{"gene":"ASB9","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2006,"finding":"ASB9 ankyrin repeat domain binds creatine kinase B (CKB) in a SOCS box-independent manner; the interaction promotes polyubiquitylation of CKB and decreases total CKB protein levels, with CKB degradation being primarily SOCS box-dependent, establishing ASB9 as a substrate receptor of an E3 ubiquitin ligase targeting CKB for proteasomal degradation.","method":"Proteomics/pull-down to identify interaction, co-immunoprecipitation to confirm binding, ubiquitination assay to measure polyubiquitylation, protein level measurements in cells with ASB9 expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction confirmed by proteomics and Co-IP, functional ubiquitination and degradation assays, independently replicated in multiple subsequent studies","pmids":["17148442"],"is_preprint":false},{"year":2010,"finding":"ASB9 interacts with ubiquitous mitochondrial creatine kinase (uMtCK) via its ankyrin repeat domain in a SOCS box-independent manner and co-localizes with uMtCK in mitochondria. Full-length ASB9 (but not the naturally occurring SOCS box-deleted variant ASB9ΔSOCS) induces ubiquitination of uMtCK, causes abnormal mitochondrial structure, decreases mitochondrial membrane potential, reduces creatine kinase activity, and reduces cell growth.","method":"Co-immunoprecipitation, co-localization (confocal microscopy), ubiquitination assay, mitochondrial membrane potential assay, creatine kinase activity assay, cell growth assay; comparison of full-length ASB9 vs. ASB9ΔSOCS","journal":"BMC biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, colocalization, functional assays) in a single study with rigorous domain-deletion controls","pmids":["20302626"],"is_preprint":false},{"year":2012,"finding":"Crystal structure of the ASB9-2 isoform (containing one ankyrin repeat domain) at 2.2-Å resolution revealed an arch shape with L-shaped cross-section. Mutagenesis (His103, Phe107) and truncation analysis showed that the first six ankyrin repeats plus the N-terminal region are essential for CKB binding.","method":"X-ray crystallography (molecular replacement, 2.2 Å), amino acid substitution analysis, docking experiments, truncation mutant binding assays","journal":"The protein journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis in single study; functional validation of binding residues by mutational analysis","pmids":["22418839"],"is_preprint":false},{"year":2013,"finding":"ASB9 is unstable alone but forms a stable ternary complex with Elongin B and Elongin C (EloBC), which then binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD, establishing selective Cullin 5 recruitment for the ECS-type CRL complex.","method":"Differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization mass spectrometry, ion-mobility mass spectrometry (IM-MS), molecular modeling","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal biophysical methods (ITC, IM-MS, DSF) providing thermodynamic and structural information in a single rigorous study","pmids":["23837592"],"is_preprint":false},{"year":2015,"finding":"One ASB9 molecule binds to a CK dimer with extremely tight affinity; the N-terminal disordered region and first ankyrin repeat of ASB9 are protected upon binding. ASB9 protects CK residues 182–203 (one side of the active site), and ASB9 N-terminal residues may occupy one CK active site, partially inhibiting CK enzymatic activity.","method":"Amide hydrogen-deuterium exchange mass spectrometry (HDXMS), enzymatic activity assays, mutational analysis, molecular docking, binding affinity measurements (deletion constructs)","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — HDXMS interface mapping plus enzymatic assays and mutagenesis in single study with multiple orthogonal methods","pmids":["25654263"],"is_preprint":false},{"year":2016,"finding":"Integrative structural modeling combined with small-angle X-ray scattering (SAXS) defined the ASB9–CK interface and constructed an atomic model of the full CK-targeting CRL. Dominant modes of motion in the correctly docked complex permit close approach of ubiquitin to the CK substrate, suggesting a dynamic mechanism for ubiquitin transfer over ~60 Å.","method":"Integrative molecular modeling, small-angle X-ray scattering (SAXS), normal-mode analysis","journal":"Structure","confidence":"Medium","confidence_rationale":"Tier 1 (structure) / Weak — single study using computational/modeling approach validated by SAXS shape fitting; no independent replication","pmids":["27396830"],"is_preprint":false},{"year":2019,"finding":"Yeast two-hybrid screening of ovarian granulosa cell cDNA library identified PAR1, TAOK1, and TNFAIP6/TSG6 as ASB9 binding partners in granulosa cells. Notably, no interaction was found between ASB9 and CKB in these granulosa cells, in contrast to other cell types.","method":"Yeast two-hybrid screening, negative result for ASB9–CKB interaction in granulosa cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid for binding partners, single lab; negative result for CKB is informative but lacks independent confirmation in GCs","pmids":["30811458"],"is_preprint":false},{"year":2019,"finding":"CRISPR/Cas9-mediated inhibition of ASB9 in ovarian granulosa cells led to increased granulosa cell proliferation and modulated expression of target genes (PAR1, TAOK1, TNFAIP6), establishing a functional role for ASB9 in suppressing GC proliferation.","method":"CRISPR/Cas9 gene inhibition, cell proliferation assay, gene expression analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined cellular phenotype; single lab, single study","pmids":["30811458"],"is_preprint":false},{"year":2020,"finding":"Cryo-EM structures of the substrate CKB bound to ASB9-ELOB/C and of full-length CUL5 bound to RBX2 revealed that ASB9 and CUL5 behave as rigid rods connected through an ELOB/C hinge. HDX-MS mapped onto the full structural model showed long-range allosteric communication from the substrate through CUL5 to the RBX2 flexible linker, proposing a revised allosteric mechanism for CUL-E3 ligase function.","method":"Cryo-EM structure determination, hydrogen-deuterium exchange mass spectrometry (HDX-MS), integrative structural