{"gene":"ASB10","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2011,"finding":"ASB10 silencing in perfused anterior segment organ culture reduced aqueous humor outflow facility by ~50% compared with control-infected anterior segments, demonstrating a functional role for ASB10 in trabecular meshwork outflow regulation.","method":"ASB10 siRNA silencing in perfused anterior segment organ culture with outflow facility measurement","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct loss-of-function experiment with defined physiological readout (outflow facility), single lab, one method","pmids":["22156576"],"is_preprint":false},{"year":2011,"finding":"ASB10 mRNA and protein are strongly expressed in trabecular meshwork, retinal ganglion cells, and ciliary body, as established by expression analysis in human ocular tissues.","method":"mRNA and protein expression analysis (immunohistochemistry/RT-PCR) in human ocular tissues","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization experiment replicated across tissue types, single lab","pmids":["22156576"],"is_preprint":false},{"year":2011,"finding":"A synonymous variant c.765C>T (Thr255Thr) in ASB10 affects an exon splice enhancer site and alters mRNA splicing in lymphoblasts of affected family members with POAG.","method":"Minigene splicing assay / mRNA analysis in patient lymphoblasts","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct splice assay in patient-derived cells, single lab, single method","pmids":["22156576"],"is_preprint":false},{"year":2013,"finding":"ASB10 localizes to intracellular vesicular structures in human trabecular meshwork (HTM) cells and co-localizes with markers of the ubiquitin-proteasomal pathway (ubiquitin, α4 subunit of 20S proteasome) and autophagic structures (LC3, p62, HDAC6, HSP70, Rab7, LAMP1), as shown by confocal and super-resolution structured illumination microscopy.","method":"Immunofluorescence confocal microscopy and super-resolution structured illumination microscopy with multiple pathway biomarkers in primary HTM cells","journal":"Molecular vision","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal fluorescent markers, quantitative Pearson colocalization, single lab","pmids":["23901248"],"is_preprint":false},{"year":2013,"finding":"ASB10 physically interacts with HSP70 and with the α4 subunit of the 20S proteasome in HTM cells, as demonstrated by co-immunoprecipitation.","method":"Co-immunoprecipitation of ASB10-transfected HTM cells","journal":"Molecular vision","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP confirmed for two binding partners, single lab, single method","pmids":["23901248"],"is_preprint":false},{"year":2013,"finding":"ASB10 itself is not ubiquitinated, despite its association with ubiquitin-mediated degradation pathway components in HTM cells.","method":"Co-immunoprecipitation and immunofluorescence in HTM cells (negative finding)","journal":"Molecular vision","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — explicitly tested and reported as negative, single lab","pmids":["23901248"],"is_preprint":false},{"year":2013,"finding":"Treatment of HTM cells with the autophagy/proteasome inhibitor MG132 significantly increased the number of small ASB10-stained vesicles, while autophagy inhibitors (wortmannin, bafilomycin A1) decreased them, indicating ASB10 vesicle dynamics are coupled to autophagic flux.","method":"Pharmacological modulation of autophagy with MG132, wortmannin, and bafilomycin A1, followed by quantitative immunofluorescence microscopy","journal":"Molecular vision","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological perturbations with quantitative readout, single lab","pmids":["23901248"],"is_preprint":false},{"year":2021,"finding":"ASB10 functions as an E3 ubiquitin ligase that ubiquitylates TEM8 (tumor endothelial marker 8) for proteasomal degradation; ASB10 deficiency in triple-negative breast cancer results in elevated homeostatic TEM8 levels.","method":"Ubiquitination assay, protein stability assay, and overexpression/knockdown of ASB10 with TEM8 protein level measurement in breast cancer cells","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ubiquitination assay with functional consequence on substrate levels, single lab, single study","pmids":["34285210"],"is_preprint":false},{"year":2025,"finding":"Asb10 binds HSP70 and competitively blocks STUB1-mediated ubiquitination