CCT4 physically interacts with Cdc20 (the APC/C activator) as shown by co-immunoprecipitation; CCT4 knockdown causes accumulation of APC/C substrates securin and Bim, leading to decreased cyclin D1, reduced Bcl-2, and activated caspase-9-mediated apoptosis in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, CCT4 shRNA knockdown, Western blot for securin, Bim, cyclin D1, Bcl-2, cleaved caspase-9, flow cytometry for apoptosis","journal":"Chinese medical journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single co-IP establishing the CCT4–Cdc20 interaction plus functional downstream readouts; single lab","pmids":["34732665"],"is_preprint":false},{"year":2025,"finding":"Monomeric CCT4 (not incorporated into TRiC oligomer) promotes tunneling nanotube (TNT) formation in mammalian cultured cells; GFP-CCT4 monomers induce actin- and tubulin-containing nanotubes, enhance microtubule dynamics, and are themselves transported intercellularly via these nanotubes.","method":"GFP-CCT4 overexpression (monomer-specific constructs), fluorescence microscopy, live-cell imaging, TNT quantification, microtubule dynamics assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — live imaging with functional constructs distinguishing monomer from oligomer; single lab, single study, no independent replication","pmids":["41104595"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM revealed an unexpected bent conformation of CCT4 within the TRiC-HDAC1 complex that may facilitate co-chaperone (prefoldin) dissociation from TRiC during the HDAC1 folding cycle; HDAC1 and HDAC3 were established as TRiC substrates that adopt near-native states within the closed TRiC chamber.","method":"Cryo-electron microscopy (cryo-EM), cross-linking mass spectrometry (XL-MS), biochemical reconstitution of TRiC-HDAC complexes","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-EM structure with XL-MS in a single preprint study; mechanistically informative but not yet peer-reviewed and not independently replicated","pmids":["bio_10.1101_2025.05.21.655272"],"is_preprint":true},{"year":1996,"finding":"The mouse Cct4 gene was chromosomally mapped to proximal chromosome 11 and shown to be biallelically expressed (not imprinted) in neonatal mice.","method":"Genetic mapping, YAC restriction mapping, pulse-field gel electrophoresis, allelic expression analysis","journal":"Genomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — chromosomal mapping and imprinting test; informative for gene regulation but no functional mechanism established","pmids":["8661059"],"is_preprint":false}],"current_model":"CCT4 is the delta subunit of the hetero-oligomeric TRiC/CCT chaperonin complex; it can uniquely form functional homo-oligomeric double rings on its own, hydrolyzes ATP with allosteric cooperativity that depends on Cys450 and Gly345, and folds substrates including actin, tubulin, mLST8, STAT3, and class I HDACs; as a monomer it additionally promotes tunneling nanotube formation and microtubule dynamics; within cells it interacts with Cdc20 to regulate APC/C activity, and its translation is promoted by YB-1 binding to the CCT4 mRNA 5'UTR to sustain mTOR signaling."},"narrative":{"mechanistic_narrative":"CCT4 is the delta subunit of the cytosolic chaperonin TRiC/CCT, a hetero-oligomeric folding machine that processes cytoskeletal and signaling clients including actin, tubulin, mLST8, STAT3, and class I HDACs [PMID:18272176, PMID:35239512, PMID:35646538, PMID:bio_10.1101_2025.05.21.655272]. CCT4 is allosterically central to the complex: the G345D mutation abolishes both intra-ring and inter-ring cooperativity in ATP binding and disrupts actin organization and cell morphology, establishing that CCT-wide allostery depends on this subunit [PMID:18272176]. Uniquely among CCT subunits, human CCT4 expressed alone assembles into homo-oligomeric back-to-back double rings that hydrolyze ATP and function as bona fide chaperonins, refolding luciferase and beta-actin and suppressing aggregation of gamma-D-crystallin and huntingtin; Cys450 is required for this oligomeric ring assembly [PMID:23612981, PMID:25124038]. Through its substrate-folding activity CCT4 supports downstream signaling and survival programs: it folds mLST8 to sustain mTORC1/mTORC2 signaling, with its own translation driven by YB-1 binding the CCT4 mRNA 5'UTR [PMID:35239512]; it folds STAT3 to maintain proteostasis in tumor cells [PMID:35646538]; and it interacts with the APC/C activator Cdc20, so that its loss stabilizes APC/C substrates and triggers caspase-9-mediated apoptosis [PMID:34732665]. Beyond the oligomeric complex, monomeric CCT4 promotes