{"gene":"OTUD7A","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":2018,"finding":"OTUD7A localizes to dendritic and spine compartments in cortical neurons, and reduced OTUD7A levels contribute to dendritic spine and dendrite outgrowth deficits in 15q13.3 microdeletion mouse models.","method":"Heterozygous deletion mouse model (Df(h15q13)/+), subcellular localization imaging, morphological analysis of cortical neurons","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — replicated across two independent mouse/cell studies in the same year with consistent findings","pmids":["29395074"],"is_preprint":false},{"year":2018,"finding":"OTUD7A localizes to dendritic spines; Otud7a knockout mice display decreased dendritic spine density and reduced frequency of miniature excitatory postsynaptic currents (mEPSCs) in the frontal cortex, demonstrating a role in regulating dendritic spine density and glutamatergic synaptic transmission.","method":"Otud7a knockout mouse model, immunofluorescence localization, electrophysiology (mEPSC recording), spine density quantification","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — clean KO with multiple orthogonal phenotypic readouts (morphology + electrophysiology), replicated across two labs","pmids":["29395075"],"is_preprint":false},{"year":2021,"finding":"OTUD7A acts as the deubiquitinase that deubiquitinates and stabilizes the EWS-FLI1 oncoprotein in Ewing sarcoma, opposing SPOP-mediated ubiquitin-dependent degradation; depletion of OTUD7A reduces EWS-FLI1 protein abundance and impedes Ewing sarcoma growth in vitro and in vivo.","method":"Co-immunoprecipitation, in vivo deubiquitination assay, OTUD7A knockdown in cell lines, xenograft mouse model, AI-based virtual drug screen with catalytic inhibitor 7Ai","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical deubiquitination assay combined with genetic KD, in vivo xenograft, and pharmacological inhibitor validation","pmids":["34060252"],"is_preprint":false},{"year":2020,"finding":"A homozygous missense variant p.(Leu233Phe) in the OTU catalytic domain of OTUD7A leads to proteasome complex formation and function impairment, as demonstrated in patient-derived fibroblasts and OTUD7A knockout HAP1 cells.","method":"Trio exome sequencing, biochemical proteasome activity assays in patient fibroblasts and OTUD7A knockout HAP1 cell line","journal":"Clinical genetics","confidence":"Medium","confidence_rationale":"Tier 2 — functional assay in patient cells and KO cell line, single study","pmids":["31997314"],"is_preprint":false},{"year":2023,"finding":"OTUD7A interacts with Ankyrin-G (Ank3) and Ankyrin-B (Ank2); the epilepsy-associated L233F variant disrupts these interactions. OTUD7A-dependent regulation prevents polyubiquitination and degradation of Ankyrin-G, maintaining its levels at the axon initial segment and in dendritic spine nanodomains. Restoring OTUD7A or Ankyrin-G expression reverses neuronal abnormalities in 15q13.3 microdeletion neurons.","method":"Neuron-specific proximity-labeling proteomics (BioID2), co-immunoprecipitation, structured illumination microscopy, polyubiquitination assays, patient iPSC-derived neurons, rescue experiments","journal":"Molecular psychiatry","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including proteomics, biochemical ubiquitination assays, super-resolution microscopy, and functional rescue","pmids":["36604605"],"is_preprint":false},{"year":2022,"finding":"OTUD7A loss-of-function in human iPSC-derived neurons reduces dendritic complexity, density of synaptic proteins GluA1 and PSD-95, and neuronal network activity; transcriptomic analysis shows downregulated genes enriched for synapse development and function pathways.","method":"CRISPR-Cas9 engineering of LoF mutation in hiPSC-derived neurons, immunofluorescence, multi-electrode array, transcriptomic analysis","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — clean CRISPR KO in human neurons with multiple phenotypic readouts, single study","pmids":["35931052"],"is_preprint":false},{"year":2025,"finding":"OTUD7a directly interacts with TAK1 (TGF-β-activated kinase 1), inhibits its ubiquitination-mediated degradation (deubiquitinase activity), and subsequently increases phosphorylation of TAK1 and its downstream targets JNK/P38, promoting pathological cardiac hypertrophy. Cardiac-specific OTUD7a knockout alleviates hypertrophy while overexpression aggravates it.","method":"Cardiac-specific knockout mice, AAV9-mediated overexpression, co-immunoprecipitation, ubiquitination assays, RNA-sequencing and interactome analysis, TAK1 inhibitor (5Z-7-oxozeaenol) rescue, TAK1 overexpression epistasis","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical deubiquitination assay plus genetic KO/OE in vivo, epistasis with TAK1 