modeling","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structures of substrate-bound complex and full-length CUL5 combined with HDX-MS; multiple orthogonal methods in single study revealing allosteric mechanism","pmids":["32513959"],"is_preprint":false},{"year":2021,"finding":"CRISPR/Cas9-mediated inhibition of ASB9 in ovarian granulosa cells increased GC number, decreased caspase-3/7 activity, CASP3 expression, and BAX/BCL2 ratio (reduced apoptosis), and increased pMAPK3/1 phosphorylation; conversely, ASB9 induction post-hCG was concomitant with decreased pMAPK3/1 levels, placing ASB9 upstream as a negative regulator of MAPK signaling in GCs.","method":"CRISPR/Cas9 inhibition, cell proliferation and apoptosis assays (caspase3/7 activity, CASP3 and BAX/BCL2 expression), phospho-MAPK3/1 western blot, in vivo hCG model","journal":"Molecular reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with multiple cellular phenotype readouts and in vivo correlation; single lab, single study","pmids":["34476862"],"is_preprint":false},{"year":2023,"finding":"ASB9 interacts with hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) in human spermatogonial stem cells (SSC line), as confirmed by protein immunoprecipitation. ASB9 overexpression inhibited SSC proliferation and increased apoptosis; re-expression of HIF1AN reversed these effects, establishing HIF1AN as the functional target of ASB9 in SSCs. CKB was tested but did not show direct interaction with ASB9 in this cell type.","method":"Protein immunoprecipitation, ASB9 overexpression in SSC line, cell proliferation and apoptosis assays, HIF1AN re-expression rescue experiment","journal":"Biological research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP plus rescue experiment establish substrate identity and functional consequence; single lab, single study","pmids":["36683111"],"is_preprint":false},{"year":2023,"finding":"CLOCK transcription factor binds to the E-box element in the ASB9 promoter and increases ASB9 expression, which in turn inhibits porcine granulosa cell proliferation, placing CLOCK upstream of ASB9 in a transcriptional regulatory pathway.","method":"CLOCK overexpression/knockdown, ASB9 promoter E-box binding assay (chromatin immunoprecipitation or promoter-reporter implied), cell proliferation assay","journal":"Journal of animal science and biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct promoter binding and downstream proliferation phenotype; single lab, single study","pmids":["37280645"],"is_preprint":false},{"year":2025,"finding":"The ASB9-CUL5 E3 ligase polyubiquitylates free histones H3 and H4 (but not H2A/H2B, or histones in nucleosomes or complexed with chaperone Asf1), generating K48 and K63 polyubiquitin chains. The ligase-histone interaction is highly electrostatic; neddylated ASB9-CRL5 binds with highest affinity. Crucially, this ubiquitylation does not require the ring-between-ring ligase ARIH2, representing the first example of CUL5-mediated ubiquitylation without a RBR helper ligase.","method":"In vitro ubiquitylation assays with reconstituted ASB9-CRL5, binding affinity measurements, lysine site mapping by mass spectrometry, nucleosome and Asf1-complex competition assays, polyubiquitin chain-type analysis","journal":"Molecular & cellular proteomics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro ubiquitylation with multiple substrate/condition controls, mechanistic insight into substrate specificity and ARIH2 independence; peer-reviewed publication","pmids":["41260500"],"is_preprint":false},{"year":2025,"finding":"ASB9-CUL5 E3 ligase polyubiquitylates free histones H3 and H4 with substrate specificity (H2A and H2B are not polyubiquitylated); histones in nucleosomes or bound by chaperone Asf1 are not ubiquitylated. This represents the first CUL5-mediated ubiquitylation not requiring an ARIH2 RBR helper ligase. (Preprint version of the same study as PMID:41260500.)","method":"In vitro reconstituted ubiquitylation assay, binding experiments, polyubiquitin chain-linkage analysis, substrate context experiments (nucleosome, Asf1 complex)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — same rigorous reconstitution data as peer-reviewed paper; preprint version; confidence not raised further as it is the same dataset","pmids":["40501794"],"is_preprint":true},{"year":2026,"finding":"ASB9 assembles a testis-specific CRL complex (TNP2-ASB9-ELOB/C-CUL5-RBX1) that mediates ubiquitin-dependent degradation of transition protein TNP2 during spermiogenesis. ASB9 deficiency in humans and mice causes TNP2 retention, failure of the histone-to-protamine transition, sperm head malformation, and male infertility.","method":"Co-immunoprecipitation to identify complex assembly, genetic knockout (mouse and human loss-of-function), ubiquitination assays, spermiogenesis phenotype analysis (sperm morphology, fertility)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — complex assembly by Co-IP, in vivo knockout phenotype in two species, mechanistic rescue implied; multiple orthogonal methods","pmids":["41915740"],"is_preprint":false}],"current_model":"ASB9 is the substrate-recognition subunit of an ECS-type Cullin 5 RING E3 ubiquitin ligase complex (ASB9–ElonginB/C–CUL5–RBX2) that uses its ankyrin repeat domain to bind substrates—including creatine kinase B (CKB), ubiquitous mitochondrial creatine kinase (uMtCK), HIF1AN, transition protein TNP2, and free histones H3/H4—and its SOCS box to recruit ElonginB/C and CUL5, driving K48/K63 polyubiquitylation and proteasomal degradation of these targets; structural and HDX-MS studies reveal that ASB9 and CUL5 act as rigid rods connected through an ElonginB/C hinge that transmits long-range allosteric signals from the bound substrate to the RBX2 E2-recruiting domain, facilitating dynamic ubiquitin transfer, while its physiological roles include inhibiting mitochondrial function and creatine kinase activity, regulating spermatogonial stem cell proliferation/apoptosis via HIF1AN, enabling the histone-to-protamine transition in spermiogenesis via TNP2 degradation, degrading free histones H3/H4 (independently of ARIH2, unlike other CUL5 complexes), and suppressing granulosa cell proliferation through MAPK signaling modulation."