tunneling-nanotube formation and microtubule dynamics and is itself trafficked intercellularly [PMID:41104595]. A C450Y missense mutation in rat Cct4 causes hereditary sensory neuropathy with loss of sensory ganglia, and the same substitution abolishes ring assembly in vitro, linking CCT4 folding competence to neuronal maintenance [PMID:12874111, PMID:25124038].","teleology":[{"year":1996,"claim":"Before functional characterization, the basic genetics of Cct4 were defined to enable downstream study of its regulation.","evidence":"Genetic and YAC restriction mapping with allelic expression analysis in mouse","pmids":["8661059"],"confidence":"Low","gaps":["No functional mechanism established","Chromosomal location does not address protein activity or complex membership"]},{"year":2003,"claim":"It was unknown whether a single CCT subunit defect produces a discrete in vivo phenotype; a rat Cct4 C450Y mutation causing hereditary sensory neuropathy showed that intact CCT4 is required for sensory neuron maintenance.","evidence":"Genetic mapping and candidate gene sequencing in a mutant rat strain","pmids":["12874111"],"confidence":"Medium","gaps":["No in vitro reconstitution of the mutant protein at this stage","Mechanistic link between subunit defect and neuron loss not yet defined"]},{"year":2008,"claim":"The role of CCT4 in chaperonin function was unresolved; a yeast G345D mutation showed that CCT4 is the allosteric linchpin governing cooperative ATP binding across the whole complex.","evidence":"Yeast ts-mutant kinetic ATP cooperativity assays plus actin/morphology phenotyping","pmids":["18272176"],"confidence":"High","gaps":["Structural basis of the cooperativity defect not resolved","Does not address whether CCT4 has activity outside the hetero-oligomer"]},{"year":2013,"claim":"Whether any single CCT subunit can fold substrates autonomously was unknown; human CCT4 alone was shown to form active homo-oligomeric chaperonin double rings.","evidence":"Recombinant E. coli expression, cryo-EM, ATPase and luciferase/crystallin refolding assays","pmids":["23612981"],"confidence":"High","gaps":["Physiological relevance of homo-oligomers in cells not established","Client repertoire of homo-oligomer versus hetero-oligomer not delineated"]},{"year":2014,"claim":"The mechanistic consequence of the neuropathy mutation was unknown; C450Y was shown to abolish ring assembly, connecting Cys450 to oligomerization and disease.","evidence":"Recombinant expression, negative-stain EM, beta-actin refolding and huntingtin aggregation assays","pmids":["25124038"],"confidence":"High","gaps":["Effect of C450Y within native hetero-oligomeric TRiC not directly tested","Cellular consequences in sensory neurons not reconstructed"]},{"year":2021,"claim":"A non-folding interaction of CCT4 was probed; CCT4 was found to bind Cdc20 and its loss stabilizes APC/C substrates to drive apoptosis.","evidence":"Co-IP and shRNA knockdown with Western blot and flow cytometry in hepatocellular carcinoma cells","pmids":["34732665"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal validation of the CCT4-Cdc20 interaction","Whether the effect requires folding activity or is direct on APC/C is unclear"]},{"year":2022,"claim":"How CCT4 feeds into oncogenic signaling and how its own expression is controlled were open; CCT4 was shown to fold mLST8 to sustain mTOR signaling, with translation promoted by YB-1 binding its 5'UTR, and to fold STAT3.","evidence":"RIP, polysome profiling, reciprocal Co-IP, folding assays and xenografts (mLST8/mTOR); drug-target binding and STAT3 folding assays with xenografts","pmids":["35239512","35646538"],"confidence":"Medium","gaps":["Structural detail of how CCT4/TRiC engages mLST8 and STAT3 not resolved","Generality beyond the tumor models tested not established"]},{"year":2025,"claim":"Whether CCT4 has functions outside the oligomeric chamber was untested; monomeric CCT4 was shown to promote tunneling-nanotube formation and microtubule dynamics, and structural work captured a bent CCT4 conformation during HDAC folding.","evidence":"GFP-monomer constructs with live imaging and TNT/microtubule assays; cryo-EM and XL-MS of TRiC-HDAC complexes (preprint)","pmids":["41104595","bio_10.1101_2025.05.21.655272"],"confidence":"Medium","gaps":["Monomer function reported by a single lab without independent replication","HDAC-folding structural model from an unreviewed preprint","Cellular abundance and regulation of free monomeric CCT4 not quantified"]},{"year":null,"claim":"How CCT4's distinct activities — hetero-oligomeric TRiC subunit, autonomous homo-oligomer, and free monomer — are partitioned