inhibitor and overexpression, multiple orthogonal methods","pmids":["41268652"],"is_preprint":false},{"year":2025,"finding":"OTUD7A (Cezanne-2) employs a modified cysteine protease catalytic mechanism involving a Cys210/His367 dyad, with an oxyanion hole in the C-loop and polarization of His367 via a conserved water bridge with Glu173; it selectively cleaves Lys11-linked polyubiquitin chains via isopeptide bond cleavage through a tetrahedral oxyanion intermediate.","method":"Molecular dynamics simulations (12 µs), QM/MM calculations, structural analysis of diubiquitin-Cez2 complex","journal":"Journal of chemical information and modeling","confidence":"Medium","confidence_rationale":"Tier 1 computational — extensive MD+QM/MM providing mechanistic detail, but no experimental mutagenesis validation in this study","pmids":["39782030"],"is_preprint":false},{"year":2013,"finding":"Cezanne2 (OTUD7A) interacts with TRAF6 and cleaves polyubiquitin chains from TRAF6 substrates, thereby modulating NF-κB signaling and downstream MMP2, MMP9, and ICAM1 levels.","method":"Co-immunoprecipitation, in vivo deubiquitination assay, reporter gene assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP plus in vivo deubiquitination assay, single study","pmids":["23792447"],"is_preprint":false},{"year":2025,"finding":"OTUD7A deubiquitinase regulates KDM5B stability in KRAS-mutant lung adenocarcinoma; OTUD7A inhibition reduces KDM5B expression, increases H4K20me3, downregulates GABPA, increases mitochondrial ROS production, and induces ferroptosis.","method":"KRAS-mutant LUAD cell lines, mouse models, organoid models, cisplatin combination treatment, molecular pathway analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 — genetic depletion with defined downstream pathway, single study without direct biochemical deubiquitination assay for KDM5B","pmids":["41422226"],"is_preprint":false}],"current_model":"OTUD7A is an OTU-family deubiquitinase (catalytic Cys/His dyad, Lys11-linkage selective) that stabilizes multiple substrates—including EWS-FLI1, TAK1, Ankyrin-G/B, and KDM5B—by removing polyubiquitin chains to prevent their proteasomal degradation; in neurons it localizes to dendritic spines and the axon initial segment where it maintains Ankyrin-G levels and is required for normal spine density, glutamatergic transmission, and neuronal network activity, while loss-of-function underlies the neurodevelopmental phenotypes of 15q13.3 microdeletion syndrome."},"narrative":{"teleology":[{"year":2013,"claim":"The initial mechanistic question—does OTUD7A function as a deubiquitinase with defined signaling targets?—was addressed by showing it interacts with TRAF6, removes its polyubiquitin chains, and thereby modulates NF-κB signaling.","evidence":"Co-immunoprecipitation, in vivo deubiquitination assay, and NF-κB reporter assay in cell lines","pmids":["23792447"],"confidence":"Medium","gaps":["Single study without reciprocal validation of the TRAF6 interaction by orthogonal methods","Linkage-type specificity of deubiquitination not determined","In vivo physiological relevance not tested"]},{"year":2018,"claim":"Whether OTUD7A has a physiological role in the nervous system was established by two independent studies showing it localizes to dendritic spines and is required for normal spine density, dendrite outgrowth, and glutamatergic synaptic transmission in the cortex, directly linking it to 15q13.3 microdeletion syndrome phenotypes.","evidence":"Heterozygous 15q13.3 deletion mouse model and Otud7a-knockout mice with immunofluorescence, spine morphometry, and mEPSC electrophysiology","pmids":["29395074","29395075"],"confidence":"High","gaps":["Molecular substrates in neurons not yet identified","Whether the catalytic activity or a scaffolding role mediates the synaptic phenotype was unknown","Human neuronal validation not performed"]},{"year":2020,"claim":"A human disease-causing mechanism was clarified when a homozygous p.Leu233Phe variant in the OTU catalytic domain was shown to impair proteasome complex formation and function, linking a specific catalytic-domain mutation to neurological disease.","evidence":"Trio exome sequencing, proteasome activity assays in patient fibroblasts and OTUD7A-knockout HAP1 cells","pmids":["31997314"],"confidence":"Medium","gaps":["Single family; replication in additional patients needed","How a deubiquitinase mutation impairs the proteasome complex remains mechanistically unclear","Whether L233F abolishes deubiquitinase catalytic activity per se was not directly tested"]},{"year":2021,"claim":"The question of whether OTUD7A stabilizes oncoproteins was answered by demonstrating it deubiquitinates and stabilizes EWS-FLI1, opposing SPOP-mediated degradation; OTUD7A depletion reduced EWS-FLI1 levels and suppressed Ewing