},"narrative":{"mechanistic_narrative":"ASB9 is the substrate-recognition subunit of an ECS-type Cullin 5 RING E3 ubiquitin ligase that selects diverse substrates through its ankyrin repeat domain and recruits the catalytic core via its SOCS box, thereby controlling the proteasomal turnover of metabolic, transcriptional, and chromatin-associated targets [PMID:17148442, PMID:23837592]. The ankyrin repeats engage substrates such as cytosolic creatine kinase B (CKB) and ubiquitous mitochondrial creatine kinase (uMtCK) in a SOCS box-independent manner, while the SOCS box drives substrate degradation; full-length ASB9 ubiquitylates uMtCK, disrupts mitochondrial structure, lowers membrane potential, and reduces creatine kinase activity [PMID:17148442, PMID:20302626]. ASB9 is unstable alone but forms a stable ternary complex with Elongin B/C that binds selectively to the Cullin 5 N-terminal domain, assembling the ASB9–EloBC–CUL5–RBX2 ligase [PMID:23837592]. Structural and HDX-MS studies show that ASB9 binds creatine kinase with very high affinity and that ASB9 and CUL5 behave as rigid rods joined by an EloB/C hinge, transmitting long-range allosteric signals from the bound substrate to the RBX2 E2-recruiting region to enable dynamic ubiquitin transfer across the complex [PMID:25654263, PMID:32513959]. The ligase polyubiquitylates free histones H3 and H4 — but not H2A/H2B, nucleosomal histones, or Asf1-bound histones — generating K48 and K63 chains, and does so without requiring an ARIH2 RBR helper ligase, distinguishing it from other CUL5 complexes [PMID:41260500]. Physiologically, ASB9 suppresses ovarian granulosa cell proliferation and apoptosis through MAPK signaling and is itself transcriptionally induced by CLOCK [PMID:34476862, PMID:37280645], restrains spermatogonial stem cell proliferation by degrading HIF1AN [PMID:36683111], and assembles a testis-specific TNP2-targeting CRL whose loss causes TNP2 retention, failed histone-to-protamine transition, and male infertility in mice and humans [PMID:41915740].","teleology":[{"year":2006,"claim":"Established ASB9 as a substrate receptor of an E3 ubiquitin ligase by identifying its first target and showing degradation depends on the SOCS box, defining the bipartite logic of substrate binding versus degradation.","evidence":"Proteomics/pull-down, Co-IP, ubiquitination assay, and cellular protein-level measurement of CKB","pmids":["17148442"],"confidence":"High","gaps":["Did not identify the Cullin/Elongin core recruited by the SOCS box","Physiological context of CKB regulation untested"]},{"year":2010,"claim":"Extended ASB9 targeting to mitochondrial uMtCK and linked its E3 activity to functional consequences for mitochondrial integrity and creatine kinase activity, using a naturally occurring SOCS-deleted variant as a degradation-deficient control.","evidence":"Co-IP, confocal co-localization, ubiquitination and mitochondrial membrane potential/CK activity assays comparing ASB9 vs ASB9ΔSOCS","pmids":["20302626"],"confidence":"High","gaps":["In vivo relevance of mitochondrial CK regulation not addressed","Tissue specificity of uMtCK targeting unknown"]},{"year":2012,"claim":"Defined the structural basis of substrate recognition by solving the ASB9 ankyrin domain and mapping the CKB-binding determinants, showing which repeats and residues mediate target engagement.","evidence":"X-ray crystallography at 2.2 Å, mutagenesis (His103/Phe107), and truncation binding assays","pmids":["22418839"],"confidence":"High","gaps":["Structure of full ligase complex not determined","Did not address how substrate binding couples to ubiquitin transfer"]},{"year":2013,"claim":"Resolved how the unstable ASB9 receptor is stabilized and which Cullin scaffold it recruits, establishing selective CUL5 over CUL2 engagement that defines the complex as an ECS-type CRL.","evidence":"DSF, ITC, native and ion-mobility mass spectrometry, and molecular modeling of ASB9–EloBC–Cul5NTD assembly","pmids":["23837592"],"confidence":"High","gaps":["Did not visualize the catalytic RBX2/E2 arm","Did not connect assembly to ubiquitin transfer geometry"]},{"year":2015,"claim":"Quantified the ASB9–creatine kinase interface and revealed that ASB9 not only targets but partially inhibits CK enzymatic activity, adding a non-degradative regulatory dimension to its function.","evidence":"HDX-MS interface mapping, enzymatic activity assays, mutagenesis, and binding affinity measurements with deletion constructs","pmids":["25654263"],"confidence":"High","gaps":["Whether enzymatic inhibition occurs independent of degradation in cells untested","Stoichiometry within full CRL not addressed"]},{"year":2016,"claim":"Built an atomic model of the full CK-targeting CRL and proposed a dynamic motion-based mechanism allowing ubiquitin to reach the substrate across ~60 Å, framing ligase function as conformationally driven.","evidence":"Integrative modeling, SAXS, and normal-mode analysis","pmids":["27396830"],"confidence":"Medium","gaps":["Computational model not validated by experimental structure at the time","Allosteric coupling not directly demonstrated"]},{"year":2020,"claim":"Provided experimental structures and HDX-MS evidence that ASB9 and CUL5 act as rigid rods hinged at EloB/C, demonstrating long-range allosteric signaling from substrate to the RBX2 catalytic linker and revising the mechanistic model of CUL-E3 ligases.","evidence":"Cryo-EM of CKB–ASB9–ELOB/C and full-length CUL5–RBX2, combined with HDX-MS and integrative modeling","pmids":["32513959"],"confidence":"High","gaps":["Allosteric pathway not dissected by point mutations","Whether allostery generalizes to non-CK substrates untested"]},{"year":2019,"claim":"Identified cell-type-specific ASB9 partners (PAR1, TAOK1, TNFAIP6) in granulosa cells and showed ASB9 suppresses their proliferation, while documenting that the canonical CKB interaction is absent in this context, indicating context-dependent substrate selection.","evidence":"Yeast two-hybrid screen and CRISPR/Cas9 inhibition with proliferation and gene-expression readouts in ovarian granulosa cells","pmids":["30811458"],"confidence":"Medium","gaps":["Y2H partners not confirmed as ubiquitylation substrates","Single