and regulated within cells remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No quantitative accounting of monomer vs oligomer pools in vivo","Determinants of substrate selection across the different CCT4 forms unknown","Structural mechanism linking allostery, ATP cooperativity, and substrate release incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,3,4,5,8]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,5,8]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,4,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6]}],"complexes":["TRiC/CCT chaperonin","CCT4 homo-oligomeric double ring"],"partners":["CDC20","YB-1","MLST8","STAT3","HDAC1","HDAC3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P50991","full_name":"T-complex protein 1 subunit delta","aliases":["CCT-delta","Chaperonin containing T-complex polypeptide 1 subunit 4","Stimulator of TAR RNA-binding"],"length_aa":539,"mass_kda":57.9,"function":"Component of the chaperonin-containing T-complex (TRiC), a molecular chaperone complex that assists the folding of actin, tubulin and other proteins upon ATP hydrolysis (PubMed:25467444, PubMed:36493755, PubMed:35449234, PubMed:37193829). The TRiC complex mediates the folding of WRAP53/TCAB1, thereby regulating telomere maintenance (PubMed:25467444). As part of the TRiC complex may play a role in the assembly of BBSome, a complex involved in ciliogenesis regulating transports vesicles to the cilia (PubMed:20080638)","subcellular_location":"Cytoplasm; Melanosome; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, cilium basal body","url":"https://www.uniprot.org/uniprotkb/P50991/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/CCT4","classification":"Common Essential","n_dependent_lines":1208,"n_total_lines":1208,"dependency_fraction":1.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000115484","cell_line_id":"CID000209","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":2}],"interactors":[{"gene":"ACTB","stoichiometry":10.0},{"gene":"CCT2","stoichiometry":10.0},{"gene":"CCT3","stoichiometry":10.0},{"gene":"CCT7","stoichiometry":10.0},{"gene":"TCP1","stoichiometry":10.0},{"gene":"CCT8","stoichiometry":10.0},{"gene":"CCT6A","stoichiometry":10.0},{"gene":"CCT5","stoichiometry":10.0},{"gene":"PDCD5","stoichiometry":10.0},{"gene":"PPP2CA","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000209","total_profiled":1310},"omim":[{"mim_id":"610150","title":"CHAPERONIN CONTAINING T-COMPLEX POLYPEPTIDE 1, SUBUNIT 5; CCT5","url":"https://www.omim.org/entry/610150"},{"mim_id":"609804","title":"CILIA- AND FLAGELLA-ASSOCIATED PROTEIN 52; CFAP52","url":"https://www.omim.org/entry/609804"},{"mim_id":"605142","title":"CHAPERONIN CONTAINING T-COMPLEX POLYPEPTIDE 1, SUBUNIT 4; CCT4","url":"https://www.omim.org/entry/605142"},{"mim_id":"605139","title":"CHAPERONIN CONTAINING T-COMPLEX POLYPEPTIDE 1, SUBUNIT 2; CCT2","url":"https://www.omim.org/entry/605139"},{"mim_id":"256840","title":"NEUROPATHY, HEREDITARY SENSORY, WITH SPASTIC PARAPLEGIA, AUTOSOMAL RECESSIVE; HSNSP","url":"https://www.omim.org/entry/256840"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CCT4"},"hgnc":{"alias_symbol":["Cctd"],"prev_symbol":[]},"alphafold":{"accession":"P50991","domains":[{"cath_id":"1.10.560.10","chopping":"27-153_422-530","consensus_level":"high","plddt":93.9978,"start":27,"end":530},{"cath_id":"3.30.260.10","chopping":"156-221_391-419","consensus_level":"medium","plddt":91.4159,"start":156,"end":419},{"cath_id":"3.50.7.10","chopping":"227-258_278-378","consensus_level":"high","plddt":90.0928,"start":227,"end":378}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P50991","model_url":"https://alphafold.ebi.ac.uk/files/AF-P50991-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P50991-F1-predicted_aligned_error_v6.png","plddt_mean":89.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CCT4","jax_strain_url":"https://www.jax.org/strain/search?query=CCT4"},"sequence":{"accession":"P50991","fasta_url":"https://rest.uniprot.org/uniprotkb/P50991.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P50991/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P50991"}},"corpus_meta":[{"pmid":"35239512","id":"PMC_35239512","title":"Upregulated YB-1 protein promotes glioblastoma growth through a YB-1/CCT4/mLST8/mTOR pathway.","date":"2022","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/35239512","citation_count":58,"is_preprint":false},{"pmid":"23612981","id":"PMC_23612981","title":"Human CCT4 and CCT5 chaperonin subunits expressed in Escherichia coli form biologically active homo-oligomers.