{"gene":"ADGB","run_date":"2026-06-09T22:02:41","timeline":{"discoveries":[{"year":2011,"finding":"Androglobin (ADGB) is a chimeric protein with a unique modular architecture comprising an N-terminal calpain-like domain homologous to catalytic domain II of human calpain-7, an internal circularly permuted globin domain, and an IQ calmodulin-binding motif. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom.","method":"Comparative genomics, domain architecture analysis, recombinant protein expression and UV-vis spectroscopy","journal":"Molecular biology and evolution","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro recombinant expression with spectroscopic characterization, single study, domain architecture identified by bioinformatics","pmids":["22115833"],"is_preprint":false},{"year":2022,"finding":"Adgb knockout mice display male infertility with impaired spermatid maturation, abnormal sperm shape, and ultrastructural defects in microtubule and mitochondrial organization. Immunoprecipitation and mass spectrometry identified septin 10 (Sept10) as an interactor of Adgb, confirmed by reciprocal co-immunoprecipitation both in vivo (testis lysates) and in vitro. Absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. In vitro data suggest Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner.","method":"Knockout mouse model, immunoprecipitation/mass spectrometry, reciprocal co-IP, immunofluorescence, electron microscopy","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP confirmed in vivo and in vitro, KO mouse with defined cellular phenotype, multiple orthogonal methods","pmids":["35700329"],"is_preprint":false},{"year":2021,"finding":"In Tetrahymena thermophila, Adgb/androglobin localizes to the C1b/C1f supercomplex of the ciliary central apparatus. Deletion of Adgb caused only minor alterations in ciliary motility, whereas loss of other C1b/C1f subunits (Spef2A or Cfap69) caused loss of the entire C1b projection and abnormal cilia motion.","method":"Gene deletion in Tetrahymena, cryo-ET/proteomics of ciliary fractions, motility assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic deletion with defined subcellular localization and motility phenotype in ciliate model, single lab","pmids":["34083607"],"is_preprint":false},{"year":2023,"finding":"Pathogenic variants in ADGB disrupt binding of ADGB to calmodulin, causing asthenozoospermia and male infertility. Mass spectrometry identified 42 candidate interacting proteins involved in sperm assembly, flagella formation, and sperm motility; CFAP69 and SPEF2 were confirmed to bind ADGB by co-immunoprecipitation.","method":"Whole-exome sequencing, Adgb knockout mice, mass spectrometry, co-immunoprecipitation, electron microscopy","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP confirmation of CFAP69 and SPEF2 interactions, KO mouse phenotype, single lab with multiple methods","pmids":["36995441"],"is_preprint":false},{"year":2023,"finding":"FOXJ1 activates the ADGB promoter in transactivation assays in vitro, establishing ADGB as a downstream transcriptional target of FOXJ1 in ciliated cells. A truncating FOXJ1 variant (p.Glu267Glyfs*12) failed to activate the ADGB promoter.","method":"Transactivation luciferase reporter assay, in vivo frog epidermis ectopic cilia assay","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assay with loss-of-function FOXJ1 variant as control, two orthogonal assays (luciferase and in vivo frog), single lab","pmids":["37158461"],"is_preprint":false},{"year":2024,"finding":"Calmodulin (CaM) interacts with ADGB via its IQ motif, and this interaction enhances the nitrite reductase activity of the ADGB heme-binding globin domain. Fluorescence quenching experiments using CaM mutants labeled at Cys41 (N-lobe) showed greater energy transfer to the heme group upon ADGB binding, consistent with predicted structural models of the Adgb-CaM complex.","method":"Recombinant protein expression, AlphaFold3/HDOCK structural prediction, fluorescence quenching assay with CaM mutants, UV-vis kinetic assay for nitrite reductase activity","journal":"RSC chemical biology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro enzymatic assay and fluorescence binding assay with mutagenesis, single lab, structural prediction only","pmids":["39719941"],"is_preprint":false},{"year":2024,"finding":"Bi-allelic deleterious ADGB variants in infertile men cause multiple acrosome and flagellum malformations in spermatozoa. Functional assays revealed structural defects associated with dysregulation of ADGB