lab; reciprocal validation lacking"]},{"year":2021,"claim":"Placed ASB9 upstream as a negative regulator of MAPK signaling in granulosa cells, linking its loss to reduced apoptosis and increased proliferation and connecting it to the ovulatory hCG response in vivo.","evidence":"CRISPR/Cas9 inhibition with apoptosis markers, phospho-MAPK3/1 western blot, and an in vivo hCG model","pmids":["34476862"],"confidence":"Medium","gaps":["Direct substrate linking ASB9 to MAPK not identified","Single lab, single study"]},{"year":2023,"claim":"Defined HIF1AN as the functional ASB9 target in spermatogonial stem cells through a rescue experiment, and showed CLOCK transcriptionally activates ASB9, embedding it in upstream and downstream regulatory circuits in reproductive tissues.","evidence":"Co-IP and HIF1AN re-expression rescue in an SSC line; CLOCK E-box promoter binding and proliferation assays in granulosa cells","pmids":["36683111","37280645"],"confidence":"Medium","gaps":["HIF1AN ubiquitylation/degradation by ASB9 not directly shown in these studies","CKB again absent as a partner, leaving context-specificity rules unresolved"]},{"year":2025,"claim":"Demonstrated that the reconstituted ASB9-CRL5 polyubiquitylates free histones H3/H4 with strict substrate-context specificity and does so without an ARIH2 helper ligase, the first such example for CUL5 complexes.","evidence":"In vitro reconstituted ubiquitylation, binding affinity measurements, lysine-site mapping, chain-type analysis, and nucleosome/Asf1 competition assays","pmids":["41260500"],"confidence":"High","gaps":["Cellular role of free histone degradation by ASB9 not established","Mechanism replacing ARIH2 function not defined"]},{"year":2026,"claim":"Established a physiological, in vivo role for ASB9 in spermiogenesis by showing it assembles a testis-specific TNP2-targeting CRL whose loss causes failed histone-to-protamine transition and male infertility in mice and humans.","evidence":"Co-IP for complex assembly, mouse and human loss-of-function genetics, ubiquitination assays, and spermiogenesis/fertility phenotyping","pmids":["41915740"],"confidence":"High","gaps":["Uses RBX1 rather than RBX2 — how core composition is selected unclear","Relationship between TNP2 degradation and the free-histone ubiquitylation activity not integrated"]},{"year":null,"claim":"How ASB9 selects among its many substrates in a tissue-specific manner, and which physiological program governs whether it targets metabolic enzymes, transcriptional regulators, or chromatin components, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model for context-dependent substrate choice","Cellular significance of free histone ubiquitylation unknown","Determinants of RBX1 vs RBX2 and core variation undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,3,12,14]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,12,14]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,12,14]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[10,14]}],"complexes":["ASB9–ElonginB/C–CUL5–RBX2 CRL","TNP2–ASB9–ElonginB/C–CUL5–RBX1 testis CRL"],"partners":["CKB","CKMT2","ELOB","ELOC","CUL5","HIF1AN","TNP2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96DX5","full_name":"Ankyrin repeat and SOCS box protein 9","aliases":[],"length_aa":294,"mass_kda":31.9,"function":"Substrate-recognition component of a cullin-5-RING E3 ubiquitin-protein ligase complex (ECS complex, also named CRL5 complex), which mediates the ubiquitination and subsequent proteasomal degradation of target proteins (PubMed:25654263, PubMed:33268465). The ECS(ASB9) complex catalyzes ubiquitination of creatine kinases CKB and CKMT1A (PubMed:20302626, PubMed:22418839, PubMed:25654263, PubMed:33268465) Does not interact with the Elongin BC complex, likely to be a negative regulator of isoform 1","subcellular_location":"Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q96DX5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ASB9","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ASB9","total_profiled":1310},"omim":[{"mim_id":"300890","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 9; ASB9","url":"https://www.omim.org/entry/300890"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"kidney","ntpm":15.7},{"tissue":"liver","ntpm":16.3},{"tissue":"testis","ntpm":24.2}],"url":"https://www.proteinatlas.org/search/ASB9"},"hgnc":{"alias_symbol":["DKFZP564L0862","MGC4954","FLJ20636"],"prev_symbol":[]},"alphafold":{"accession":"Q96DX5","domains":[{"cath_id":"1.25.40.20","chopping":"21-256","consensus_level":"medium","plddt":95.0967,"start":21,"end":256},{"cath_id":"1.10.750.20","chopping":"257-294","consensus_level":"medium","plddt":96.8853,"start":257,"end":294}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96DX5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96DX5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96DX5-F1-predicted_aligned_error_v6.png","plddt_mean":91.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ASB9","jax_strain_url":"https://www.jax.org/strain/search?query=ASB9"},"sequence":{"accession":"Q96DX5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96DX5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96DX5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96DX5"}},"corpus_meta":[{"pmid":"20302626","id":"PMC_20302626","title":"ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function.","date":"2010","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/20302626","citation_count":50,"is_preprint":false},{"pmid":"17148442","id":"PMC_17148442","title":"Ankyrin repeat and suppressors of cytokine signaling box protein asb-9 targets creatine kinase B for degradation.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17148442","citation_count":43,"is_preprint":false},{"pmid":"32513959","id":"PMC_32513959","title":"Structure and dynamics of the ASB9 CUL-RING E3 Ligase.