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23612981","citation_count":55,"is_preprint":false},{"pmid":"12874111","id":"PMC_12874111","title":"Hereditary sensory neuropathy is caused by a mutation in the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct4 ) gene.","date":"2003","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12874111","citation_count":49,"is_preprint":false},{"pmid":"18272176","id":"PMC_18272176","title":"ATP-induced allostery in the eukaryotic chaperonin CCT is abolished by the mutation G345D in CCT4 that renders yeast temperature-sensitive for growth.","date":"2008","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18272176","citation_count":46,"is_preprint":false},{"pmid":"25124038","id":"PMC_25124038","title":"Biochemical characterization of mutants in chaperonin proteins CCT4 and CCT5 associated with hereditary sensory neuropathy.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25124038","citation_count":29,"is_preprint":false},{"pmid":"35646538","id":"PMC_35646538","title":"Anticarin-β shows a promising anti-osteosarcoma effect by specifically inhibiting CCT4 to impair proteostasis.","date":"2022","source":"Acta pharmaceutica Sinica. 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gynecology","url":"https://pubmed.ncbi.nlm.nih.gov/38216268","citation_count":8,"is_preprint":false},{"pmid":"37058032","id":"PMC_37058032","title":"Role of CCT4/ErbB signaling in nephroblastoma: Implications for a biomarker of Wilms tumor.","date":"2023","source":"Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37058032","citation_count":6,"is_preprint":false},{"pmid":"37143143","id":"PMC_37143143","title":"Propofol inhibits myocardial injury induced by microvesicles derived from hypoxia-reoxygenated endothelial cells via lncCCT4-2/CCT4 signaling.","date":"2023","source":"Biological research","url":"https://pubmed.ncbi.nlm.nih.gov/37143143","citation_count":6,"is_preprint":false},{"pmid":"8543170","id":"PMC_8543170","title":"Cloning and sequencing of the chaperonin-encoding Cctd gene from Fugu rubripes rubripes.","date":"1995","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/8543170","citation_count":5,"is_preprint":false},{"pmid":"8661059","id":"PMC_8661059","title":"Chromosomal assignment and imprinting tests for the mouse delta subunit of the cytosolic chaperonin containing TCP-1 (Cct4) gene to proximal chromosome 11.","date":"1996","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8661059","citation_count":2,"is_preprint":false},{"pmid":"41104595","id":"PMC_41104595","title":"CCT4 promotes tunneling nanotube formation.","date":"2025","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/41104595","citation_count":0,"is_preprint":false},{"pmid":"41403933","id":"PMC_41403933","title":"Pan-cancer multi-omics analysis of CCT4 in tumor progression and cancer immunity, with focus on lung adenocarcinoma.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41403933","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.21.655272","title":"TRiC-assisted folding of class I HDAC family proteins regulated by distinct co-chaperone and cofactor networks","date":"2025-05-21","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.21.655272","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11446,"output_tokens":2679,"usd":0.037262,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9881,"output_tokens":3172,"usd":0.064353,"stage2_stop_reason":"end_turn"},"total_usd":0.101615,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"Human CCT4 expressed alone in E. coli forms homo-oligomeric double-ring complexes (two back-to-back rings of eight subunits, ~20S) that hydrolyze ATP at rates similar to the hetero-oligomeric TRiC complex and are active as chaperonins, capable of refolding luciferase and suppressing aggregation/refolding human γD-crystallin.\",\n      \"method\": \"Recombinant expression in E. coli, sucrose gradient sedimentation, negative stain and cryo-electron microscopy, ATPase assay, luciferase refolding assay, γD-crystallin aggregation suppression assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with multiple orthogonal functional assays (ATPase, luciferase refolding, crystallin aggregation suppression) and structural characterization by cryo-EM in a single rigorous study\",\n      \"pmids\": [\"23612981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The G345D mutation in CCT4 (anc2-1 allele in S. cerevisiae) abolishes both intra-ring and inter-ring cooperativity in ATP binding by the CCT chaperonin complex, demonstrating that allostery in CCT depends critically on CCT4 and is required for normal actin organization, budding, and cell shape.