{"gene":"AHSA1","run_date":"2026-06-09T22:02:42","timeline":{"discoveries":[{"year":2024,"finding":"Metazoan AHSA1 possesses an intrinsic chaperone domain (ICD), a ~20 amino acid peptide preceding the conserved NxNNWHW motif, that diminishes Hsp90 ATPase stimulation by interfering with the NxNNWHW motif function. The NxNNWHW motif stimulates Hsp90 ATPase activity and modulates Hsp90's apparent affinity for Ahsa1 and ATP. The ICD controls regulated recruitment of Hsp90 in cells, and its deletion results in loss of interaction with Hsp90 and the glucocorticoid receptor.","method":"In vitro ATPase assays, mutagenesis of ICD and NxNNWHW motif, co-immunoprecipitation in cells","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro ATPase assay combined with domain mutagenesis and cellular co-IP, multiple orthogonal methods in one study","pmids":["38937628"],"is_preprint":false},{"year":2022,"finding":"AHSA1 acts as a co-chaperone of HSP90A to activate CDK6 and PSMD2 in multiple myeloma cells. AHSA1-K137 was identified as the specific binding site for the inhibitor Bufalin; mutation of K137 decreased the interaction of AHSA1 with HSP90A and suppressed AHSA1-mediated activation of CDK6 and PSMD2.","method":"Co-immunoprecipitation, mass spectrometry, site-directed mutagenesis, microscale thermophoresis assay, xenograft model","journal":"Journal of experimental & clinical cancer research : CR","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP, mass spectrometry, site mutagenesis, and in vivo model; multiple orthogonal methods establishing binding site and functional consequence","pmids":["34991674"],"is_preprint":false},{"year":2015,"finding":"Silencing AHSA1 in osteosarcoma cells decreased HSP90 ATPase activity, establishing that AHSA1 is required for HSP90 ATPase stimulation in cancer cells. AHSA1 knockdown also increased levels of Wnt/β-catenin negative regulators Axin-2 and GSK3β while decreasing Wnt-5a and β-catenin, placing AHSA1 upstream of Wnt/β-catenin signaling.","method":"siRNA knockdown, ATPase activity assay, western blot for pathway components","journal":"Biomedicine & pharmacotherapy","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — ATPase assay plus western blot pathway analysis; single lab, two orthogonal methods","pmids":["26796264"],"is_preprint":false},{"year":2022,"finding":"AHSA1 recruits ERK1/2 and promotes phosphorylation and inactivation of CALD1 (caldesmon) in hepatocellular carcinoma cells, independent of HSP90 and MEK1/2, thereby promoting proliferation and EMT.","method":"Co-immunoprecipitation, ERK1/2 phosphorylation inhibitor (SCH772984) rescue, CALD1 knockdown epistasis, gain- and loss-of-function studies in vitro and in vivo","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP showing complex, inhibitor rescue, and epistasis knockdown; single lab, multiple orthogonal methods","pmids":["36230524"],"is_preprint":false},{"year":2025,"finding":"The AHSA1/Hsp90α complex forms in microglia and targets the mitochondrial import protein TOMM70, facilitating mitophagy in a Parkinson disease mouse model. Knockdown of AHSA1 or inhibition of Hsp90α with geldanamycin suppressed microglial mitophagy and attenuated dopaminergic neuronal death.","method":"Co-immunoprecipitation, immunofluorescence, siRNA knockdown, geldanamycin pharmacological inhibition, MPTP mouse model, microglia/dopaminergic neuron co-culture","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP confirming complex, knockdown and pharmacological inhibition with defined mitophagy readout; single lab, two orthogonal approaches","pmids":["40685075"],"is_preprint":false},{"year":2026,"finding":"CHK1 directly interacts with AHSA1 and suppresses TRIM8-mediated ubiquitination and degradation of AHSA1, thereby stabilizing the AHSA1-HSP90 complex and enhancing HSP90 ATPase activity to activate mitophagy in cardiomyocytes.","method":"Immunoprecipitation and mass spectrometry (IP-MS), cardiomyocyte-specific CHK1 overexpression and knockout mouse models, western blot","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS identifying direct interaction, in vivo genetic models with functional readout; single lab but multiple orthogonal methods","pmids":["42229233"],"is_preprint":false},{"year":2025,"finding":"The AHSA1-HSP90AA1 complex stabilizes IFI6 and TGFB1 proteins in EGFR-mutated lung adenocarcinoma cells, with IFI6 stabilization enhancing mitochondrial function and Akt phosphorylation