and multiple spermatogenesis proteins. CaM deficiency (but normal PLCζ) was detected in sperm from ADGB-deficient patients, suggesting ADGB is required for calmodulin localization or stability in sperm.","method":"Whole-exome sequencing, Sanger sequencing, electron microscopy, immunofluorescence, western blot","journal":"Andrology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (EM, IF, WB) in human patient samples, single lab","pmids":["38385883"],"is_preprint":false},{"year":2025,"finding":"RFX3 regulates ADGB promoter-driven luciferase activity and endogenous ADGB expression levels, identifying RFX3 as a transcriptional regulator of ADGB. Stable ADGB overexpression in A549 lung cancer cells caused transcriptomic changes indicative of increased cell motility and extracellular matrix remodeling.","method":"Promoter-luciferase reporter assay, CRISPRa endogenous expression induction, stable overexpression with transcriptome analysis","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase assay and endogenous expression validation with CRISPRa, single lab, two orthogonal methods","pmids":["41138754"],"is_preprint":false},{"year":2026,"finding":"ADGB localizes to the acrosome and flagella of spermatogenic cells in humans and mice, with high expression after puberty. Co-immunoprecipitation experiments confirmed TTC29 and CFAP47 as interacting proteins of ADGB. Compound heterozygous pathogenic ADGB mutations cause oligoasthenoteratozoospermia with acrosome loss, disorganized mitochondrial sheath, and disrupted axonemal '9+2' microtubule structure.","method":"Immunofluorescence localization, co-immunoprecipitation, transmission electron microscopy, western blot, whole-exome sequencing","journal":"Sichuan da xue xue bao. Yi xue ban","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with TTC29 and CFAP47, direct localization by IF, multiple orthogonal methods, single lab","pmids":["41834962"],"is_preprint":false}],"current_model":"Androglobin (ADGB) is a chimeric metazoan protein with an N-terminal calpain-like domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif; it is highly expressed in postmeiotic spermatids and cells bearing motile cilia/flagella, where it is required for sperm head shaping and flagellum formation by interacting with septin 10 (Sept10), CFAP69, SPEF2, TTC29, and CFAP47, with calmodulin binding via the IQ motif regulating its nitrite reductase activity and Sept10 proteolysis; transcriptionally, it is controlled by FOXJ1 and RFX3, and its loss causes male infertility with defective manchette assembly, sperm annulus formation, and acrosomal development."},"narrative":{"mechanistic_narrative":"Androglobin (ADGB) is a chimeric metazoan protein that functions in the assembly of motile cilia/flagella and in spermatid maturation [PMID:35700329, PMID:34083607]. Its unique modular architecture combines an N-terminal calpain-like catalytic domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif [PMID:22115833]. Calmodulin binds the IQ motif and enhances the nitrite reductase activity of the heme-binding globin domain [PMID:39719941], and ADGB is in turn required for proper calmodulin localization or stability in sperm [PMID:38385883]. In spermatogenesis, ADGB localizes to the acrosome and flagella [PMID:41834962] and is required for sperm head shaping, manchette and annulus formation, and flagellum integrity, acting through interactions with cytoskeletal and ciliary assembly factors including septin 10, CFAP69, SPEF2, TTC29, and CFAP47 [PMID:35700329, PMID:36995441, PMID:41834962]; loss of ADGB drives mislocalization of Sept10 and contributes to its proteolysis in a calmodulin-dependent manner [PMID:35700329]. In the ciliary central apparatus of Tetrahymena, ADGB associates with the C1b/C1f supercomplex [PMID:34083607]. ADGB expression is transcriptionally controlled by the ciliogenic regulators FOXJ1 and RFX3 [PMID:37158461, PMID:41138754]. Bi-allelic pathogenic ADGB variants cause male infertility presenting as asthenozoospermia and oligoasthenoteratozoospermia with acrosome, mitochondrial sheath, and axonemal defects [PMID:36995441, PMID:38385883, PMID:41834962].","teleology":[{"year":2011,"claim":"Established that ADGB is a structurally unprecedented chimeric protein, raising the question of how a calpain-like protease, a permuted globin, and a calmodulin-binding motif could function together.","evidence":"Comparative genomics, domain architecture analysis, and UV-vis spectroscopy of the recombinant globin domain","pmids":["22115833"],"confidence":"Medium","gaps":["Heme