sarcoma growth in vivo.","evidence":"Co-IP, in vivo deubiquitination assay, knockdown in Ewing sarcoma cell lines, xenograft model, catalytic inhibitor 7Ai","pmids":["34060252"],"confidence":"High","gaps":["Whether 7Ai is specific to OTUD7A versus other OTU-family members not fully resolved","Structural basis of EWS-FLI1 recognition unknown","Linkage specificity for EWS-FLI1 deubiquitination not determined"]},{"year":2022,"claim":"Translation to human neurons was achieved by showing that CRISPR-engineered OTUD7A loss-of-function in iPSC-derived neurons reduces dendritic complexity, synaptic protein levels (GluA1, PSD-95), and network activity, with transcriptomics confirming enrichment for synapse development pathways.","evidence":"CRISPR-Cas9 knockout in human iPSC-derived neurons, immunofluorescence, multi-electrode array, transcriptomic analysis","pmids":["35931052"],"confidence":"Medium","gaps":["Single isogenic pair; additional clonal lines not reported","Direct OTUD7A substrates responsible for the transcriptomic changes not identified","Whether phenotypes are rescue-able by catalytic-active OTUD7A was not tested"]},{"year":2023,"claim":"The critical neuronal substrate question was resolved by identifying Ankyrin-G and Ankyrin-B as direct OTUD7A interactors; OTUD7A prevents Ankyrin-G polyubiquitination and degradation at the axon initial segment and dendritic spine nanodomains, and the L233F epilepsy variant disrupts these interactions. Rescue of Ankyrin-G reversed neuronal deficits in 15q13.3 microdeletion neurons.","evidence":"BioID2 proximity-labeling proteomics, co-IP, structured illumination microscopy, polyubiquitination assays, patient iPSC-derived neurons with rescue experiments","pmids":["36604605"],"confidence":"High","gaps":["Whether additional neuronal substrates contribute to the phenotype beyond Ankyrins is unknown","The ubiquitin linkage type on Ankyrin-G cleaved by OTUD7A was not determined","In vivo rescue in mouse models not performed"]},{"year":2025,"claim":"A cardiac role was established: OTUD7A deubiquitinates and stabilizes TAK1, activating JNK/P38 signaling to drive pathological cardiac hypertrophy, as demonstrated by cardiac-specific knockout (protective) and overexpression (aggravating) with TAK1-dependent epistasis.","evidence":"Cardiac-specific Otud7a knockout and AAV9-mediated overexpression in mice, co-IP, ubiquitination assays, TAK1 inhibitor and overexpression epistasis","pmids":["41268652"],"confidence":"High","gaps":["Whether OTUD7A regulation of TAK1 extends to inflammatory contexts beyond the heart is unknown","The ubiquitin ligase opposing OTUD7A on TAK1 was not identified","Whether the Lys11-linkage preference applies to TAK1 deubiquitination specifically was not tested"]},{"year":2025,"claim":"The catalytic mechanism was elucidated at atomic resolution: OTUD7A uses a Cys210/His367 dyad (not a classical catalytic triad), with His367 polarized through a water bridge to Glu173 and an oxyanion hole in the C-loop, selectively cleaving Lys11-linked polyubiquitin via a tetrahedral intermediate.","evidence":"12 µs molecular dynamics simulations and QM/MM calculations on the diubiquitin–Cez2 complex","pmids":["39782030"],"confidence":"Medium","gaps":["Computational predictions lack experimental mutagenesis validation","Whether the Lys11 selectivity mechanism applies to all substrates in cells is untested","Crystal structure of a substrate-bound OTUD7A complex not available"]},{"year":2025,"claim":"An oncogenic substrate-stabilization role in lung cancer was demonstrated: OTUD7A stabilizes the histone demethylase KDM5B in KRAS-mutant lung adenocarcinoma, and its inhibition triggers H4K20me3 increase, GABPA downregulation, mitochondrial ROS accumulation, and ferroptosis.","evidence":"KRAS-mutant LUAD cell lines, mouse and organoid models, cisplatin combination treatment, pathway analysis","pmids":["41422226"],"confidence":"Medium","gaps":["Direct biochemical deubiquitination assay for KDM5B by OTUD7A not shown","Whether OTUD7A inhibition sensitizes non-KRAS-mutant tumors is unknown","The ferroptosis pathway link requires independent replication"]},{"year":null,"claim":"Key unresolved questions include the full substrate repertoire of OTUD7A in neurons, whether its Lys11-linkage selectivity governs all physiological substrates, and the structural basis for substrate recognition.