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32513959","citation_count":36,"is_preprint":false},{"pmid":"23837592","id":"PMC_23837592","title":"Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin 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substrate-recognition of CKB.","date":"2012","source":"The protein journal","url":"https://pubmed.ncbi.nlm.nih.gov/22418839","citation_count":11,"is_preprint":false},{"pmid":"27396830","id":"PMC_27396830","title":"Model of the Ankyrin and SOCS Box Protein, ASB9, E3 Ligase Reveals a Mechanism for Dynamic Ubiquitin Transfer.","date":"2016","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/27396830","citation_count":8,"is_preprint":false},{"pmid":"37280645","id":"PMC_37280645","title":"CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.","date":"2023","source":"Journal of animal science and biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/37280645","citation_count":7,"is_preprint":false},{"pmid":"40501794","id":"PMC_40501794","title":"The Mechanism of Histone Ubiquitylation by the ASB9-CUL5 Ubiquitin Ligase.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40501794","citation_count":0,"is_preprint":false},{"pmid":"41915740","id":"PMC_41915740","title":"ASB9 promotes ubiquitin-mediated degradation of TNP2 to facilitate histone-to-protamine transition in humans and mice.","date":"2026","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/41915740","citation_count":0,"is_preprint":false},{"pmid":"41260500","id":"PMC_41260500","title":"The Mechanism of Histone Ubiquitylation by the ASB9-CUL5 Ubiquitin Ligase.","date":"2025","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/41260500","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.07.18.24310640","title":"Chromosome X-Wide Common Variant Association Study (XWAS) in Autism Spectrum Disorder","date":"2024-07-18","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.18.24310640","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10523,"output_tokens":4276,"usd":0.047855,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12152,"output_tokens":4373,"usd":0.085042,"stage2_stop_reason":"end_turn"},"total_usd":0.132897,"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\": 2006,\n      \"finding\": \"ASB9 ankyrin repeat domain binds creatine kinase B (CKB) in a SOCS box-independent manner; the interaction promotes polyubiquitylation of CKB and decreases total CKB protein levels, with CKB degradation being primarily SOCS box-dependent, establishing ASB9 as a substrate receptor of an E3 ubiquitin ligase targeting CKB for proteasomal degradation.\",\n      \"method\": \"Proteomics/pull-down to identify interaction, co-immunoprecipitation to confirm binding, ubiquitination assay to measure polyubiquitylation, protein level measurements in cells with ASB9 expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction confirmed by proteomics and Co-IP, functional ubiquitination and degradation assays, independently replicated in multiple subsequent studies\",\n      \"pmids\": [\"17148442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ASB9 interacts with ubiquitous mitochondrial creatine kinase (uMtCK) via its ankyrin repeat domain in a SOCS box-independent manner and co-localizes with uMtCK in mitochondria. Full-length ASB9 (but not the naturally occurring SOCS box-deleted variant ASB9ΔSOCS) induces ubiquitination of uMtCK, causes abnormal mitochondrial structure, decreases mitochondrial membrane potential, reduces creatine kinase activity, and reduces cell growth.\",\n      \"method\": \"Co-immunoprecipitation, co-localization (confocal microscopy), ubiquitination assay, mitochondrial membrane potential assay, creatine kinase activity assay, cell growth assay; comparison of full-length ASB9 vs. ASB9ΔSOCS\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, colocalization, functional assays) in a single study with rigorous domain-deletion controls\",\n      \"pmids\": [\"20302626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Crystal structure of the ASB9-2 isoform (containing one ankyrin repeat domain) at 2.2-Å resolution revealed an arch shape with L-shaped cross-section. Mutagenesis (His103, Phe107) and truncation analysis showed that the first six ankyrin repeats plus the N-terminal region are essential for CKB binding.\",\n      \"method\": \"X-ray crystallography (molecular replacement, 2.2 Å), amino acid substitution analysis, docking experiments, truncation mutant binding assays\",\n      \"journal\": \"The protein journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis in single study; functional validation of binding residues by mutational analysis\",\n      \"pmids\": [\"22418839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ASB9 is unstable alone but forms a stable ternary complex with Elongin B and Elongin C (EloBC), which then binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD, establishing selective Cullin 5 recruitment for the ECS-type CRL complex.\",\n      \"method\": \"Differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization mass spectrometry, ion-mobility mass spectrometry (IM-MS), molecular modeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal biophysical methods (ITC, IM-MS, DSF) providing thermodynamic and structural information in a single rigorous study\",\n      \"pmids\": [\"23837592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"One ASB9 molecule binds to a CK dimer with extremely tight affinity; the N-terminal disordered region and first ankyrin repeat of ASB9 are protected upon binding. ASB9 protects CK residues 182–203 (one side of the active site), and ASB9 N-terminal residues may occupy one CK active site, partially inhibiting CK enzymatic activity.\",\n      \"method\": \"Amide hydrogen-deuterium exchange mass spectrometry (HDXMS), enzymatic activity assays, mutational analysis, molecular docking, binding affinity measurements (deletion constructs)\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — HDXMS interface mapping plus enzymatic assays and mutagenesis in single study with multiple orthogonal methods\",\n      \"pmids\": [\"25654263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Integrative structural modeling combined with small-angle X-ray scattering (SAXS) defined the ASB9–CK interface and constructed an atomic model of the full CK-targeting CRL. Dominant modes of motion in the correctly docked complex permit close approach of ubiquitin to the CK substrate, suggesting a dynamic mechanism for ubiquitin transfer over ~60 Å.