to promote Osimertinib resistance.","method":"Co-immunoprecipitation, western blot, overexpression/knockdown functional assays, pharmacological inhibition","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP demonstrating complex and client stabilization, knockdown epistasis; single lab, two orthogonal methods","pmids":["40234395"],"is_preprint":false},{"year":2026,"finding":"AHSA1 binds DNAJB4 (Co-IP), and AHSA1 overexpression enhances DNAJB4 protein levels, suppresses ERAD pathway protein expression (XBP-1s, ATF4, CHOP, GADD34), reduces apoptosis, and promotes endometrial cancer cell colony formation; these effects were abolished by DNAJB4 deletion, establishing DNAJB4 as a downstream effector of AHSA1 in ERAD regulation.","method":"Co-immunoprecipitation, DNAJB4 knockdown epistasis, overexpression functional assays, western blot for ERAD markers","journal":"Journal of reproductive immunology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus epistasis knockdown with defined ERAD readout; single lab, two orthogonal methods","pmids":["41990440"],"is_preprint":false},{"year":2013,"finding":"In a zebrafish model of HDR syndrome, Ahsa1 and Hsp90 activity genetically promoted more severe craniofacial phenotypes caused by Gata3 mutation, placing Ahsa1/Hsp90 as modifiers of transcription-factor-driven developmental defect severity.","method":"Zebrafish forward genetic screen, genetic epistasis in gata3 mutant background, live imaging","journal":"Disease models & mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in a vertebrate model with defined developmental phenotype; single lab","pmids":["23720234"],"is_preprint":false}],"current_model":"AHSA1 (hAha1) is a co-chaperone that binds to HSP90 via its conserved NxNNWHW motif to stimulate HSP90 ATPase activity; in metazoans, an intrinsic chaperone domain (ICD) autoinhibits this stimulation and controls regulated recruitment to HSP90 and clients such as the glucocorticoid receptor. AHSA1 acts as the critical activating subunit of the AHSA1–HSP90 complex, which stabilizes and activates client proteins including CDK6, PSMD2, IFI6, TGFB1, and TOMM70, thereby driving cell proliferation, drug resistance, mitophagy, and EMT across multiple cellular contexts. AHSA1 protein stability is itself regulated by CHK1-dependent suppression of TRIM8-mediated ubiquitination. AHSA1 additionally engages non-HSP90 pathways by recruiting ERK1/2 to phosphorylate and inactivate CALD1, and by binding DNAJB4 to suppress ER-associated degradation signaling."},"narrative":{"mechanistic_narrative":"AHSA1 is a co-chaperone that activates the HSP90 ATPase cycle and thereby controls the maturation of HSP90 client proteins across diverse cellular contexts [PMID:38937628, PMID:26796264]. It engages HSP90 through its conserved NxNNWHW motif, which stimulates HSP90 ATPase activity and tunes HSP90's apparent affinity for ATP; in metazoans an intrinsic chaperone domain (ICD) immediately preceding this motif autoinhibits stimulation and governs regulated recruitment of HSP90 to clients such as the glucocorticoid receptor, with ICD deletion abolishing both HSP90 and receptor binding [PMID:38937628]. AHSA1 is required for HSP90 ATPase stimulation in cancer cells, where loss of AHSA1 lowers HSP90 activity [PMID:26796264]. As the activating subunit of AHSA1–HSP90 complexes, it stabilizes and matures client proteins including CDK6 and PSMD2, IFI6 and TGFB1, and the mitochondrial import receptor TOMM70, driving proliferation, drug resistance, and mitophagy [PMID:34991674, PMID:40234395, PMID:40685075]. AHSA1 protein levels are themselves controlled by CHK1, which interacts with AHSA1 and suppresses TRIM8-mediated ubiquitination to stabilize the AHSA1–HSP90 complex [PMID:42229233]. Beyond HSP90, AHSA1 recruits ERK1/2 to phosphorylate and inactivate caldesmon (CALD1) independently of HSP90 and MEK1/2 [PMID:36230524], and binds DNAJB4 to suppress ER-associated degradation signaling [PMID:41990440].","teleology":[{"year":2013,"claim":"Established that Ahsa1, acting through Hsp90, can modify the severity of a transcription-factor-driven developmental defect, linking the co-chaperone to client-dependent phenotypic outcomes in a whole vertebrate.","evidence":"Zebrafish genetic epistasis in a gata3 mutant HDR-syndrome model with live imaging","pmids":["23720234"],"confidence":"Medium","gaps":["Does not identify the molecular client mediating the modifier effect","Genetic, not