redox chemistry and a physiological substrate for the globin domain were not defined","Catalytic activity of the calpain-like domain not demonstrated","No cellular function or expression context established"]},{"year":2021,"claim":"Placed ADGB within the ciliary central apparatus, addressing where the protein acts in motile cilia, while showing it is dispensable for the structural integrity of the C1b projection.","evidence":"Gene deletion in Tetrahymena thermophila with cryo-ET/proteomics of ciliary fractions and motility assays","pmids":["34083607"],"confidence":"Medium","gaps":["Only minor motility phenotype, leaving its functional contribution unclear","Mechanism of recruitment to C1b/C1f unknown","Ciliate findings not directly mapped to mammalian sperm flagella"]},{"year":2022,"claim":"Defined ADGB as essential for spermatid maturation and identified Sept10 as a direct partner, linking ADGB to manchette/annulus formation and to calmodulin-dependent proteolysis.","evidence":"Adgb knockout mouse, IP/MS with reciprocal co-IP in vivo and in vitro, immunofluorescence, electron microscopy","pmids":["35700329"],"confidence":"High","gaps":["Direct demonstration that the calpain-like domain cleaves Sept10 in vivo not shown","Whether globin redox state regulates proteolysis unresolved","Full substrate repertoire unknown"]},{"year":2023,"claim":"Connected ADGB to human disease and expanded its interactome, establishing that calmodulin binding and flagellar assembly factors are required for fertility.","evidence":"Whole-exome sequencing of infertile men, Adgb KO mice, MS interactome, co-IP confirming CFAP69 and SPEF2","pmids":["36995441"],"confidence":"Medium","gaps":["Stoichiometry and hierarchy among CFAP69/SPEF2/ADGB interactions undefined","How variants mechanistically abolish CaM binding not resolved at structural level"]},{"year":2023,"claim":"Identified FOXJ1 as a transcriptional activator of ADGB, embedding ADGB in the master ciliogenic gene-expression program.","evidence":"Transactivation luciferase reporter with a loss-of-function FOXJ1 variant and an in vivo frog epidermis ciliogenesis assay","pmids":["37158461"],"confidence":"Medium","gaps":["Direct FOXJ1 occupancy of the endogenous ADGB locus not shown","Co-regulators and tissue specificity of regulation undefined"]},{"year":2024,"claim":"Mechanistically linked calmodulin binding to the globin enzymatic activity, showing CaM enhances ADGB nitrite reductase activity via conformational coupling to the heme.","evidence":"Recombinant protein, AlphaFold3/HDOCK modeling, fluorescence quenching with CaM Cys41 mutants, UV-vis nitrite reductase kinetics","pmids":["39719941"],"confidence":"Medium","gaps":["Structural model is computational, not experimentally solved","Physiological relevance of nitrite reductase activity in sperm not demonstrated","Link between redox activity and Sept10 proteolysis untested"]},{"year":2024,"claim":"Extended the human phenotype to acrosome and flagellum malformations and showed ADGB is required for calmodulin abundance in sperm, suggesting a reciprocal ADGB–CaM dependency.","evidence":"Whole-exome/Sanger sequencing, electron microscopy, immunofluorescence, western blot on patient sperm","pmids":["38385883"],"confidence":"Medium","gaps":["Mechanism by which ADGB stabilizes or localizes CaM not defined","Single-lab patient cohort"]},{"year":2025,"claim":"Added RFX3 as a second ciliogenic transcriptional regulator of ADGB and raised a non-sperm role in cell motility and ECM remodeling.","evidence":"Promoter-luciferase reporter, CRISPRa endogenous induction, stable overexpression transcriptomics in A549 cells","pmids":["41138754"],"confidence":"Medium","gaps":["Functional significance of the cancer-cell motility/ECM signature unestablished","Whether RFX3 acts directly or via the cilia program unclear"]},{"year":2026,"claim":"Refined ADGB subcellular localization to acrosome and flagella and added TTC29 and CFAP47 to the flagellar interactome, consolidating ADGB as a hub for axoneme and mitochondrial sheath organization.","evidence":"Immunofluorescence localization, co-IP for TTC29 and CFAP47, transmission EM, western blot, whole-exome sequencing","pmids":["41834962"],"confidence":"Medium","gaps":["Whether interactions are direct or scaffold-mediated not resolved","Order of assembly events ADGB participates in unknown"]},{"year":null,"claim":"How the calpain-like protease, globin redox chemistry, and calmodulin sensing are integrated into a single coordinated activity governing flagellar/ciliary