and proteasomal degradation of HSP70, thereby stabilizing HSP70 protein levels and exacerbating cardiac hypertrophic growth.","method":"Immunoprecipitation-mass spectrometry and co-immunoprecipitation; adenoviral Asb10 overexpression in NRVMs; siRNA and pharmacological HSP70 inhibition; AAV9-Asb10 overexpression and shAsb10 knockdown in mouse TAC model","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — IP-MS identification of binding partner, reciprocal co-IP, in vitro ubiquitination competition, in vivo rescue with both OE and KD, multiple orthogonal methods in single study","pmids":["40399264"],"is_preprint":false},{"year":2025,"finding":"Asb10 overexpression in vivo (AAV9-Asb10) worsens cardiac function and increases interstitial fibrosis after transverse aortic constriction (TAC), while Asb10 knockdown (AAV9-shAsb10) improves outcomes, establishing Asb10 as a pro-hypertrophic factor in pressure-overload heart failure.","method":"AAV9-mediated cardiac-specific overexpression and shRNA knockdown in mouse TAC surgical model with echocardiographic and histological assessment","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — bidirectional genetic manipulation (OE and KD) in vivo with multiple functional readouts in a single rigorous study","pmids":["40399264"],"is_preprint":false},{"year":2025,"finding":"The pro-hypertrophic effects of Asb10 are mediated through HSP70 stabilization leading to cardiac inflammation and activation of phospho-HDAC2 at Ser394 (pHDAC2S394).","method":"siRNA and pharmacological inhibition of HSP70 in Asb10-overexpressing NRVMs; measurement of inflammatory markers and pHDAC2S394 levels","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via HSP70 inhibition partially reverses Asb10 phenotype, single lab, single study","pmids":["40399264"],"is_preprint":false}],"current_model":"ASB10 is an E3 ubiquitin ligase whose ankyrin repeat domain binds substrate proteins (including TEM8 and HSP70) and whose SOCS box recruits ubiquitin ligase machinery; in trabecular meshwork cells it associates with both the proteasomal and autophagic-lysosomal degradation pathways and is required for normal aqueous humor outflow, while in cardiomyocytes it stabilizes HSP70 by blocking STUB1-mediated ubiquitination, thereby exacerbating pressure-overload cardiac hypertrophy."},"narrative":{"mechanistic_narrative":"ASB10 is a substrate-recognition E3 ubiquitin ligase that controls the abundance of specific client proteins and thereby regulates tissue homeostasis in the eye, vasculature, and heart [PMID:34285210, PMID:40399264]. As an E3 ligase it ubiquitylates the tumor endothelial marker TEM8, targeting it for proteasomal degradation so that loss of ASB10 elevates homeostatic TEM8 levels [PMID:34285210]. In the heart, ASB10 acts in the opposite direction on a different client: it binds HSP70 and competitively blocks STUB1-mediated ubiquitination of HSP70, stabilizing HSP70 and driving inflammation and accumulation of Ser394-phosphorylated HDAC2, which together exacerbate pressure-overload cardiac hypertrophy and fibrosis [PMID:40399264]. In trabecular meshwork cells ASB10 localizes to intracellular vesicles where it physically associates with HSP70 and with proteasomal (20S α4 subunit) and autophagic-lysosomal machinery, and its vesicle dynamics track autophagic flux, while ASB10 itself is not ubiquitinated [PMID:23901248]; silencing ASB10 reduces aqueous humor outflow facility, and a splice-altering coding variant links ASB10 to primary open-angle glaucoma [PMID:22156576]. A unified enzymatic mechanism connecting substrate selection across these tissues to a single biochemical activity has not been fully resolved in the available corpus.","teleology":[{"year":2011,"claim":"Established that ASB10 is functionally required for a defined physiological process, aqueous humor outflow, and is genetically linked to glaucoma, motivating mechanistic study of an otherwise uncharacterized protein.","evidence":"siRNA silencing in perfused anterior segment organ culture with outflow facility readout; ocular tissue expression analysis; minigene/patient lymphoblast splicing assay","pmids":["22156576"],"confidence":"Medium","gaps":["Did not identify the molecular activity of ASB10 or any substrate","Mechanism linking outflow regulation to a biochemical function unknown","Disease causality rests on splice alteration without protein-level mechanism"]},{"year":2013,"claim":"Resolved