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal structure of full-length OTUD7A with a bound substrate","Comprehensive substrate identification across tissues not performed","Therapeutic window for OTUD7A inhibition in cancer versus neurological toxicity unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[2,4,6,7,8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,4,6,8]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,1,4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,4,6,7,8]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,1,4,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,9]}],"complexes":[],"partners":["ANK3","ANK2","MAP3K7","TRAF6","EWSR1-FLI1","KDM5B"],"other_free_text":[]},"mechanistic_narrative":"OTUD7A is an OTU-family deubiquitinase that employs a Cys210/His367 catalytic dyad with selectivity for Lys11-linked polyubiquitin chains, stabilizing diverse substrates—including Ankyrin-G, TAK1, EWS-FLI1, TRAF6, and KDM5B—by opposing their ubiquitin-dependent proteasomal degradation [PMID:39782030, PMID:34060252, PMID:36604605, PMID:41268652]. In cortical neurons, OTUD7A localizes to dendritic spines and the axon initial segment, where it deubiquitinates and maintains Ankyrin-G levels; loss of OTUD7A reduces dendritic spine density, decreases glutamatergic synaptic transmission, and disrupts neuronal network activity, establishing it as a critical regulator of excitatory synapse structure and function [PMID:29395075, PMID:36604605, PMID:35931052]. Loss-of-function mutations in OTUD7A, including the catalytic-domain variant p.Leu233Phe and haploinsufficiency from 15q13.3 microdeletions, cause neurodevelopmental phenotypes including epilepsy and intellectual disability [PMID:29395074, PMID:31997314, PMID:36604605]. Outside the nervous system, OTUD7A stabilizes TAK1 to promote JNK/P38 signaling in pathological cardiac hypertrophy and stabilizes EWS-FLI1 to sustain Ewing sarcoma growth [PMID:41268652, PMID:34060252]."},"prefetch_data":{"uniprot":{"accession":"Q8TE49","full_name":"OTU domain-containing protein 7A","aliases":["Zinc finger protein Cezanne 2"],"length_aa":926,"mass_kda":100.7,"function":"Deubiquitinase, which cleaves 'Lys-11'-linked polyubiquitin chains. Might be required for PA28-20S proteasome assembly (Probable)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q8TE49/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/OTUD7A","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/OTUD7A","total_profiled":1310},"omim":[{"mim_id":"620790","title":"NEURODEVELOPMENTAL DISORDER WITH HYPOTONIA AND SEIZURES; NEDHS","url":"https://www.omim.org/entry/620790"},{"mim_id":"612626","title":"CHROMOSOME 15q26-qter DELETION SYNDROME","url":"https://www.omim.org/entry/612626"},{"mim_id":"612024","title":"OTU DOMAIN-CONTAINING PROTEIN 7A; OTUD7A","url":"https://www.omim.org/entry/612024"},{"mim_id":"612001","title":"CHROMOSOME 15q13.3 DELETION SYNDROME","url":"https://www.omim.org/entry/612001"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":9.8}],"url":"https://www.proteinatlas.org/search/OTUD7A"},"hgnc":{"alias_symbol":["CEZANNE2"],"prev_symbol":["C15orf16","OTUD7"]},"alphafold":{"accession":"Q8TE49","domains":[{"cath_id":"3.90.70.80","chopping":"142-259_267-278_301-447_640-667","consensus_level":"high","plddt":91.5254,"start":142,"end":667},{"cath_id":"-","chopping":"890-924","consensus_level":"high","plddt":86.6509,"start":890,"end":924}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TE49","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TE49-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TE49-F1-predicted_aligned_error_v6.png","plddt_mean":64.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=OTUD7A","jax_strain_url":"https://www.jax.org/strain/search?query=OTUD7A"},"sequence":{"accession":"Q8TE49","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TE49.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TE49/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TE49"}},"corpus_meta":[{"pmid":"29395074","id":"PMC_29395074","title":"OTUD7A Regulates Neurodevelopmental Phenotypes in the 15q13.3 Microdeletion Syndrome.","date":"2018","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29395074","citation_count":79,"is_preprint":false},{"pmid":"34060252","id":"PMC_34060252","title":"SPOP and OTUD7A Control EWS-FLI1 Protein Stability to Govern Ewing Sarcoma Growth.","date":"2021","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/34060252","citation_count":59,"is_preprint":false},{"pmid":"29395075","id":"PMC_29395075","title":"Otud7a Knockout Mice Recapitulate Many Neurological Features of 15q13.3 Microdeletion Syndrome.","date":"2018","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29395075","citation_count":59,"is_preprint":false},{"pmid":"23792447","id":"PMC_23792447","title":"Snail1-dependent transcriptional repression of Cezanne2 in hepatocellular carcinoma.","date":"2013","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/23792447","citation_count":30,"is_preprint":false},{"pmid":"31997314","id":"PMC_31997314","title":"Report of the first patient with a homozygous OTUD7A variant responsible for epileptic encephalopathy and related proteasome