assembly remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental structure of full-length ADGB or its complexes","In vivo proteolytic substrates beyond Sept10 unconfirmed","Physiological role of globin nitrite reductase activity in sperm not demonstrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1]},{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[5]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,3,8]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[2,8]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,8]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[1,3,8]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[2]}],"complexes":["C1b/C1f ciliary central apparatus supercomplex"],"partners":["SEPT10","CFAP69","SPEF2","TTC29","CFAP47","CALM1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8N7X0","full_name":"Androglobin","aliases":[],"length_aa":1667,"mass_kda":189.7,"function":"Probable chimeric globin with a bis-histidyl six-coordinate heme-iron atom through which it could bind dioxygen, carbon monoxide and nitric oxide (PubMed:22115833). Required for sperm flagellum formation and maturation of elongating spermatids, thus playing an essential role in male fertility (PubMed:36995441)","subcellular_location":"Cell projection, cilium, flagellum","url":"https://www.uniprot.org/uniprotkb/Q8N7X0/entry"},"depmap":{"release":"DepMap","has_data":false,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ADGB"},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ADGB","total_profiled":1310},"omim":[{"mim_id":"614630","title":"ANDROGLOBIN; ADGB","url":"https://www.omim.org/entry/614630"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mid piece","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"choroid plexus","ntpm":4.7},{"tissue":"fallopian tube","ntpm":7.1},{"tissue":"testis","ntpm":24.4}],"url":"https://www.proteinatlas.org/search/ADGB"},"hgnc":{"alias_symbol":["FLJ23121","dJ408K24.1","CAPN16"],"prev_symbol":["C6orf103"]},"alphafold":{"accession":"Q8N7X0","domains":[{"cath_id":"-","chopping":"46-62_79-265","consensus_level":"medium","plddt":86.0416,"start":46,"end":265},{"cath_id":"3.90.70.10","chopping":"273-299_413-468_503-515_629-654","consensus_level":"medium","plddt":84.2771,"start":273,"end":654},{"cath_id":"2.60.120.380","chopping":"655-698_707-784","consensus_level":"medium","plddt":87.4215,"start":655,"end":784},{"cath_id":"-","chopping":"785-893_906-982","consensus_level":"medium","plddt":83.3914,"start":785,"end":982},{"cath_id":"2.60.120.380","chopping":"1103-1178_1251-1275_1356-1389","consensus_level":"medium","plddt":88.3827,"start":1103,"end":1389}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N7X0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N7X0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N7X0-F1-predicted_aligned_error_v6.png","plddt_mean":66.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ADGB","jax_strain_url":"https://www.jax.org/strain/search?query=ADGB"},"sequence":{"accession":"Q8N7X0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N7X0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N7X0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N7X0"}},"corpus_meta":[{"pmid":"22115833","id":"PMC_22115833","title":"Androglobin: 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Yi xue ban = Journal of Sichuan University. 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The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom.\",\n      \"method\": \"Comparative genomics, domain architecture analysis, recombinant protein expression and UV-vis spectroscopy\",\n      \"journal\": \"Molecular biology and evolution\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro recombinant expression with spectroscopic characterization, single study, domain architecture identified by bioinformatics\",\n      \"pmids\": [\"22115833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Adgb knockout mice display male infertility with impaired spermatid maturation, abnormal sperm shape, and ultrastructural defects in microtubule and mitochondrial organization. Immunoprecipitation and mass spectrometry identified septin 10 (Sept10) as an interactor of Adgb, confirmed by reciprocal co-immunoprecipitation both in vivo (testis lysates) and in vitro. Absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. In vitro data suggest Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner.\",\n      \"method\": \"Knockout mouse model, immunoprecipitation/mass spectrometry, reciprocal co-IP, immunofluorescence, electron microscopy\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP confirmed in vivo and in vitro, KO mouse with defined cellular phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"35700329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In Tetrahymena thermophila, Adgb/androglobin localizes to the C1b/C1f supercomplex of the ciliary central apparatus. Deletion of Adgb caused only minor alterations in ciliary motility, whereas loss of other C1b/C1f subunits (Spef2A or Cfap69) caused loss of the entire C1b projection and abnormal cilia motion.\",\n      \"method\": \"Gene deletion in Tetrahymena, cryo-ET/proteomics of ciliary fractions, motility assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic deletion with defined subcellular localization and motility phenotype in ciliate model, single lab\",\n      \"pmids\": [\"34083607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Pathogenic variants in ADGB disrupt binding of ADGB to calmodulin, causing asthenozoospermia and male infertility. Mass spectrometry identified 42 candidate interacting proteins involved in sperm assembly, flagella formation, and sperm motility; CFAP69 and SPEF2 were confirmed to bind ADGB by co-immunoprecipitation.\",\n      \"method\": \"Whole-exome sequencing, Adgb knockout mice, mass spectrometry, co-immunoprecipitation, electron microscopy\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP confirmation of CFAP69 and SPEF2 interactions, KO mouse phenotype, single lab with multiple methods\",\n      \"pmids\": [\"36995441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FOXJ1 activates the ADGB promoter in transactivation assays in vitro, establishing ADGB as a downstream transcriptional target of FOXJ1 in ciliated cells. A truncating FOXJ1 variant (p.Glu267Glyfs*12) failed to activate the ADGB promoter.\",\n      \"method\": \"Transactivation luciferase reporter assay, in vivo frog epidermis ectopic cilia assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assay with loss-of-function FOXJ1 variant as control, two orthogonal assays (luciferase and in vivo frog), single lab\",\n      \"pmids\": [\"37158461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Calmodulin (CaM) interacts with ADGB via its IQ motif, and this interaction enhances the nitrite reductase activity of the ADGB heme-binding globin domain. Fluorescence quenching experiments using CaM mutants labeled at Cys41 (N-lobe) showed greater energy transfer to the heme group upon ADGB binding, consistent with predicted structural models of the Adgb-CaM complex.\",\n      \"method\": \"Recombinant protein expression, AlphaFold3/HDOCK structural prediction, fluorescence quenching assay with CaM mutants, UV-vis kinetic assay for nitrite reductase activity\",\n      \"journal\": \"RSC chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro enzymatic assay and fluorescence binding assay with mutagenesis, single lab, structural prediction only\",\n      \"pmids\": [\"39719941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Bi-allelic deleterious ADGB variants in infertile men cause multiple acrosome and flagellum malformations in spermatozoa. Functional assays revealed structural defects associated with dysregulation of ADGB and multiple spermatogenesis proteins. CaM deficiency (but normal PLCζ) was detected in sperm from ADGB-deficient patients, suggesting ADGB is required for calmodulin localization or stability in sperm.\",\n      \"method\": \"Whole-exome sequencing, Sanger sequencing, electron microscopy, immunofluorescence, western blot\",\n      \"journal\": \"Andrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (EM, IF, WB) in human patient samples, single lab\",\n      \"pmids\": [\"38385883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RFX3 regulates ADGB promoter-driven luciferase activity and endogenous ADGB expression levels, identifying RFX3 as a transcriptional regulator of ADGB. Stable ADGB overexpression in A549 lung cancer cells caused transcriptomic changes indicative of increased cell motility and extracellular matrix remodeling.\",\n      \"method\": \"Promoter-luciferase reporter assay, CRISPRa endogenous expression induction, stable overexpression with transcriptome analysis\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase assay and endogenous expression validation with CRISPRa, single lab, two orthogonal methods\",\n      \"pmids\": [\"41138754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ADGB localizes to the acrosome and flagella of spermatogenic cells in humans and mice, with high expression after puberty. Co-immunoprecipitation experiments confirmed TTC29 and CFAP47 as interacting proteins of ADGB. Compound heterozygous pathogenic ADGB mutations cause oligoasthenoteratozoospermia with acrosome loss, disorganized mitochondrial sheath, and disrupted axonemal '9+2' microtubule structure.