where ASB10 acts and what it binds, placing it at protein-degradation machinery and identifying HSP70 and the proteasome as partners, while showing ASB10 is not itself a degradation substrate.","evidence":"Confocal/super-resolution colocalization with proteasomal and autophagic markers, co-IP of HSP70 and 20S α4 subunit, and pharmacological autophagy/proteasome modulation in primary HTM cells","pmids":["23901248"],"confidence":"Medium","gaps":["No catalytic ubiquitin-ligase activity demonstrated","No substrate ubiquitination shown in this system","Functional consequence of HSP70/proteasome association in outflow not established"]},{"year":2021,"claim":"Demonstrated ASB10's enzymatic identity directly, showing it is an E3 ligase that ubiquitylates TEM8 for proteasomal degradation and controls its homeostatic levels.","evidence":"In vitro ubiquitination and protein stability assays with ASB10 overexpression/knockdown and TEM8 level measurement in triple-negative breast cancer cells","pmids":["34285210"],"confidence":"Medium","gaps":["Whether TEM8 is a substrate in ocular or cardiac tissue not addressed","SOCS-box/ankyrin domain requirements for catalysis not dissected","Single study, single cancer cell context"]},{"year":2025,"claim":"Defined a tissue-specific, non-degradative mechanism in which ASB10 competitively shields HSP70 from STUB1-mediated ubiquitination, and showed bidirectional in vivo control of cardiac hypertrophy through this axis.","evidence":"IP-MS and reciprocal co-IP, in vitro ubiquitination competition, adenoviral overexpression in NRVMs with HSP70 inhibition, and AAV9 overexpression and shRNA knockdown in the mouse TAC model with echocardiographic/histological and pHDAC2S394 readouts","pmids":["40399264"],"confidence":"High","gaps":["How ASB10 acts as a ligase on TEM8 yet as a competitive protector of HSP70 from another ligase is unreconciled","Direct measurement of HSP70 ubiquitination kinetics in vivo not shown","pHDAC2S394 link is epistatic rather than fully causal"]},{"year":null,"claim":"It remains unresolved how ASB10 selects between degradative ubiquitination (TEM8) and protective substrate shielding (HSP70), and whether a single domain architecture explains both behaviors across eye, heart, and tumor contexts.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate engagement","No unified rule for substrate fate","Ocular substrate(s) underlying outflow regulation unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[7]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[7,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[3,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[7,8]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[3,6]}],"complexes":[],"partners":["HSP70","STUB1","TEM8","PSMA7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8WXI3","full_name":"Ankyrin repeat and SOCS box protein 10","aliases":[],"length_aa":467,"mass_kda":50.9,"function":"May be a substrate-recognition component of a SCF-like ECS (Elongin-Cullin-SOCS-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q8WXI3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ASB10","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ASB10","total_profiled":1310},"omim":[{"mim_id":"615054","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 10; ASB10","url":"https://www.omim.org/entry/615054"},{"mim_id":"603383","title":"GLAUCOMA 1, OPEN ANGLE, F; GLC1F","url":"https://www.omim.org/entry/603383"},{"mim_id":"137760","title":"GLAUCOMA, PRIMARY OPEN ANGLE; POAG","url":"https://www.omim.org/entry/137760"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"heart muscle","ntpm":25.0},{"tissue":"skeletal muscle","ntpm":73.0},{"tissue":"tongue","ntpm":13.3}],"url":"https://www.proteinatlas.org/search/ASB10"},"hgnc":{"alias_symbol":[],"prev_symbol":["GLC1F"]},"alphafold":{"accession":"Q8WXI3","domains":[{"cath_id":"1.25.40.20","chopping":"46-93_112-207","consensus_level":"medium","plddt":89.1795,"start":46,"end":207},{"cath_id":"1.25.40.20","chopping":"208-360","consensus_level":"medium","plddt":90.5856,"start":208,"end":360},{"cath_id":"-","chopping":"362-467","consensus_level":"medium","plddt":86.0791,"start":362,"end":467}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WXI3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WXI3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WXI3-F1-predicted_aligned_error_v6.png","plddt_mean":79.