dysfunction.","date":"2020","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31997314","citation_count":23,"is_preprint":false},{"pmid":"36604605","id":"PMC_36604605","title":"Impaired OTUD7A-dependent Ankyrin regulation mediates neuronal dysfunction in mouse and human models of the 15q13.3 microdeletion syndrome.","date":"2023","source":"Molecular psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/36604605","citation_count":18,"is_preprint":false},{"pmid":"33830472","id":"PMC_33830472","title":"CircRNA_OTUD7A upregulates FOXP1 expression to facilitate the progression of diffuse large B-cell lymphoma via acting as a sponge of miR-431-5p.","date":"2021","source":"Genes & genomics","url":"https://pubmed.ncbi.nlm.nih.gov/33830472","citation_count":17,"is_preprint":false},{"pmid":"35931052","id":"PMC_35931052","title":"Loss of function of OTUD7A in the schizophrenia- associated 15q13.3 deletion impairs synapse development and function in human neurons.","date":"2022","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35931052","citation_count":16,"is_preprint":false},{"pmid":"41422226","id":"PMC_41422226","title":"Suppressing the OTUD7A/KDM5B/GABPA axis enhances the sensitivity of cisplatin through inducing ferroptosis in KRAS-mutant LUAD.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/41422226","citation_count":1,"is_preprint":false},{"pmid":"40049827","id":"PMC_40049827","title":"Genetic counseling of prenatally detected familial 15q13.2q13.3 microdeletion encompassing CHRNA7 and OTUD7A with asymptomatic carriers in the family.","date":"2025","source":"Taiwanese journal of obstetrics & gynecology","url":"https://pubmed.ncbi.nlm.nih.gov/40049827","citation_count":1,"is_preprint":false},{"pmid":"41268652","id":"PMC_41268652","title":"OTUD7a Accelerates Pathological Cardiac Hypertrophy via TAK1 Activation.","date":"2025","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/41268652","citation_count":0,"is_preprint":false},{"pmid":"39782030","id":"PMC_39782030","title":"Activation and Reactivity of the Deubiquitinylase OTU Cezanne-2 from MD Simulations and QM/MM Calculations.","date":"2025","source":"Journal of chemical information and modeling","url":"https://pubmed.ncbi.nlm.nih.gov/39782030","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8274,"output_tokens":2549,"usd":0.031529},"stage2":{"model":"claude-opus-4-6","input_tokens":5882,"output_tokens":2970,"usd":0.15549},"total_usd":0.187019,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"OTUD7A localizes to dendritic and spine compartments in cortical neurons, and reduced OTUD7A levels contribute to dendritic spine and dendrite outgrowth deficits in 15q13.3 microdeletion mouse models.\",\n      \"method\": \"Heterozygous deletion mouse model (Df(h15q13)/+), subcellular localization imaging, morphological analysis of cortical neurons\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — replicated across two independent mouse/cell studies in the same year with consistent findings\",\n      \"pmids\": [\"29395074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OTUD7A localizes to dendritic spines; Otud7a knockout mice display decreased dendritic spine density and reduced frequency of miniature excitatory postsynaptic currents (mEPSCs) in the frontal cortex, demonstrating a role in regulating dendritic spine density and glutamatergic synaptic transmission.\",\n      \"method\": \"Otud7a knockout mouse model, immunofluorescence localization, electrophysiology (mEPSC recording), spine density quantification\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple orthogonal phenotypic readouts (morphology + electrophysiology), replicated across two labs\",\n      \"pmids\": [\"29395075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUD7A acts as the deubiquitinase that deubiquitinates and stabilizes the EWS-FLI1 oncoprotein in Ewing sarcoma, opposing SPOP-mediated ubiquitin-dependent degradation; depletion of OTUD7A reduces EWS-FLI1 protein abundance and impedes Ewing sarcoma growth in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assay, OTUD7A knockdown in cell lines, xenograft mouse model, AI-based virtual drug screen with catalytic inhibitor 7Ai\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical deubiquitination assay combined with genetic KD, in vivo xenograft, and pharmacological inhibitor validation\",\n      \"pmids\": [\"34060252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A homozygous missense variant p.(Leu233Phe) in the OTU catalytic domain of OTUD7A leads to proteasome complex formation and function impairment, as demonstrated in patient-derived fibroblasts and OTUD7A knockout HAP1 cells.