\",\n      \"method\": \"Integrative molecular modeling, small-angle X-ray scattering (SAXS), normal-mode analysis\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 (structure) / Weak — single study using computational/modeling approach validated by SAXS shape fitting; no independent replication\",\n      \"pmids\": [\"27396830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Yeast two-hybrid screening of ovarian granulosa cell cDNA library identified PAR1, TAOK1, and TNFAIP6/TSG6 as ASB9 binding partners in granulosa cells. Notably, no interaction was found between ASB9 and CKB in these granulosa cells, in contrast to other cell types.\",\n      \"method\": \"Yeast two-hybrid screening, negative result for ASB9–CKB interaction in granulosa cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid for binding partners, single lab; negative result for CKB is informative but lacks independent confirmation in GCs\",\n      \"pmids\": [\"30811458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CRISPR/Cas9-mediated inhibition of ASB9 in ovarian granulosa cells led to increased granulosa cell proliferation and modulated expression of target genes (PAR1, TAOK1, TNFAIP6), establishing a functional role for ASB9 in suppressing GC proliferation.\",\n      \"method\": \"CRISPR/Cas9 gene inhibition, cell proliferation assay, gene expression analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined cellular phenotype; single lab, single study\",\n      \"pmids\": [\"30811458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cryo-EM structures of the substrate CKB bound to ASB9-ELOB/C and of full-length CUL5 bound to RBX2 revealed that ASB9 and CUL5 behave as rigid rods connected through an ELOB/C hinge. HDX-MS mapped onto the full structural model showed long-range allosteric communication from the substrate through CUL5 to the RBX2 flexible linker, proposing a revised allosteric mechanism for CUL-E3 ligase function.\",\n      \"method\": \"Cryo-EM structure determination, hydrogen-deuterium exchange mass spectrometry (HDX-MS), integrative structural modeling\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structures of substrate-bound complex and full-length CUL5 combined with HDX-MS; multiple orthogonal methods in single study revealing allosteric mechanism\",\n      \"pmids\": [\"32513959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CRISPR/Cas9-mediated inhibition of ASB9 in ovarian granulosa cells increased GC number, decreased caspase-3/7 activity, CASP3 expression, and BAX/BCL2 ratio (reduced apoptosis), and increased pMAPK3/1 phosphorylation; conversely, ASB9 induction post-hCG was concomitant with decreased pMAPK3/1 levels, placing ASB9 upstream as a negative regulator of MAPK signaling in GCs.\",\n      \"method\": \"CRISPR/Cas9 inhibition, cell proliferation and apoptosis assays (caspase3/7 activity, CASP3 and BAX/BCL2 expression), phospho-MAPK3/1 western blot, in vivo hCG model\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with multiple cellular phenotype readouts and in vivo correlation; single lab, single study\",\n      \"pmids\": [\"34476862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ASB9 interacts with hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) in human spermatogonial stem cells (SSC line), as confirmed by protein immunoprecipitation. ASB9 overexpression inhibited SSC proliferation and increased apoptosis; re-expression of HIF1AN reversed these effects, establishing HIF1AN as the functional target of ASB9 in SSCs. CKB was tested but did not show direct interaction with ASB9 in this cell type.\",\n      \"method\": \"Protein immunoprecipitation, ASB9 overexpression in SSC line, cell proliferation and apoptosis assays, HIF1AN re-expression rescue experiment\",\n      \"journal\": \"Biological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP plus rescue experiment establish substrate identity and functional consequence; single lab, single study\",\n      \"pmids\": [\"36683111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CLOCK transcription factor binds to the E-box element in the ASB9 promoter and increases ASB9 expression, which in turn inhibits porcine granulosa cell proliferation, placing CLOCK upstream of ASB9 in a transcriptional regulatory pathway.\",\n      \"method\": \"CLOCK overexpression/knockdown, ASB9 promoter E-box binding assay (chromatin immunoprecipitation or promoter-reporter implied), cell proliferation assay\",\n      \"journal\": \"Journal of animal science and biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct promoter binding and downstream proliferation phenotype; single lab, single study\",\n      \"pmids\": [\"37280645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The ASB9-CUL5 E3 ligase polyubiquitylates free histones H3 and H4 (but not H2A/H2B, or histones in nucleosomes or complexed with chaperone Asf1), generating K48 and K63 polyubiquitin chains. The ligase-histone interaction is highly electrostatic; neddylated ASB9-CRL5 binds with highest affinity. Crucially, this ubiquitylation does not require the ring-between-ring ligase ARIH2, representing the first example of CUL5-mediated ubiquitylation without a RBR helper ligase.\",\n      \"method\": \"In vitro ubiquitylation assays with reconstituted ASB9-CRL5, binding affinity measurements, lysine site mapping by mass spectrometry, nucleosome and Asf1-complex competition assays, polyubiquitin chain-type analysis\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro ubiquitylation with multiple substrate/condition controls, mechanistic insight into substrate specificity and ARIH2 independence; peer-reviewed publication\",\n      \"pmids\": [\"41260500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ASB9-CUL5 E3 ligase polyubiquitylates free histones H3 and H4 with substrate specificity (H2A and H2B are not polyubiquitylated); histones in nucleosomes or bound by chaperone Asf1 are not ubiquitylated. This represents the first CUL5-mediated ubiquitylation not requiring an ARIH2 RBR helper ligase. (Preprint version of the same study as PMID:41260500.)