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ASB10","jax_strain_url":"https://www.jax.org/strain/search?query=ASB10"},"sequence":{"accession":"Q8WXI3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WXI3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WXI3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WXI3"}},"corpus_meta":[{"pmid":"10037570","id":"PMC_10037570","title":"GLC1F, a new primary open-angle glaucoma locus, maps to 7q35-q36.","date":"1999","source":"Archives of ophthalmology (Chicago, Ill. : 1960)","url":"https://pubmed.ncbi.nlm.nih.gov/10037570","citation_count":143,"is_preprint":false},{"pmid":"22156576","id":"PMC_22156576","title":"Variants in ASB10 are associated with open-angle glaucoma.","date":"2011","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22156576","citation_count":66,"is_preprint":false},{"pmid":"34285210","id":"PMC_34285210","title":"TEM8 marks neovasculogenic tumor-initiating cells in triple-negative breast cancer.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34285210","citation_count":61,"is_preprint":false},{"pmid":"17210857","id":"PMC_17210857","title":"A new locus (GLC1H) for adult-onset primary open-angle glaucoma maps to the 2p15-p16 region.","date":"2007","source":"Archives of ophthalmology (Chicago, Ill. : 1960)","url":"https://pubmed.ncbi.nlm.nih.gov/17210857","citation_count":39,"is_preprint":false},{"pmid":"26473621","id":"PMC_26473621","title":"Identification of Rare Variants in ATP8B4 as a Risk Factor for Systemic Sclerosis by Whole-Exome Sequencing.","date":"2016","source":"Arthritis & rheumatology (Hoboken, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/26473621","citation_count":38,"is_preprint":false},{"pmid":"34968235","id":"PMC_34968235","title":"Epigenetics of Skeletal Muscle-Associated Genes in the ASB, LRRC, TMEM, and OSBPL Gene Families.","date":"2020","source":"Epigenomes","url":"https://pubmed.ncbi.nlm.nih.gov/34968235","citation_count":32,"is_preprint":false},{"pmid":"23901248","id":"PMC_23901248","title":"Ankyrin repeat and suppressor of cytokine signaling box containing protein-10 is associated with ubiquitin-mediated degradation pathways in trabecular meshwork cells.","date":"2013","source":"Molecular vision","url":"https://pubmed.ncbi.nlm.nih.gov/23901248","citation_count":25,"is_preprint":false},{"pmid":"11436127","id":"PMC_11436127","title":"Genetic linkage of autosomal dominant primary open angle glaucoma to chromosome 3q in a Greek pedigree.","date":"2001","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/11436127","citation_count":22,"is_preprint":false},{"pmid":"22798626","id":"PMC_22798626","title":"Analysis of ASB10 variants in open angle glaucoma.","date":"2012","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22798626","citation_count":21,"is_preprint":false},{"pmid":"12847422","id":"PMC_12847422","title":"Genetic, ophthalmic, morphometric and histopathological analysis of the Retinopathy Globe Enlarged (rge) chicken.","date":"2003","source":"Molecular vision","url":"https://pubmed.ncbi.nlm.nih.gov/12847422","citation_count":20,"is_preprint":false},{"pmid":"27296073","id":"PMC_27296073","title":"Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma.","date":"2016","source":"Experimental eye research","url":"https://pubmed.ncbi.nlm.nih.gov/27296073","citation_count":14,"is_preprint":false},{"pmid":"30237808","id":"PMC_30237808","title":"Epigenome-Wide Analyses Identify Two Novel Associations With Recurrent Stroke in the Vitamin Intervention for Stroke Prevention Clinical Trial.","date":"2018","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30237808","citation_count":14,"is_preprint":false},{"pmid":"26713451","id":"PMC_26713451","title":"Variants in the ASB10 Gene Are Associated with Primary Open Angle Glaucoma.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26713451","citation_count":10,"is_preprint":false},{"pmid":"37950974","id":"PMC_37950974","title":"Ubiquitin proteasome system and glaucoma: A survey of genetics and molecular biology studies supporting a link with pathogenic and therapeutic relevance.","date":"2023","source":"Molecular aspects of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37950974","citation_count":9,"is_preprint":false},{"pmid":"15073581","id":"PMC_15073581","title":"Exclusion of 14 candidate loci for primary open angle glaucoma in Finnish families.","date":"2004","source":"Molecular vision","url":"https://pubmed.ncbi.nlm.nih.gov/15073581","citation_count":9,"is_preprint":false},{"pmid":"31522561","id":"PMC_31522561","title":"Lack of Correlation between ASB10 and Normal-tension Glaucoma in a Population from the Republic of Korea.","date":"2019","source":"Current eye research","url":"https://pubmed.ncbi.nlm.nih.gov/31522561","citation_count":3,"is_preprint":false},{"pmid":"40399264","id":"PMC_40399264","title":"Asb10 accelerates pathological cardiac remodeling by stabilizing HSP70.