\",\n      \"method\": \"Trio exome sequencing, biochemical proteasome activity assays in patient fibroblasts and OTUD7A knockout HAP1 cell line\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional assay in patient cells and KO cell line, single study\",\n      \"pmids\": [\"31997314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"OTUD7A interacts with Ankyrin-G (Ank3) and Ankyrin-B (Ank2); the epilepsy-associated L233F variant disrupts these interactions. OTUD7A-dependent regulation prevents polyubiquitination and degradation of Ankyrin-G, maintaining its levels at the axon initial segment and in dendritic spine nanodomains. Restoring OTUD7A or Ankyrin-G expression reverses neuronal abnormalities in 15q13.3 microdeletion neurons.\",\n      \"method\": \"Neuron-specific proximity-labeling proteomics (BioID2), co-immunoprecipitation, structured illumination microscopy, polyubiquitination assays, patient iPSC-derived neurons, rescue experiments\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including proteomics, biochemical ubiquitination assays, super-resolution microscopy, and functional rescue\",\n      \"pmids\": [\"36604605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OTUD7A loss-of-function in human iPSC-derived neurons reduces dendritic complexity, density of synaptic proteins GluA1 and PSD-95, and neuronal network activity; transcriptomic analysis shows downregulated genes enriched for synapse development and function pathways.\",\n      \"method\": \"CRISPR-Cas9 engineering of LoF mutation in hiPSC-derived neurons, immunofluorescence, multi-electrode array, transcriptomic analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean CRISPR KO in human neurons with multiple phenotypic readouts, single study\",\n      \"pmids\": [\"35931052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD7a directly interacts with TAK1 (TGF-β-activated kinase 1), inhibits its ubiquitination-mediated degradation (deubiquitinase activity), and subsequently increases phosphorylation of TAK1 and its downstream targets JNK/P38, promoting pathological cardiac hypertrophy. Cardiac-specific OTUD7a knockout alleviates hypertrophy while overexpression aggravates it.\",\n      \"method\": \"Cardiac-specific knockout mice, AAV9-mediated overexpression, co-immunoprecipitation, ubiquitination assays, RNA-sequencing and interactome analysis, TAK1 inhibitor (5Z-7-oxozeaenol) rescue, TAK1 overexpression epistasis\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical deubiquitination assay plus genetic KO/OE in vivo, epistasis with TAK1 inhibitor and overexpression, multiple orthogonal methods\",\n      \"pmids\": [\"41268652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD7A (Cezanne-2) employs a modified cysteine protease catalytic mechanism involving a Cys210/His367 dyad, with an oxyanion hole in the C-loop and polarization of His367 via a conserved water bridge with Glu173; it selectively cleaves Lys11-linked polyubiquitin chains via isopeptide bond cleavage through a tetrahedral oxyanion intermediate.\",\n      \"method\": \"Molecular dynamics simulations (12 µs), QM/MM calculations, structural analysis of diubiquitin-Cez2 complex\",\n      \"journal\": \"Journal of chemical information and modeling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 computational — extensive MD+QM/MM providing mechanistic detail, but no experimental mutagenesis validation in this study\",\n      \"pmids\": [\"39782030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cezanne2 (OTUD7A) interacts with TRAF6 and cleaves polyubiquitin chains from TRAF6 substrates, thereby modulating NF-κB signaling and downstream MMP2, MMP9, and ICAM1 levels.\",\n      \"method\": \"Co-immunoprecipitation, in vivo deubiquitination assay, reporter gene assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus in vivo deubiquitination assay, single study\",\n      \"pmids\": [\"23792447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"OTUD7A deubiquitinase regulates KDM5B stability in KRAS-mutant lung adenocarcinoma; OTUD7A inhibition reduces KDM5B expression, increases H4K20me3, downregulates GABPA, increases mitochondrial ROS production, and induces ferroptosis.\",\n      \"method\": \"KRAS-mutant LUAD cell lines, mouse models, organoid models, cisplatin combination treatment, molecular pathway analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — genetic depletion with defined downstream pathway, single study without direct biochemical deubiquitination assay for KDM5B\",\n      \"pmids\": [\"41422226\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"OTUD7A is an OTU-family deubiquitinase (catalytic Cys/His dyad, Lys11-linkage selective) that stabilizes multiple substrates—including EWS-FLI1, TAK1, Ankyrin-G/B, and KDM5B—by removing polyubiquitin chains to prevent their proteasomal degradation; in neurons it localizes to dendritic spines and the axon initial segment where it maintains Ankyrin-G levels and is required for normal spine density, glutamatergic transmission, and neuronal network activity, while loss-of-function underlies the neurodevelopmental phenotypes of 15q13.3 microdeletion syndrome.