biochemical, evidence for the Ahsa1–Hsp90 interaction in this context"]},{"year":2015,"claim":"Showed that AHSA1 is required for HSP90 ATPase activity in cancer cells and positioned it upstream of Wnt/β-catenin signaling, connecting the co-chaperone to a defined oncogenic pathway.","evidence":"siRNA knockdown with HSP90 ATPase assay and western blot of Wnt pathway components in osteosarcoma cells","pmids":["26796264"],"confidence":"Medium","gaps":["Wnt regulation is correlative — no direct AHSA1/HSP90 client in the pathway identified","Single cell-line context"]},{"year":2022,"claim":"Defined AHSA1 as the activating subunit that channels HSP90 toward specific clients (CDK6, PSMD2) and mapped a druggable binding site (K137) required for HSP90 engagement, providing a structural and therapeutic handle.","evidence":"Reciprocal Co-IP, mass spectrometry, K137 mutagenesis, microscale thermophoresis, and xenograft in multiple myeloma","pmids":["34991674"],"confidence":"High","gaps":["Whether CDK6/PSMD2 stabilization is direct or secondary to global HSP90 activation","K137 role in client selectivity versus general HSP90 binding not separated"]},{"year":2022,"claim":"Revealed an HSP90-independent function for AHSA1 as a scaffold recruiting ERK1/2 to phosphorylate and inactivate CALD1, expanding its role beyond classical co-chaperone activity.","evidence":"Co-IP, ERK1/2 inhibitor (SCH772984) rescue, and CALD1 knockdown epistasis in hepatocellular carcinoma in vitro and in vivo","pmids":["36230524"],"confidence":"Medium","gaps":["Mechanism of AHSA1–ERK1/2 recruitment unresolved","MEK1/2 independence of ERK activation not mechanistically explained"]},{"year":2025,"claim":"Extended the AHSA1/HSP90α complex to neuroprotective mitophagy by showing it targets the mitochondrial import receptor TOMM70 in microglia, linking the co-chaperone to mitochondrial quality control.","evidence":"Co-IP, immunofluorescence, siRNA knockdown, geldanamycin inhibition, and MPTP Parkinson mouse model with co-culture","pmids":["40685075"],"confidence":"Medium","gaps":["Whether TOMM70 is a direct HSP90 client or an indirect effector","Mechanistic basis of mitophagy initiation downstream of TOMM70"]},{"year":2025,"claim":"Demonstrated that AHSA1–HSP90AA1 stabilizes IFI6 and TGFB1 to drive Osimertinib resistance, mechanistically connecting co-chaperone activity to acquired drug resistance via client stabilization and Akt signaling.","evidence":"Co-IP, western blot, overexpression/knockdown, and pharmacological inhibition in EGFR-mutated lung adenocarcinoma","pmids":["40234395"],"confidence":"Medium","gaps":["Direct versus indirect stabilization of IFI6/TGFB1 not distinguished","Single tumor-type context"]},{"year":2024,"claim":"Resolved how metazoan AHSA1 activity is intrinsically autoregulated by identifying the ICD that suppresses NxNNWHW-driven HSP90 ATPase stimulation and controls regulated recruitment to HSP90 and the glucocorticoid receptor.","evidence":"In vitro ATPase assays, ICD/NxNNWHW mutagenesis, and cellular Co-IP","pmids":["38937628"],"confidence":"High","gaps":["Signal or modification that relieves ICD autoinhibition in cells unknown","Structural basis of ICD–NxNNWHW interference not defined"]},{"year":2026,"claim":"Identified upstream control of AHSA1 protein stability, showing CHK1 suppresses TRIM8-mediated ubiquitination to stabilize the AHSA1–HSP90 complex and sustain ATPase-driven mitophagy in cardiomyocytes.","evidence":"IP-MS, cardiomyocyte-specific CHK1 overexpression and knockout mouse models, western blot","pmids":["42229233"],"confidence":"Medium","gaps":["TRIM8 ubiquitination site on AHSA1 not mapped","How CHK1 mechanistically blocks TRIM8 unresolved"]},{"year":2026,"claim":"Established a further non-canonical axis in which AHSA1 binds DNAJB4 to suppress ERAD signaling and apoptosis, broadening its effector repertoire beyond HSP90 clients.","evidence":"Co-IP, DNAJB4 knockdown epistasis, overexpression assays, and western blot of ERAD markers in endometrial cancer","pmids":["41990440"],"confidence":"Medium","gaps":["Whether DNAJB4 binding is HSP90-dependent not tested","Direct versus indirect ERAD marker suppression unresolved"]},{"year":null,"claim":"The physiological signals that toggle AHSA1 between ICD-autoinhibited and active states, and the rules determining which HSP90 clients AHSA1 selectively matures across tissues, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the ICD-active