\",\n      \"method\": \"Yeast temperature-sensitive mutant analysis, kinetic ATP-binding cooperativity assays (Hill coefficient measurements), actin structure and morphology phenotyping of anc2-1 mutant cells\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical reconstitution of allosteric kinetics combined with in vivo genetic phenotyping in a single focused study; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"18272176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A C450Y missense mutation in rat Cct4 causes hereditary sensory neuropathy (mutilated foot phenotype) with severe loss of sensory ganglia and fibers, establishing that a functional CCT4 subunit is required for normal sensory neuron maintenance, consistent with its role in folding tubulin, actin, and other cytosolic proteins.\",\n      \"method\": \"Genetic mapping in Sprague-Dawley rat mutant strain, candidate gene sequencing, identification of 1349G>A (C450Y) mutation in Cct4\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mapping and sequencing established causation in a well-characterized animal model; no in vitro reconstitution but strong in vivo genetic evidence\",\n      \"pmids\": [\"12874111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The C450Y CCT4 mutation reduces recovery of soluble homo-oligomeric complexes and abolishes formation of ring-shaped structures as seen by electron microscopy, indicating that Cys450 is critical for proper CCT4 oligomeric assembly; wild-type CCT4 homo-oligomers support β-actin refolding in vitro.\",\n      \"method\": \"Recombinant expression of C450Y CCT4 in E. coli, sucrose gradient centrifugation, negative stain electron microscopy, β-actin refolding assay, huntingtin aggregation suppression assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with structural (EM) and functional (refolding) assays characterizing the mechanistic consequence of the neuropathy-associated mutation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25124038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"YB-1 binds the 5'UTR of CCT4 mRNA to promote CCT4 translation; CCT4 in turn acts as a component of the CCT chaperone complex to promote folding of mLST8, an essential component of both mTORC1 and mTORC2, thereby activating mTOR signaling in glioblastoma.\",\n      \"method\": \"RNA immunoprecipitation, polysome profiling, co-immunoprecipitation, CCT4 knockdown/overexpression with mTOR pathway readouts (p-S6K, p-4EBP1, p-AKT), mLST8 folding assay, mouse xenograft model\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, polysome profiling, functional rescue experiments, and in vivo xenograft validation across multiple orthogonal methods establishing the pathway position of CCT4\",\n      \"pmids\": [\"35239512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Anticarin-β specifically binds CCT4 and inhibits CCT4-mediated STAT3 maturation/folding, thereby impairing proteostasis and suppressing osteosarcoma cell survival.\",\n      \"method\": \"Drug-target binding assays, CCT4 knockdown, STAT3 folding/maturation assays, orthotopic and patient-derived xenograft tumor models\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct drug-target binding combined with functional substrate (STAT3) folding assays and in vivo models; single lab\",\n      \"pmids\": [\"35646538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CCT4 physically interacts with Cdc20 (the APC/C activator) as shown by co-immunoprecipitation; CCT4 knockdown causes accumulation of APC/C substrates securin and Bim, leading to decreased cyclin D1, reduced Bcl-2, and activated caspase-9-mediated apoptosis in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, CCT4 shRNA knockdown, Western blot for securin, Bim, cyclin D1, Bcl-2, cleaved caspase-9, flow cytometry for apoptosis\",\n      \"journal\": \"Chinese medical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single co-IP establishing the CCT4–Cdc20 interaction plus functional downstream readouts; single lab\",\n      \"pmids\": [\"34732665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Monomeric CCT4 (not incorporated into TRiC oligomer) promotes tunneling nanotube (TNT) formation in mammalian cultured cells; GFP-CCT4 monomers induce actin- and tubulin-containing nanotubes, enhance microtubule dynamics, and are themselves transported intercellularly via these nanotubes.