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/40399264","citation_count":3,"is_preprint":false},{"pmid":"17361544","id":"PMC_17361544","title":"Glaucoma in Costa Rica. Initial approaches.","date":"2004","source":"Revista de biologia tropical","url":"https://pubmed.ncbi.nlm.nih.gov/17361544","citation_count":2,"is_preprint":false},{"pmid":"15646497","id":"PMC_15646497","title":"[Genetic ground of primary open angle glaucoma].","date":"2004","source":"Klinika oczna","url":"https://pubmed.ncbi.nlm.nih.gov/15646497","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12383,"output_tokens":2355,"usd":0.036237,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9485,"output_tokens":2256,"usd":0.051913,"stage2_stop_reason":"end_turn"},"total_usd":0.08815,"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\": 2011,\n      \"finding\": \"ASB10 silencing in perfused anterior segment organ culture reduced aqueous humor outflow facility by ~50% compared with control-infected anterior segments, demonstrating a functional role for ASB10 in trabecular meshwork outflow regulation.\",\n      \"method\": \"ASB10 siRNA silencing in perfused anterior segment organ culture with outflow facility measurement\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct loss-of-function experiment with defined physiological readout (outflow facility), single lab, one method\",\n      \"pmids\": [\"22156576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ASB10 mRNA and protein are strongly expressed in trabecular meshwork, retinal ganglion cells, and ciliary body, as established by expression analysis in human ocular tissues.\",\n      \"method\": \"mRNA and protein expression analysis (immunohistochemistry/RT-PCR) in human ocular tissues\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization experiment replicated across tissue types, single lab\",\n      \"pmids\": [\"22156576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A synonymous variant c.765C>T (Thr255Thr) in ASB10 affects an exon splice enhancer site and alters mRNA splicing in lymphoblasts of affected family members with POAG.\",\n      \"method\": \"Minigene splicing assay / mRNA analysis in patient lymphoblasts\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct splice assay in patient-derived cells, single lab, single method\",\n      \"pmids\": [\"22156576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASB10 localizes to intracellular vesicular structures in human trabecular meshwork (HTM) cells and co-localizes with markers of the ubiquitin-proteasomal pathway (ubiquitin, α4 subunit of 20S proteasome) and autophagic structures (LC3, p62, HDAC6, HSP70, Rab7, LAMP1), as shown by confocal and super-resolution structured illumination microscopy.\",\n      \"method\": \"Immunofluorescence confocal microscopy and super-resolution structured illumination microscopy with multiple pathway biomarkers in primary HTM cells\",\n      \"journal\": \"Molecular vision\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal fluorescent markers, quantitative Pearson colocalization, single lab\",\n      \"pmids\": [\"23901248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASB10 physically interacts with HSP70 and with the α4 subunit of the 20S proteasome in HTM cells, as demonstrated by co-immunoprecipitation.\",\n      \"method\": \"Co-immunoprecipitation of ASB10-transfected HTM cells\",\n      \"journal\": \"Molecular vision\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP confirmed for two binding partners, single lab, single method\",\n      \"pmids\": [\"23901248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASB10 itself is not ubiquitinated, despite its association with ubiquitin-mediated degradation pathway components in HTM cells.