transition in a cellular context","Client-selectivity determinants undefined","Integration of HSP90-dependent and HSP90-independent (ERK1/2, DNAJB4) functions unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,7]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,6]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[4,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3]}],"complexes":["AHSA1–HSP90 co-chaperone complex"],"partners":["HSP90AA1","HSP90","CHK1","TRIM8","DNAJB4","ERK1/2","TOMM70"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95433","full_name":"Activator of 90 kDa heat shock protein ATPase homolog 1","aliases":["p38"],"length_aa":338,"mass_kda":38.3,"function":"Acts as a co-chaperone of HSP90AA1 (PubMed:29127155). Activates the ATPase activity of HSP90AA1 leading to increase in its chaperone activity (PubMed:29127155). Competes with the inhibitory co-chaperone FNIP1 for binding to HSP90AA1, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins (PubMed:27353360). Competes with the inhibitory co-chaperone TSC1 for binding to HSP90AA1, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins (PubMed:29127155)","subcellular_location":"Cytoplasm, cytosol; Endoplasmic reticulum","url":"https://www.uniprot.org/uniprotkb/O95433/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AHSA1","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000100591","cell_line_id":"CID000003","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"FKBP5","stoichiometry":4.0},{"gene":"PLA2G4A","stoichiometry":4.0},{"gene":"RNF219","stoichiometry":0.2},{"gene":"HSP90AB2P","stoichiometry":0.2},{"gene":"RICTOR","stoichiometry":0.2},{"gene":"KPNB1","stoichiometry":0.2},{"gene":"APLP2","stoichiometry":0.2},{"gene":"GDAP2","stoichiometry":0.2},{"gene":"ECT2","stoichiometry":0.2},{"gene":"AP2M1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000003","total_profiled":1310},"omim":[{"mim_id":"611219","title":"UNC45 MYOSIN CHAPERONE A; UNC45A","url":"https://www.omim.org/entry/611219"},{"mim_id":"608466","title":"ACTIVATOR OF HEAT-SHOCK 90-KD PROTEIN ATPase 1; AHSA1","url":"https://www.omim.org/entry/608466"},{"mim_id":"602421","title":"CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR; CFTR","url":"https://www.omim.org/entry/602421"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AHSA1"},"hgnc":{"alias_symbol":["p38","Aha1","hAha1"],"prev_symbol":["C14orf3"]},"alphafold":{"accession":"O95433","domains":[{"cath_id":"3.15.10.20","chopping":"27-155","consensus_level":"high","plddt":92.0371,"start":27,"end":155},{"cath_id":"3.30.530.20","chopping":"206-330","consensus_level":"high","plddt":91.861,"start":206,"end":330}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95433","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95433-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95433-F1-predicted_aligned_error_v6.png","plddt_mean":80.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AHSA1","jax_strain_url":"https://www.jax.org/strain/search?query=AHSA1"},"sequence":{"accession":"O95433","fasta_url":"https://rest.uniprot.org/uniprotkb/O95433.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95433/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95433"}},"corpus_meta":[{"pmid":"26796264","id":"PMC_26796264","title":"AHSA1 regulates proliferation, apoptosis, migration, and invasion of osteosarcoma.","date":"2015","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/26796264","citation_count":30,"is_preprint":false},{"pmid":"34991674","id":"PMC_34991674","title":"AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma.","date":"2022","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/34991674","citation_count":30,"is_preprint":false},{"pmid":"23720234","id":"PMC_23720234","title":"Ahsa1 and Hsp90 activity confers more severe craniofacial phenotypes in a zebrafish model of hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR).","date":"2013","source":"Disease models & mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/23720234","citation_count":24,"is_preprint":false},{"pmid":"36230524","id":"PMC_36230524","title":"AHSA1 Promotes Proliferation and EMT by Regulating ERK/CALD1 Axis in Hepatocellular Carcinoma.","date":"2022","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/36230524","citation_count":16,"is_preprint":false},{"pmid":"20818668","id":"PMC_20818668","title":"Solution structure and function of YndB, an AHSA1 protein from Bacillus subtilis.","date":"2010","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/20818668","citation_count":11,"is_preprint":false},{"pmid":"34316513","id":"PMC_34316513","title":"Silencing of LINC00707 suppresses cell proliferation, migration, and invasion of osteosarcoma cells by modulating miR-338-3p/AHSA1 axis.","date":"2021","source":"Open life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34316513","citation_count":10,"is_preprint":false},{"pmid":"40234395","id":"PMC_40234395","title":"AHSA1-HSP90AA1 complex stabilized IFI6 and TGFB1 promotes mitochondrial stability and EMT in EGFR-mutated lung adenocarcinoma under Osimertinib pressure.