\",\n      \"method\": \"GFP-CCT4 overexpression (monomer-specific constructs), fluorescence microscopy, live-cell imaging, TNT quantification, microtubule dynamics assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — live imaging with functional constructs distinguishing monomer from oligomer; single lab, single study, no independent replication\",\n      \"pmids\": [\"41104595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM revealed an unexpected bent conformation of CCT4 within the TRiC-HDAC1 complex that may facilitate co-chaperone (prefoldin) dissociation from TRiC during the HDAC1 folding cycle; HDAC1 and HDAC3 were established as TRiC substrates that adopt near-native states within the closed TRiC chamber.\",\n      \"method\": \"Cryo-electron microscopy (cryo-EM), cross-linking mass spectrometry (XL-MS), biochemical reconstitution of TRiC-HDAC complexes\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-EM structure with XL-MS in a single preprint study; mechanistically informative but not yet peer-reviewed and not independently replicated\",\n      \"pmids\": [\"bio_10.1101_2025.05.21.655272\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The mouse Cct4 gene was chromosomally mapped to proximal chromosome 11 and shown to be biallelically expressed (not imprinted) in neonatal mice.\",\n      \"method\": \"Genetic mapping, YAC restriction mapping, pulse-field gel electrophoresis, allelic expression analysis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — chromosomal mapping and imprinting test; informative for gene regulation but no functional mechanism established\",\n      \"pmids\": [\"8661059\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CCT4 is the delta subunit of the hetero-oligomeric TRiC/CCT chaperonin complex; it can uniquely form functional homo-oligomeric double rings on its own, hydrolyzes ATP with allosteric cooperativity that depends on Cys450 and Gly345, and folds substrates including actin, tubulin, mLST8, STAT3, and class I HDACs; as a monomer it additionally promotes tunneling nanotube formation and microtubule dynamics; within cells it interacts with Cdc20 to regulate APC/C activity, and its translation is promoted by YB-1 binding to the CCT4 mRNA 5'UTR to sustain mTOR signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CCT4 is the delta subunit of the cytosolic chaperonin TRiC/CCT, a hetero-oligomeric folding machine that processes cytoskeletal and signaling clients including actin, tubulin, mLST8, STAT3, and class I HDACs [#1, #4, #5, #8]. CCT4 is allosterically central to the complex: the G345D mutation abolishes both intra-ring and inter-ring cooperativity in ATP binding and disrupts actin organization and cell morphology, establishing that CCT-wide allostery depends on this subunit [#1]. Uniquely among CCT subunits, human CCT4 expressed alone assembles into homo-oligomeric back-to-back double rings that hydrolyze ATP and function as bona fide chaperonins, refolding luciferase and beta-actin and suppressing aggregation of gamma-D-crystallin and huntingtin; Cys450 is required for this oligomeric ring assembly [#0, #3]. Through its substrate-folding activity CCT4 supports downstream signaling and survival programs: it folds mLST8 to sustain mTORC1/mTORC2 signaling, with its own translation driven by YB-1 binding the CCT4 mRNA 5'UTR [#4]; it folds STAT3 to maintain proteostasis in tumor cells [#5]; and it interacts with the APC/C activator Cdc20, so that its loss stabilizes APC/C substrates and triggers caspase-9-mediated apoptosis [#6]. Beyond the oligomeric complex, monomeric CCT4 promotes tunneling-nanotube formation and microtubule dynamics and is itself trafficked intercellularly [#7]. A C450Y missense mutation in rat Cct4 causes hereditary sensory neuropathy with loss of sensory ganglia, and the same substitution abolishes ring assembly in vitro, linking CCT4 folding competence to neuronal maintenance [#2, #3].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Before functional characterization, the basic genetics of Cct4 were defined to enable downstream study of its regulation.\",\n      \"evidence\": \"Genetic and YAC restriction mapping with allelic expression analysis in mouse\",\n      \"pmids\": [\"8661059\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No functional mechanism established\", \"Chromosomal location does not address protein activity or complex membership\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"It was unknown whether a single CCT subunit defect produces a discrete in vivo phenotype; a rat Cct4 C450Y mutation causing hereditary sensory neuropathy showed that intact CCT4 is required for sensory neuron maintenance.