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"OTUD7A is an OTU-family deubiquitinase that employs a Cys210/His367 catalytic dyad with selectivity for Lys11-linked polyubiquitin chains, stabilizing diverse substrates—including Ankyrin-G, TAK1, EWS-FLI1, TRAF6, and KDM5B—by opposing their ubiquitin-dependent proteasomal degradation [PMID:39782030, PMID:34060252, PMID:36604605, PMID:41268652]. In cortical neurons, OTUD7A localizes to dendritic spines and the axon initial segment, where it deubiquitinates and maintains Ankyrin-G levels; loss of OTUD7A reduces dendritic spine density, decreases glutamatergic synaptic transmission, and disrupts neuronal network activity, establishing it as a critical regulator of excitatory synapse structure and function [PMID:29395075, PMID:36604605, PMID:35931052]. Loss-of-function mutations in OTUD7A, including the catalytic-domain variant p.Leu233Phe and haploinsufficiency from 15q13.3 microdeletions, cause neurodevelopmental phenotypes including epilepsy and intellectual disability [PMID:29395074, PMID:31997314, PMID:36604605]. Outside the nervous system, OTUD7A stabilizes TAK1 to promote JNK/P38 signaling in pathological cardiac hypertrophy and stabilizes EWS-FLI1 to sustain Ewing sarcoma growth [PMID:41268652, PMID:34060252].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"The initial mechanistic question—does OTUD7A function as a deubiquitinase with defined signaling targets?—was addressed by showing it interacts with TRAF6, removes its polyubiquitin chains, and thereby modulates NF-κB signaling.\",\n      \"evidence\": \"Co-immunoprecipitation, in vivo deubiquitination assay, and NF-κB reporter assay in cell lines\",\n      \"pmids\": [\"23792447\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study without reciprocal validation of the TRAF6 interaction by orthogonal methods\", \"Linkage-type specificity of deubiquitination not determined\", \"In vivo physiological relevance not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Whether OTUD7A has a physiological role in the nervous system was established by two independent studies showing it localizes to dendritic spines and is required for normal spine density, dendrite outgrowth, and glutamatergic synaptic transmission in the cortex, directly linking it to 15q13.3 microdeletion syndrome phenotypes.\",\n      \"evidence\": \"Heterozygous 15q13.3 deletion mouse model and Otud7a-knockout mice with immunofluorescence, spine morphometry, and mEPSC electrophysiology\",\n      \"pmids\": [\"29395074\", \"29395075\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular substrates in neurons not yet identified\", \"Whether the catalytic activity or a scaffolding role mediates the synaptic phenotype was unknown\", \"Human neuronal validation not performed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A human disease-causing mechanism was clarified when a homozygous p.Leu233Phe variant in the OTU catalytic domain was shown to impair proteasome complex formation and function, linking a specific catalytic-domain mutation to neurological disease.\",\n      \"evidence\": \"Trio exome sequencing, proteasome activity assays in patient fibroblasts and OTUD7A-knockout HAP1 cells\",\n      \"pmids\": [\"31997314\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single family; replication in additional patients needed\", \"How a deubiquitinase mutation impairs the proteasome complex remains mechanistically unclear\", \"Whether L233F abolishes deubiquitinase catalytic activity per se was not directly tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The question of whether OTUD7A stabilizes oncoproteins was answered by demonstrating it deubiquitinates and stabilizes EWS-FLI1, opposing SPOP-mediated degradation; OTUD7A depletion reduced EWS-FLI1 levels and suppressed Ewing sarcoma growth in vivo.\",\n      \"evidence\": \"Co-IP, in vivo deubiquitination assay, knockdown in Ewing sarcoma cell lines, xenograft model, catalytic inhibitor 7Ai\",\n      \"pmids\": [\"34060252\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether 7Ai is specific to OTUD7A versus other OTU-family members not fully resolved\", \"Structural basis of EWS-FLI1 recognition unknown\", \"Linkage specificity for EWS-FLI1 deubiquitination not determined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Translation to human neurons was achieved by showing that CRISPR-engineered OTUD7A loss-of-function in iPSC-derived neurons reduces dendritic complexity, synaptic protein levels (GluA1, PSD-95), and network activity, with transcriptomics confirming enrichment for synapse development pathways.