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/40234395","citation_count":9,"is_preprint":false},{"pmid":"38937628","id":"PMC_38937628","title":"Recruitment of Ahsa1 to Hsp90 is regulated by a conserved peptide that inhibits ATPase stimulation.","date":"2024","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/38937628","citation_count":9,"is_preprint":false},{"pmid":"38022728","id":"PMC_38022728","title":"AHSA1 Regulates Hepatocellular Carcinoma Progression via the TGF-β/Akt-Cyclin D1/CDK6 Pathway.","date":"2023","source":"Journal of hepatocellular carcinoma","url":"https://pubmed.ncbi.nlm.nih.gov/38022728","citation_count":5,"is_preprint":false},{"pmid":"29318533","id":"PMC_29318533","title":"Chemical shift assignments of CHU_1110: an AHSA1-like protein from Cytophaga hutchinsonii.","date":"2018","source":"Biomolecular NMR assignments","url":"https://pubmed.ncbi.nlm.nih.gov/29318533","citation_count":1,"is_preprint":false},{"pmid":"30368907","id":"PMC_30368907","title":"Solution NMR structure of CHU_1110 from Cytophaga hutchinsonii, an AHSA1 protein potentially involved in metal ion stress response.","date":"2018","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/30368907","citation_count":1,"is_preprint":false},{"pmid":"40685075","id":"PMC_40685075","title":"AHSA1/Hsp90α Complex Facilitates Microglial Mitophagy by Targeting TOMM70 in Parkinson Disease.","date":"2025","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/40685075","citation_count":0,"is_preprint":false},{"pmid":"40358702","id":"PMC_40358702","title":"UPP1 and AHSA1 as emerging biomarkers and targets in pancreatic cancer: A proteomic approach.","date":"2025","source":"Biomolecules & biomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/40358702","citation_count":0,"is_preprint":false},{"pmid":"40625056","id":"PMC_40625056","title":"AHSA1 promotes the progression of lung cancer by enhancing the expression of HSP90α.","date":"2025","source":"Histology and histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/40625056","citation_count":0,"is_preprint":false},{"pmid":"41990440","id":"PMC_41990440","title":"AHSA1-DNAJB4 axis: A regulatory mechanism that initiates the ERAD pathway to facilitate endometrial cancer progression.","date":"2026","source":"Journal of reproductive 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Biomarkers","date":"2025-09-17","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.17.676758","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11059,"output_tokens":2502,"usd":0.035354,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9673,"output_tokens":3386,"usd":0.066507,"stage2_stop_reason":"end_turn"},"total_usd":0.101861,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2024,\n      \"finding\": \"Metazoan AHSA1 possesses an intrinsic chaperone domain (ICD), a ~20 amino acid peptide preceding the conserved NxNNWHW motif, that diminishes Hsp90 ATPase stimulation by interfering with the NxNNWHW motif function. The NxNNWHW motif stimulates Hsp90 ATPase activity and modulates Hsp90's apparent affinity for Ahsa1 and ATP. The ICD controls regulated recruitment of Hsp90 in cells, and its deletion results in loss of interaction with Hsp90 and the glucocorticoid receptor.\",\n      \"method\": \"In vitro ATPase assays, mutagenesis of ICD and NxNNWHW motif, co-immunoprecipitation in cells\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro ATPase assay combined with domain mutagenesis and cellular co-IP, multiple orthogonal methods in one study\",\n      \"pmids\": [\"38937628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AHSA1 acts as a co-chaperone of HSP90A to activate CDK6 and PSMD2 in multiple myeloma cells. AHSA1-K137 was identified as the specific binding site for the inhibitor Bufalin; mutation of K137 decreased the interaction of AHSA1 with HSP90A and suppressed AHSA1-mediated activation of CDK6 and PSMD2.