\",\n      \"method\": \"In vitro reconstituted ubiquitylation assay, binding experiments, polyubiquitin chain-linkage analysis, substrate context experiments (nucleosome, Asf1 complex)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — same rigorous reconstitution data as peer-reviewed paper; preprint version; confidence not raised further as it is the same dataset\",\n      \"pmids\": [\"40501794\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ASB9 assembles a testis-specific CRL complex (TNP2-ASB9-ELOB/C-CUL5-RBX1) that mediates ubiquitin-dependent degradation of transition protein TNP2 during spermiogenesis. ASB9 deficiency in humans and mice causes TNP2 retention, failure of the histone-to-protamine transition, sperm head malformation, and male infertility.\",\n      \"method\": \"Co-immunoprecipitation to identify complex assembly, genetic knockout (mouse and human loss-of-function), ubiquitination assays, spermiogenesis phenotype analysis (sperm morphology, fertility)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — complex assembly by Co-IP, in vivo knockout phenotype in two species, mechanistic rescue implied; multiple orthogonal methods\",\n      \"pmids\": [\"41915740\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ASB9 is the substrate-recognition subunit of an ECS-type Cullin 5 RING E3 ubiquitin ligase complex (ASB9–ElonginB/C–CUL5–RBX2) that uses its ankyrin repeat domain to bind substrates—including creatine kinase B (CKB), ubiquitous mitochondrial creatine kinase (uMtCK), HIF1AN, transition protein TNP2, and free histones H3/H4—and its SOCS box to recruit ElonginB/C and CUL5, driving K48/K63 polyubiquitylation and proteasomal degradation of these targets; structural and HDX-MS studies reveal that ASB9 and CUL5 act as rigid rods connected through an ElonginB/C hinge that transmits long-range allosteric signals from the bound substrate to the RBX2 E2-recruiting domain, facilitating dynamic ubiquitin transfer, while its physiological roles include inhibiting mitochondrial function and creatine kinase activity, regulating spermatogonial stem cell proliferation/apoptosis via HIF1AN, enabling the histone-to-protamine transition in spermiogenesis via TNP2 degradation, degrading free histones H3/H4 (independently of ARIH2, unlike other CUL5 complexes), and suppressing granulosa cell proliferation through MAPK signaling modulation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ASB9 is the substrate-recognition subunit of an ECS-type Cullin 5 RING E3 ubiquitin ligase that selects diverse substrates through its ankyrin repeat domain and recruits the catalytic core via its SOCS box, thereby controlling the proteasomal turnover of metabolic, transcriptional, and chromatin-associated targets [#0, #3]. The ankyrin repeats engage substrates such as cytosolic creatine kinase B (CKB) and ubiquitous mitochondrial creatine kinase (uMtCK) in a SOCS box-independent manner, while the SOCS box drives substrate degradation; full-length ASB9 ubiquitylates uMtCK, disrupts mitochondrial structure, lowers membrane potential, and reduces creatine kinase activity [#0, #1]. ASB9 is unstable alone but forms a stable ternary complex with Elongin B/C that binds selectively to the Cullin 5 N-terminal domain, assembling the ASB9–EloBC–CUL5–RBX2 ligase [#3]. Structural and HDX-MS studies show that ASB9 binds creatine kinase with very high affinity and that ASB9 and CUL5 behave as rigid rods joined by an EloB/C hinge, transmitting long-range allosteric signals from the bound substrate to the RBX2 E2-recruiting region to enable dynamic ubiquitin transfer across the complex [#4, #8]. The ligase polyubiquitylates free histones H3 and H4 — but not H2A/H2B, nucleosomal histones, or Asf1-bound histones — generating K48 and K63 chains, and does so without requiring an ARIH2 RBR helper ligase, distinguishing it from other CUL5 complexes [#12]. Physiologically, ASB9 suppresses ovarian granulosa cell proliferation and apoptosis through MAPK signaling and is itself transcriptionally induced by CLOCK [#9, #11], restrains spermatogonial stem cell proliferation by degrading HIF1AN [#10], and assembles a testis-specific TNP2-targeting CRL whose loss causes TNP2 retention, failed histone-to-protamine transition, and male infertility in mice and humans [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established ASB9 as a substrate receptor of an E3 ubiquitin ligase by identifying its first target and showing degradation depends on the SOCS box, defining the bipartite logic of substrate binding versus degradation.\",\n      \"evidence\": \"Proteomics/pull-down, Co-IP, ubiquitination assay, and cellular protein-level measurement of CKB\",\n      \"pmids\": [\"17148442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the Cullin/Elongin core recruited by the SOCS box\", \"Physiological context of CKB regulation untested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended ASB9 targeting to mitochondrial uMtCK and linked its E3 activity to functional consequences for mitochondrial integrity and creatine kinase activity, using a naturally occurring SOCS-deleted variant as a degradation-deficient control.\",\n      \"evidence\": \"Co-IP, confocal co-localization, ubiquitination and mitochondrial membrane potential/CK activity assays comparing ASB9 vs ASB9ΔSOCS\",\n      \"pmids\": [\"20302626\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of mitochondrial CK regulation not addressed\", \"Tissue specificity of uMtCK targeting unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the structural basis of substrate recognition by solving the ASB9 ankyrin domain and mapping the CKB-binding determinants, showing which repeats and residues mediate target engagement.\",\n      \"evidence\": \"X-ray crystallography at 2.2 Å, mutagenesis (His103/Phe107), and truncation binding assays\",\n      \"pmids\": [\"22418839\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full ligase complex not determined\", \"Did not address how substrate binding couples to ubiquitin transfer\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved how the unstable ASB9 receptor is stabilized and which Cullin scaffold it recruits, establishing selective CUL5 over CUL2 engagement that defines the complex as an ECS-type CRL.