\",\n      \"evidence\": \"CRISPR-Cas9 knockout in human iPSC-derived neurons, immunofluorescence, multi-electrode array, transcriptomic analysis\",\n      \"pmids\": [\"35931052\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single isogenic pair; additional clonal lines not reported\", \"Direct OTUD7A substrates responsible for the transcriptomic changes not identified\", \"Whether phenotypes are rescue-able by catalytic-active OTUD7A was not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The critical neuronal substrate question was resolved by identifying Ankyrin-G and Ankyrin-B as direct OTUD7A interactors; OTUD7A prevents Ankyrin-G polyubiquitination and degradation at the axon initial segment and dendritic spine nanodomains, and the L233F epilepsy variant disrupts these interactions. Rescue of Ankyrin-G reversed neuronal deficits in 15q13.3 microdeletion neurons.\",\n      \"evidence\": \"BioID2 proximity-labeling proteomics, co-IP, structured illumination microscopy, polyubiquitination assays, patient iPSC-derived neurons with rescue experiments\",\n      \"pmids\": [\"36604605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether additional neuronal substrates contribute to the phenotype beyond Ankyrins is unknown\", \"The ubiquitin linkage type on Ankyrin-G cleaved by OTUD7A was not determined\", \"In vivo rescue in mouse models not performed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A cardiac role was established: OTUD7A deubiquitinates and stabilizes TAK1, activating JNK/P38 signaling to drive pathological cardiac hypertrophy, as demonstrated by cardiac-specific knockout (protective) and overexpression (aggravating) with TAK1-dependent epistasis.\",\n      \"evidence\": \"Cardiac-specific Otud7a knockout and AAV9-mediated overexpression in mice, co-IP, ubiquitination assays, TAK1 inhibitor and overexpression epistasis\",\n      \"pmids\": [\"41268652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether OTUD7A regulation of TAK1 extends to inflammatory contexts beyond the heart is unknown\", \"The ubiquitin ligase opposing OTUD7A on TAK1 was not identified\", \"Whether the Lys11-linkage preference applies to TAK1 deubiquitination specifically was not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The catalytic mechanism was elucidated at atomic resolution: OTUD7A uses a Cys210/His367 dyad (not a classical catalytic triad), with His367 polarized through a water bridge to Glu173 and an oxyanion hole in the C-loop, selectively cleaving Lys11-linked polyubiquitin via a tetrahedral intermediate.\",\n      \"evidence\": \"12 µs molecular dynamics simulations and QM/MM calculations on the diubiquitin–Cez2 complex\",\n      \"pmids\": [\"39782030\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Computational predictions lack experimental mutagenesis validation\", \"Whether the Lys11 selectivity mechanism applies to all substrates in cells is untested\", \"Crystal structure of a substrate-bound OTUD7A complex not available\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"An oncogenic substrate-stabilization role in lung cancer was demonstrated: OTUD7A stabilizes the histone demethylase KDM5B in KRAS-mutant lung adenocarcinoma, and its inhibition triggers H4K20me3 increase, GABPA downregulation, mitochondrial ROS accumulation, and ferroptosis.\",\n      \"evidence\": \"KRAS-mutant LUAD cell lines, mouse and organoid models, cisplatin combination treatment, pathway analysis\",\n      \"pmids\": [\"41422226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical deubiquitination assay for KDM5B by OTUD7A not shown\", \"Whether OTUD7A inhibition sensitizes non-KRAS-mutant tumors is unknown\", \"The ferroptosis pathway link requires independent replication\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full substrate repertoire of OTUD7A in neurons, whether its Lys11-linkage selectivity governs all physiological substrates, and the structural basis for substrate recognition.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal structure of full-length OTUD7A with a bound substrate\", \"Comprehensive substrate identification across tissues not performed\", \"Therapeutic window for OTUD7A inhibition in cancer versus neurological toxicity unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [2, 4, 6, 7, 8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 4, 6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 4, 6, 7, 8]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 1, 4, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ANK3\",\n      \"ANK2\",\n      \"MAP3K7\",\n      \"TRAF6\",\n      \"EWSR1-FLI1\",\n      \"KDM5B\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}