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, site-directed mutagenesis, microscale thermophoresis assay, xenograft model\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP, mass spectrometry, site mutagenesis, and in vivo model; multiple orthogonal methods establishing binding site and functional consequence\",\n      \"pmids\": [\"34991674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Silencing AHSA1 in osteosarcoma cells decreased HSP90 ATPase activity, establishing that AHSA1 is required for HSP90 ATPase stimulation in cancer cells. AHSA1 knockdown also increased levels of Wnt/β-catenin negative regulators Axin-2 and GSK3β while decreasing Wnt-5a and β-catenin, placing AHSA1 upstream of Wnt/β-catenin signaling.\",\n      \"method\": \"siRNA knockdown, ATPase activity assay, western blot for pathway components\",\n      \"journal\": \"Biomedicine & pharmacotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — ATPase assay plus western blot pathway analysis; single lab, two orthogonal methods\",\n      \"pmids\": [\"26796264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AHSA1 recruits ERK1/2 and promotes phosphorylation and inactivation of CALD1 (caldesmon) in hepatocellular carcinoma cells, independent of HSP90 and MEK1/2, thereby promoting proliferation and EMT.\",\n      \"method\": \"Co-immunoprecipitation, ERK1/2 phosphorylation inhibitor (SCH772984) rescue, CALD1 knockdown epistasis, gain- and loss-of-function studies in vitro and in vivo\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP showing complex, inhibitor rescue, and epistasis knockdown; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36230524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The AHSA1/Hsp90α complex forms in microglia and targets the mitochondrial import protein TOMM70, facilitating mitophagy in a Parkinson disease mouse model. Knockdown of AHSA1 or inhibition of Hsp90α with geldanamycin suppressed microglial mitophagy and attenuated dopaminergic neuronal death.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, siRNA knockdown, geldanamycin pharmacological inhibition, MPTP mouse model, microglia/dopaminergic neuron co-culture\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP confirming complex, knockdown and pharmacological inhibition with defined mitophagy readout; single lab, two orthogonal approaches\",\n      \"pmids\": [\"40685075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CHK1 directly interacts with AHSA1 and suppresses TRIM8-mediated ubiquitination and degradation of AHSA1, thereby stabilizing the AHSA1-HSP90 complex and enhancing HSP90 ATPase activity to activate mitophagy in cardiomyocytes.\",\n      \"method\": \"Immunoprecipitation and mass spectrometry (IP-MS), cardiomyocyte-specific CHK1 overexpression and knockout mouse models, western blot\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS identifying direct interaction, in vivo genetic models with functional readout; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"42229233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The AHSA1-HSP90AA1 complex stabilizes IFI6 and TGFB1 proteins in EGFR-mutated lung adenocarcinoma cells, with IFI6 stabilization enhancing mitochondrial function and Akt phosphorylation to promote Osimertinib resistance.\",\n      \"method\": \"Co-immunoprecipitation, western blot, overexpression/knockdown functional assays, pharmacological inhibition\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP demonstrating complex and client stabilization, knockdown epistasis; single lab, two orthogonal methods\",\n      \"pmids\": [\"40234395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"AHSA1 binds DNAJB4 (Co-IP), and AHSA1 overexpression enhances DNAJB4 protein levels, suppresses ERAD pathway protein expression (XBP-1s, ATF4, CHOP, GADD34), reduces apoptosis, and promotes endometrial cancer cell colony formation; these effects were abolished by DNAJB4 deletion, establishing DNAJB4 as a downstream effector of AHSA1 in ERAD regulation.