\",\n      \"method\": \"Co-immunoprecipitation and immunofluorescence in HTM cells (negative finding)\",\n      \"journal\": \"Molecular vision\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — explicitly tested and reported as negative, single lab\",\n      \"pmids\": [\"23901248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Treatment of HTM cells with the autophagy/proteasome inhibitor MG132 significantly increased the number of small ASB10-stained vesicles, while autophagy inhibitors (wortmannin, bafilomycin A1) decreased them, indicating ASB10 vesicle dynamics are coupled to autophagic flux.\",\n      \"method\": \"Pharmacological modulation of autophagy with MG132, wortmannin, and bafilomycin A1, followed by quantitative immunofluorescence microscopy\",\n      \"journal\": \"Molecular vision\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological perturbations with quantitative readout, single lab\",\n      \"pmids\": [\"23901248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ASB10 functions as an E3 ubiquitin ligase that ubiquitylates TEM8 (tumor endothelial marker 8) for proteasomal degradation; ASB10 deficiency in triple-negative breast cancer results in elevated homeostatic TEM8 levels.\",\n      \"method\": \"Ubiquitination assay, protein stability assay, and overexpression/knockdown of ASB10 with TEM8 protein level measurement in breast cancer cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ubiquitination assay with functional consequence on substrate levels, single lab, single study\",\n      \"pmids\": [\"34285210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Asb10 binds HSP70 and competitively blocks STUB1-mediated ubiquitination and proteasomal degradation of HSP70, thereby stabilizing HSP70 protein levels and exacerbating cardiac hypertrophic growth.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry and co-immunoprecipitation; adenoviral Asb10 overexpression in NRVMs; siRNA and pharmacological HSP70 inhibition; AAV9-Asb10 overexpression and shAsb10 knockdown in mouse TAC model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — IP-MS identification of binding partner, reciprocal co-IP, in vitro ubiquitination competition, in vivo rescue with both OE and KD, multiple orthogonal methods in single study\",\n      \"pmids\": [\"40399264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Asb10 overexpression in vivo (AAV9-Asb10) worsens cardiac function and increases interstitial fibrosis after transverse aortic constriction (TAC), while Asb10 knockdown (AAV9-shAsb10) improves outcomes, establishing Asb10 as a pro-hypertrophic factor in pressure-overload heart failure.\",\n      \"method\": \"AAV9-mediated cardiac-specific overexpression and shRNA knockdown in mouse TAC surgical model with echocardiographic and histological assessment\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — bidirectional genetic manipulation (OE and KD) in vivo with multiple functional readouts in a single rigorous study\",\n      \"pmids\": [\"40399264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The pro-hypertrophic effects of Asb10 are mediated through HSP70 stabilization leading to cardiac inflammation and activation of phospho-HDAC2 at Ser394 (pHDAC2S394).\",\n      \"method\": \"siRNA and pharmacological inhibition of HSP70 in Asb10-overexpressing NRVMs; measurement of inflammatory markers and pHDAC2S394 levels\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via HSP70 inhibition partially reverses Asb10 phenotype, single lab, single study\",\n      \"pmids\": [\"40399264\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ASB10 is an E3 ubiquitin ligase whose ankyrin repeat domain binds substrate proteins (including TEM8 and HSP70) and whose SOCS box recruits ubiquitin ligase machinery; in trabecular meshwork cells it associates with both the proteasomal and autophagic-lysosomal degradation pathways and is required for normal aqueous humor outflow, while in cardiomyocytes it stabilizes HSP70 by blocking STUB1-mediated ubiquitination, thereby exacerbating pressure-overload cardiac hypertrophy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ASB10 is a substrate-recognition E3 ubiquitin ligase that controls the abundance of specific client proteins and thereby regulates tissue homeostasis in the eye, vasculature, and heart [#7, #8]. As an E3 ligase it ubiquitylates the tumor endothelial marker TEM8, targeting it for proteasomal degradation so that loss of ASB10 elevates homeostatic TEM8 levels [#7]. In the heart, ASB10 acts in the opposite direction on