\",\n      \"method\": \"Immunofluorescence localization, co-immunoprecipitation, transmission electron microscopy, western blot, whole-exome sequencing\",\n      \"journal\": \"Sichuan da xue xue bao. Yi xue ban\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with TTC29 and CFAP47, direct localization by IF, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"41834962\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Androglobin (ADGB) is a chimeric metazoan protein with an N-terminal calpain-like domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif; it is highly expressed in postmeiotic spermatids and cells bearing motile cilia/flagella, where it is required for sperm head shaping and flagellum formation by interacting with septin 10 (Sept10), CFAP69, SPEF2, TTC29, and CFAP47, with calmodulin binding via the IQ motif regulating its nitrite reductase activity and Sept10 proteolysis; transcriptionally, it is controlled by FOXJ1 and RFX3, and its loss causes male infertility with defective manchette assembly, sperm annulus formation, and acrosomal development.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Androglobin (ADGB) is a chimeric metazoan protein that functions in the assembly of motile cilia/flagella and in spermatid maturation [#1, #2]. Its unique modular architecture combines an N-terminal calpain-like catalytic domain, a circularly permuted hexacoordinated globin domain, and an IQ calmodulin-binding motif [#0]. Calmodulin binds the IQ motif and enhances the nitrite reductase activity of the heme-binding globin domain [#5], and ADGB is in turn required for proper calmodulin localization or stability in sperm [#6]. In spermatogenesis, ADGB localizes to the acrosome and flagella [#8] and is required for sperm head shaping, manchette and annulus formation, and flagellum integrity, acting through interactions with cytoskeletal and ciliary assembly factors including septin 10, CFAP69, SPEF2, TTC29, and CFAP47 [#1, #3, #8]; loss of ADGB drives mislocalization of Sept10 and contributes to its proteolysis in a calmodulin-dependent manner [#1]. In the ciliary central apparatus of Tetrahymena, ADGB associates with the C1b/C1f supercomplex [#2]. ADGB expression is transcriptionally controlled by the ciliogenic regulators FOXJ1 and RFX3 [#4, #7]. Bi-allelic pathogenic ADGB variants cause male infertility presenting as asthenozoospermia and oligoasthenoteratozoospermia with acrosome, mitochondrial sheath, and axonemal defects [#3, #6, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that ADGB is a structurally unprecedented chimeric protein, raising the question of how a calpain-like protease, a permuted globin, and a calmodulin-binding motif could function together.\",\n      \"evidence\": \"Comparative genomics, domain architecture analysis, and UV-vis spectroscopy of the recombinant globin domain\",\n      \"pmids\": [\"22115833\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Heme redox chemistry and a physiological substrate for the globin domain were not defined\", \"Catalytic activity of the calpain-like domain not demonstrated\", \"No cellular function or expression context established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed ADGB within the ciliary central apparatus, addressing where the protein acts in motile cilia, while showing it is dispensable for the structural integrity of the C1b projection.\",\n      \"evidence\": \"Gene deletion in Tetrahymena thermophila with cryo-ET/proteomics of ciliary fractions and motility assays\",\n      \"pmids\": [\"34083607\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Only minor motility phenotype, leaving its functional contribution unclear\", \"Mechanism of recruitment to C1b/C1f unknown\", \"Ciliate findings not directly mapped to mammalian sperm flagella\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined ADGB as essential for spermatid maturation and identified Sept10 as a direct partner, linking ADGB to manchette/annulus formation and to calmodulin-dependent proteolysis.