\",\n      \"method\": \"Co-immunoprecipitation, DNAJB4 knockdown epistasis, overexpression functional assays, western blot for ERAD markers\",\n      \"journal\": \"Journal of reproductive immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus epistasis knockdown with defined ERAD readout; single lab, two orthogonal methods\",\n      \"pmids\": [\"41990440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In a zebrafish model of HDR syndrome, Ahsa1 and Hsp90 activity genetically promoted more severe craniofacial phenotypes caused by Gata3 mutation, placing Ahsa1/Hsp90 as modifiers of transcription-factor-driven developmental defect severity.\",\n      \"method\": \"Zebrafish forward genetic screen, genetic epistasis in gata3 mutant background, live imaging\",\n      \"journal\": \"Disease models & mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in a vertebrate model with defined developmental phenotype; single lab\",\n      \"pmids\": [\"23720234\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AHSA1 (hAha1) is a co-chaperone that binds to HSP90 via its conserved NxNNWHW motif to stimulate HSP90 ATPase activity; in metazoans, an intrinsic chaperone domain (ICD) autoinhibits this stimulation and controls regulated recruitment to HSP90 and clients such as the glucocorticoid receptor. AHSA1 acts as the critical activating subunit of the AHSA1–HSP90 complex, which stabilizes and activates client proteins including CDK6, PSMD2, IFI6, TGFB1, and TOMM70, thereby driving cell proliferation, drug resistance, mitophagy, and EMT across multiple cellular contexts. AHSA1 protein stability is itself regulated by CHK1-dependent suppression of TRIM8-mediated ubiquitination. AHSA1 additionally engages non-HSP90 pathways by recruiting ERK1/2 to phosphorylate and inactivate CALD1, and by binding DNAJB4 to suppress ER-associated degradation signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AHSA1 is a co-chaperone that activates the HSP90 ATPase cycle and thereby controls the maturation of HSP90 client proteins across diverse cellular contexts [#0, #2]. It engages HSP90 through its conserved NxNNWHW motif, which stimulates HSP90 ATPase activity and tunes HSP90's apparent affinity for ATP; in metazoans an intrinsic chaperone domain (ICD) immediately preceding this motif autoinhibits stimulation and governs regulated recruitment of HSP90 to clients such as the glucocorticoid receptor, with ICD deletion abolishing both HSP90 and receptor binding [#0]. AHSA1 is required for HSP90 ATPase stimulation in cancer cells, where loss of AHSA1 lowers HSP90 activity [#2]. As the activating subunit of AHSA1–HSP90 complexes, it stabilizes and matures client proteins including CDK6 and PSMD2, IFI6 and TGFB1, and the mitochondrial import receptor TOMM70, driving proliferation, drug resistance, and mitophagy [#1, #6, #4]. AHSA1 protein levels are themselves controlled by CHK1, which interacts with AHSA1 and suppresses TRIM8-mediated ubiquitination to stabilize the AHSA1–HSP90 complex [#5]. Beyond HSP90, AHSA1 recruits ERK1/2 to phosphorylate and inactivate caldesmon (CALD1) independently of HSP90 and MEK1/2 [#3], and binds DNAJB4 to suppress ER-associated degradation signaling [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established that Ahsa1, acting through Hsp90, can modify the severity of a transcription-factor-driven developmental defect, linking the co-chaperone to client-dependent phenotypic outcomes in a whole vertebrate.\",\n      \"evidence\": \"Zebrafish genetic epistasis in a gata3 mutant HDR-syndrome model with live imaging\",\n      \"pmids\": [\"23720234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not identify the molecular client mediating the modifier effect\", \"Genetic, not biochemical, evidence for the Ahsa1–Hsp90 interaction in this context\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed that AHSA1 is required for HSP90 ATPase activity in cancer cells and positioned it upstream of Wnt/β-catenin signaling, connecting the co-chaperone to a defined oncogenic pathway.\",\n      \"evidence\": \"siRNA knockdown with HSP90 ATPase assay and western blot of Wnt pathway components in osteosarcoma cells\",\n      \"pmids\": [\"26796264\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Wnt regulation is correlative — no direct AHSA1/HSP90 client in the pathway identified\", \"Single cell-line context\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined AHSA1 as the activating subunit that channels HSP90 toward specific clients (CDK6, PSMD2) and mapped a druggable binding site (K137) required for HSP90 engagement, providing a structural and therapeutic handle.