\",\n      \"evidence\": \"Genetic mapping and candidate gene sequencing in a mutant rat strain\",\n      \"pmids\": [\"12874111\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution of the mutant protein at this stage\", \"Mechanistic link between subunit defect and neuron loss not yet defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The role of CCT4 in chaperonin function was unresolved; a yeast G345D mutation showed that CCT4 is the allosteric linchpin governing cooperative ATP binding across the whole complex.\",\n      \"evidence\": \"Yeast ts-mutant kinetic ATP cooperativity assays plus actin/morphology phenotyping\",\n      \"pmids\": [\"18272176\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the cooperativity defect not resolved\", \"Does not address whether CCT4 has activity outside the hetero-oligomer\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Whether any single CCT subunit can fold substrates autonomously was unknown; human CCT4 alone was shown to form active homo-oligomeric chaperonin double rings.\",\n      \"evidence\": \"Recombinant E. coli expression, cryo-EM, ATPase and luciferase/crystallin refolding assays\",\n      \"pmids\": [\"23612981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of homo-oligomers in cells not established\", \"Client repertoire of homo-oligomer versus hetero-oligomer not delineated\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The mechanistic consequence of the neuropathy mutation was unknown; C450Y was shown to abolish ring assembly, connecting Cys450 to oligomerization and disease.\",\n      \"evidence\": \"Recombinant expression, negative-stain EM, beta-actin refolding and huntingtin aggregation assays\",\n      \"pmids\": [\"25124038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effect of C450Y within native hetero-oligomeric TRiC not directly tested\", \"Cellular consequences in sensory neurons not reconstructed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A non-folding interaction of CCT4 was probed; CCT4 was found to bind Cdc20 and its loss stabilizes APC/C substrates to drive apoptosis.\",\n      \"evidence\": \"Co-IP and shRNA knockdown with Western blot and flow cytometry in hepatocellular carcinoma cells\",\n      \"pmids\": [\"34732665\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation of the CCT4-Cdc20 interaction\", \"Whether the effect requires folding activity or is direct on APC/C is unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"How CCT4 feeds into oncogenic signaling and how its own expression is controlled were open; CCT4 was shown to fold mLST8 to sustain mTOR signaling, with translation promoted by YB-1 binding its 5'UTR, and to fold STAT3.\",\n      \"evidence\": \"RIP, polysome profiling, reciprocal Co-IP, folding assays and xenografts (mLST8/mTOR); drug-target binding and STAT3 folding assays with xenografts\",\n      \"pmids\": [\"35239512\", \"35646538\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural detail of how CCT4/TRiC engages mLST8 and STAT3 not resolved\", \"Generality beyond the tumor models tested not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Whether CCT4 has functions outside the oligomeric chamber was untested; monomeric CCT4 was shown to promote tunneling-nanotube formation and microtubule dynamics, and structural work captured a bent CCT4 conformation during HDAC folding.\",\n      \"evidence\": \"GFP-monomer constructs with live imaging and TNT/microtubule assays; cryo-EM and XL-MS of TRiC-HDAC complexes (preprint)\",\n      \"pmids\": [\"41104595\", \"bio_10.1101_2025.05.21.655272\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Monomer function reported by a single lab without independent replication\", \"HDAC-folding structural model from an unreviewed preprint\", \"Cellular abundance and regulation of free monomeric CCT4 not quantified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CCT4's distinct activities — hetero-oligomeric TRiC subunit, autonomous homo-oligomer, and free monomer — are partitioned and regulated within cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No quantitative accounting of monomer vs oligomer pools in vivo\", \"Determinants of substrate selection across the different CCT4 forms unknown\", \"Structural mechanism linking allostery, ATP cooperativity, and substrate release incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 3, 4, 5, 8]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 5, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 4, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"TRiC/CCT chaperonin\",\n      \"CCT4 homo-oligomeric double ring\"\n    ],\n    \"partners\": [\n      \"Cdc20\",\n      \"YB-1\",\n      \"mLST8\",\n      \"STAT3\",\n      \"HDAC1\",\n      \"HDAC3\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}