\",\n      \"evidence\": \"Adgb knockout mouse, IP/MS with reciprocal co-IP in vivo and in vitro, immunofluorescence, electron microscopy\",\n      \"pmids\": [\"35700329\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct demonstration that the calpain-like domain cleaves Sept10 in vivo not shown\", \"Whether globin redox state regulates proteolysis unresolved\", \"Full substrate repertoire unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected ADGB to human disease and expanded its interactome, establishing that calmodulin binding and flagellar assembly factors are required for fertility.\",\n      \"evidence\": \"Whole-exome sequencing of infertile men, Adgb KO mice, MS interactome, co-IP confirming CFAP69 and SPEF2\",\n      \"pmids\": [\"36995441\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Stoichiometry and hierarchy among CFAP69/SPEF2/ADGB interactions undefined\", \"How variants mechanistically abolish CaM binding not resolved at structural level\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified FOXJ1 as a transcriptional activator of ADGB, embedding ADGB in the master ciliogenic gene-expression program.\",\n      \"evidence\": \"Transactivation luciferase reporter with a loss-of-function FOXJ1 variant and an in vivo frog epidermis ciliogenesis assay\",\n      \"pmids\": [\"37158461\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct FOXJ1 occupancy of the endogenous ADGB locus not shown\", \"Co-regulators and tissue specificity of regulation undefined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mechanistically linked calmodulin binding to the globin enzymatic activity, showing CaM enhances ADGB nitrite reductase activity via conformational coupling to the heme.\",\n      \"evidence\": \"Recombinant protein, AlphaFold3/HDOCK modeling, fluorescence quenching with CaM Cys41 mutants, UV-vis nitrite reductase kinetics\",\n      \"pmids\": [\"39719941\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural model is computational, not experimentally solved\", \"Physiological relevance of nitrite reductase activity in sperm not demonstrated\", \"Link between redox activity and Sept10 proteolysis untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the human phenotype to acrosome and flagellum malformations and showed ADGB is required for calmodulin abundance in sperm, suggesting a reciprocal ADGB–CaM dependency.\",\n      \"evidence\": \"Whole-exome/Sanger sequencing, electron microscopy, immunofluorescence, western blot on patient sperm\",\n      \"pmids\": [\"38385883\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism by which ADGB stabilizes or localizes CaM not defined\", \"Single-lab patient cohort\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Added RFX3 as a second ciliogenic transcriptional regulator of ADGB and raised a non-sperm role in cell motility and ECM remodeling.\",\n      \"evidence\": \"Promoter-luciferase reporter, CRISPRa endogenous induction, stable overexpression transcriptomics in A549 cells\",\n      \"pmids\": [\"41138754\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Functional significance of the cancer-cell motility/ECM signature unestablished\", \"Whether RFX3 acts directly or via the cilia program unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Refined ADGB subcellular localization to acrosome and flagella and added TTC29 and CFAP47 to the flagellar interactome, consolidating ADGB as a hub for axoneme and mitochondrial sheath organization.\",\n      \"evidence\": \"Immunofluorescence localization, co-IP for TTC29 and CFAP47, transmission EM, western blot, whole-exome sequencing\",\n      \"pmids\": [\"41834962\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether interactions are direct or scaffold-mediated not resolved\", \"Order of assembly events ADGB participates in unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the calpain-like protease, globin redox chemistry, and calmodulin sensing are integrated into a single coordinated activity governing flagellar/ciliary assembly remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No experimental structure of full-length ADGB or its complexes\", \"In vivo proteolytic substrates beyond Sept10 unconfirmed\", \"Physiological role of globin nitrite reductase activity in sperm not demonstrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 3, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [2, 8]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [1, 3, 8]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\"C1b/C1f ciliary central apparatus supercomplex\"],\n    \"partners\": [\"SEPT10\", \"CFAP69\", \"SPEF2\", \"TTC29\", \"CFAP47\", \"CALM1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}