\",\n      \"evidence\": \"Reciprocal Co-IP, mass spectrometry, K137 mutagenesis, microscale thermophoresis, and xenograft in multiple myeloma\",\n      \"pmids\": [\"34991674\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CDK6/PSMD2 stabilization is direct or secondary to global HSP90 activation\", \"K137 role in client selectivity versus general HSP90 binding not separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed an HSP90-independent function for AHSA1 as a scaffold recruiting ERK1/2 to phosphorylate and inactivate CALD1, expanding its role beyond classical co-chaperone activity.\",\n      \"evidence\": \"Co-IP, ERK1/2 inhibitor (SCH772984) rescue, and CALD1 knockdown epistasis in hepatocellular carcinoma in vitro and in vivo\",\n      \"pmids\": [\"36230524\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of AHSA1–ERK1/2 recruitment unresolved\", \"MEK1/2 independence of ERK activation not mechanistically explained\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the AHSA1/HSP90α complex to neuroprotective mitophagy by showing it targets the mitochondrial import receptor TOMM70 in microglia, linking the co-chaperone to mitochondrial quality control.\",\n      \"evidence\": \"Co-IP, immunofluorescence, siRNA knockdown, geldanamycin inhibition, and MPTP Parkinson mouse model with co-culture\",\n      \"pmids\": [\"40685075\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TOMM70 is a direct HSP90 client or an indirect effector\", \"Mechanistic basis of mitophagy initiation downstream of TOMM70\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that AHSA1–HSP90AA1 stabilizes IFI6 and TGFB1 to drive Osimertinib resistance, mechanistically connecting co-chaperone activity to acquired drug resistance via client stabilization and Akt signaling.\",\n      \"evidence\": \"Co-IP, western blot, overexpression/knockdown, and pharmacological inhibition in EGFR-mutated lung adenocarcinoma\",\n      \"pmids\": [\"40234395\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct versus indirect stabilization of IFI6/TGFB1 not distinguished\", \"Single tumor-type context\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved how metazoan AHSA1 activity is intrinsically autoregulated by identifying the ICD that suppresses NxNNWHW-driven HSP90 ATPase stimulation and controls regulated recruitment to HSP90 and the glucocorticoid receptor.\",\n      \"evidence\": \"In vitro ATPase assays, ICD/NxNNWHW mutagenesis, and cellular Co-IP\",\n      \"pmids\": [\"38937628\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal or modification that relieves ICD autoinhibition in cells unknown\", \"Structural basis of ICD–NxNNWHW interference not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified upstream control of AHSA1 protein stability, showing CHK1 suppresses TRIM8-mediated ubiquitination to stabilize the AHSA1–HSP90 complex and sustain ATPase-driven mitophagy in cardiomyocytes.\",\n      \"evidence\": \"IP-MS, cardiomyocyte-specific CHK1 overexpression and knockout mouse models, western blot\",\n      \"pmids\": [\"42229233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TRIM8 ubiquitination site on AHSA1 not mapped\", \"How CHK1 mechanistically blocks TRIM8 unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established a further non-canonical axis in which AHSA1 binds DNAJB4 to suppress ERAD signaling and apoptosis, broadening its effector repertoire beyond HSP90 clients.\",\n      \"evidence\": \"Co-IP, DNAJB4 knockdown epistasis, overexpression assays, and western blot of ERAD markers in endometrial cancer\",\n      \"pmids\": [\"41990440\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether DNAJB4 binding is HSP90-dependent not tested\", \"Direct versus indirect ERAD marker suppression unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The physiological signals that toggle AHSA1 between ICD-autoinhibited and active states, and the rules determining which HSP90 clients AHSA1 selectively matures across tissues, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the ICD-active transition in a cellular context\", \"Client-selectivity determinants undefined\", \"Integration of HSP90-dependent and HSP90-independent (ERK1/2, DNAJB4) functions unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 7]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\"AHSA1–HSP90 co-chaperone complex\"],\n    \"partners\": [\"HSP90AA1\", \"HSP90\", \"CHK1\", \"TRIM8\", \"DNAJB4\", \"ERK1/2\", \"TOMM70\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}