a different client: it binds HSP70 and competitively blocks STUB1-mediated ubiquitination of HSP70, stabilizing HSP70 and driving inflammation and accumulation of Ser394-phosphorylated HDAC2, which together exacerbate pressure-overload cardiac hypertrophy and fibrosis [#8, #10, #9]. In trabecular meshwork cells ASB10 localizes to intracellular vesicles where it physically associates with HSP70 and with proteasomal (20S \\u03b14 subunit) and autophagic-lysosomal machinery, and its vesicle dynamics track autophagic flux, while ASB10 itself is not ubiquitinated [#3, #4, #5, #6]; silencing ASB10 reduces aqueous humor outflow facility, and a splice-altering coding variant links ASB10 to primary open-angle glaucoma [#0, #2]. A unified enzymatic mechanism connecting substrate selection across these tissues to a single biochemical activity has not been fully resolved in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that ASB10 is functionally required for a defined physiological process, aqueous humor outflow, and is genetically linked to glaucoma, motivating mechanistic study of an otherwise uncharacterized protein.\",\n      \"evidence\": \"siRNA silencing in perfused anterior segment organ culture with outflow facility readout; ocular tissue expression analysis; minigene/patient lymphoblast splicing assay\",\n      \"pmids\": [\"22156576\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the molecular activity of ASB10 or any substrate\", \"Mechanism linking outflow regulation to a biochemical function unknown\", \"Disease causality rests on splice alteration without protein-level mechanism\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved where ASB10 acts and what it binds, placing it at protein-degradation machinery and identifying HSP70 and the proteasome as partners, while showing ASB10 is not itself a degradation substrate.\",\n      \"evidence\": \"Confocal/super-resolution colocalization with proteasomal and autophagic markers, co-IP of HSP70 and 20S \\u03b14 subunit, and pharmacological autophagy/proteasome modulation in primary HTM cells\",\n      \"pmids\": [\"23901248\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic ubiquitin-ligase activity demonstrated\", \"No substrate ubiquitination shown in this system\", \"Functional consequence of HSP70/proteasome association in outflow not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated ASB10's enzymatic identity directly, showing it is an E3 ligase that ubiquitylates TEM8 for proteasomal degradation and controls its homeostatic levels.\",\n      \"evidence\": \"In vitro ubiquitination and protein stability assays with ASB10 overexpression/knockdown and TEM8 level measurement in triple-negative breast cancer cells\",\n      \"pmids\": [\"34285210\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TEM8 is a substrate in ocular or cardiac tissue not addressed\", \"SOCS-box/ankyrin domain requirements for catalysis not dissected\", \"Single study, single cancer cell context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a tissue-specific, non-degradative mechanism in which ASB10 competitively shields HSP70 from STUB1-mediated ubiquitination, and showed bidirectional in vivo control of cardiac hypertrophy through this axis.\",\n      \"evidence\": \"IP-MS and reciprocal co-IP, in vitro ubiquitination competition, adenoviral overexpression in NRVMs with HSP70 inhibition, and AAV9 overexpression and shRNA knockdown in the mouse TAC model with echocardiographic/histological and pHDAC2S394 readouts\",\n      \"pmids\": [\"40399264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ASB10 acts as a ligase on TEM8 yet as a competitive protector of HSP70 from another ligase is unreconciled\", \"Direct measurement of HSP70 ubiquitination kinetics in vivo not shown\", \"pHDAC2S394 link is epistatic rather than fully causal\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how ASB10 selects between degradative ubiquitination (TEM8) and protective substrate shielding (HSP70), and whether a single domain architecture explains both behaviors across eye, heart, and tumor contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of substrate engagement\", \"No unified rule for substrate fate\", \"Ocular substrate(s) underlying outflow regulation unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"HSP70\", \"STUB1\", \"TEM8\", \"PSMA7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}