\",\n      \"evidence\": \"DSF, ITC, native and ion-mobility mass spectrometry, and molecular modeling of ASB9–EloBC–Cul5NTD assembly\",\n      \"pmids\": [\"23837592\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not visualize the catalytic RBX2/E2 arm\", \"Did not connect assembly to ubiquitin transfer geometry\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Quantified the ASB9–creatine kinase interface and revealed that ASB9 not only targets but partially inhibits CK enzymatic activity, adding a non-degradative regulatory dimension to its function.\",\n      \"evidence\": \"HDX-MS interface mapping, enzymatic activity assays, mutagenesis, and binding affinity measurements with deletion constructs\",\n      \"pmids\": [\"25654263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether enzymatic inhibition occurs independent of degradation in cells untested\", \"Stoichiometry within full CRL not addressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Built an atomic model of the full CK-targeting CRL and proposed a dynamic motion-based mechanism allowing ubiquitin to reach the substrate across ~60 Å, framing ligase function as conformationally driven.\",\n      \"evidence\": \"Integrative modeling, SAXS, and normal-mode analysis\",\n      \"pmids\": [\"27396830\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Computational model not validated by experimental structure at the time\", \"Allosteric coupling not directly demonstrated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided experimental structures and HDX-MS evidence that ASB9 and CUL5 act as rigid rods hinged at EloB/C, demonstrating long-range allosteric signaling from substrate to the RBX2 catalytic linker and revising the mechanistic model of CUL-E3 ligases.\",\n      \"evidence\": \"Cryo-EM of CKB–ASB9–ELOB/C and full-length CUL5–RBX2, combined with HDX-MS and integrative modeling\",\n      \"pmids\": [\"32513959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Allosteric pathway not dissected by point mutations\", \"Whether allostery generalizes to non-CK substrates untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified cell-type-specific ASB9 partners (PAR1, TAOK1, TNFAIP6) in granulosa cells and showed ASB9 suppresses their proliferation, while documenting that the canonical CKB interaction is absent in this context, indicating context-dependent substrate selection.\",\n      \"evidence\": \"Yeast two-hybrid screen and CRISPR/Cas9 inhibition with proliferation and gene-expression readouts in ovarian granulosa cells\",\n      \"pmids\": [\"30811458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Y2H partners not confirmed as ubiquitylation substrates\", \"Single lab; reciprocal validation lacking\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed ASB9 upstream as a negative regulator of MAPK signaling in granulosa cells, linking its loss to reduced apoptosis and increased proliferation and connecting it to the ovulatory hCG response in vivo.\",\n      \"evidence\": \"CRISPR/Cas9 inhibition with apoptosis markers, phospho-MAPK3/1 western blot, and an in vivo hCG model\",\n      \"pmids\": [\"34476862\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct substrate linking ASB9 to MAPK not identified\", \"Single lab, single study\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined HIF1AN as the functional ASB9 target in spermatogonial stem cells through a rescue experiment, and showed CLOCK transcriptionally activates ASB9, embedding it in upstream and downstream regulatory circuits in reproductive tissues.\",\n      \"evidence\": \"Co-IP and HIF1AN re-expression rescue in an SSC line; CLOCK E-box promoter binding and proliferation assays in granulosa cells\",\n      \"pmids\": [\"36683111\", \"37280645\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"HIF1AN ubiquitylation/degradation by ASB9 not directly shown in these studies\", \"CKB again absent as a partner, leaving context-specificity rules unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that the reconstituted ASB9-CRL5 polyubiquitylates free histones H3/H4 with strict substrate-context specificity and does so without an ARIH2 helper ligase, the first such example for CUL5 complexes.\",\n      \"evidence\": \"In vitro reconstituted ubiquitylation, binding affinity measurements, lysine-site mapping, chain-type analysis, and nucleosome/Asf1 competition assays\",\n      \"pmids\": [\"41260500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular role of free histone degradation by ASB9 not established\", \"Mechanism replacing ARIH2 function not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established a physiological, in vivo role for ASB9 in spermiogenesis by showing it assembles a testis-specific TNP2-targeting CRL whose loss causes failed histone-to-protamine transition and male infertility in mice and humans.\",\n      \"evidence\": \"Co-IP for complex assembly, mouse and human loss-of-function genetics, ubiquitination assays, and spermiogenesis/fertility phenotyping\",\n      \"pmids\": [\"41915740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Uses RBX1 rather than RBX2 — how core composition is selected unclear\", \"Relationship between TNP2 degradation and the free-histone ubiquitylation activity not integrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ASB9 selects among its many substrates in a tissue-specific manner, and which physiological program governs whether it targets metabolic enzymes, transcriptional regulators, or chromatin components, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model for context-dependent substrate choice\", \"Cellular significance of free histone ubiquitylation unknown\", \"Determinants of RBX1 vs RBX2 and core variation undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 3, 12, 14]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 12, 14]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 12, 14]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [10, 14]}\n    ],\n    \"complexes\": [\n      \"ASB9–ElonginB/C–CUL5–RBX2 CRL\",\n      \"TNP2–ASB9–ElonginB/C–CUL5–RBX1 testis CRL\"\n    ],\n    \"partners\": [\n      \"CKB\",\n      \"CKMT2\",\n      \"ELOB\",\n      \"ELOC\",\n      \"CUL5\",\n      \"HIF1AN\",\n      \"TNP2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}