{"gene":"AK8","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2011,"finding":"Human AK8 is a novel adenylate kinase with two complete and independently active AK domains within a single polypeptide chain. Full-length AK8, as well as each domain separately (AK8p1 and AK8p2), all exhibit AK enzyme activity. AK8 phosphorylates AMP, CMP, dAMP, and dCMP with ATP as phosphate donor, and AMP, CMP, and dCMP with GTP as phosphate donor, showing highest affinity for AMP with ATP. AK8 localizes to the cytosol, demonstrated by GFP-fusion protein imaging.","method":"In vitro enzyme activity assays, substrate specificity assays, GFP-fusion protein subcellular localization imaging","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic characterization with multiple substrates, domain mutagenesis/truncation, and direct localization experiment; single lab but multiple orthogonal methods","pmids":["21080915"],"is_preprint":false},{"year":2014,"finding":"AK8 is present in the axoneme compartment of the mouse sperm flagellum (a third flagellar compartment distinct from outer dense fibers and mitochondrial sheath where AK1 and AK2 reside), as shown by immunolocalization. Addition of AMP to ATP-containing medium recapitulated the ADP-driven flagellar waveform, consistent with AK-mediated nucleotide interconversion in the axoneme.","method":"Immunolocalization/immunofluorescence in mouse sperm, detergent-modeled sperm motility assays with defined nucleotide substrates, digital image analysis of flagellar waveforms","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment placing AK8 in the axoneme with functional nucleotide experiments; single lab, two orthogonal approaches","pmids":["24740601"],"is_preprint":false},{"year":2011,"finding":"Knockout of Ak8 in mice results in autosomal recessive congenital hydrocephalus, with dysfunctional motile cilia identified as the underlying pathogenetic mechanism, placing AK8 as required for proper motile cilia function in vivo.","method":"Knockout mouse model, neuropathological analysis","journal":"Veterinary pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockout with defined phenotypic readout (hydrocephalus) linked to ciliary dysfunction; single study, histopathology-based mechanistic placement","pmids":["21746835"],"is_preprint":false},{"year":2020,"finding":"CFAP45 structural modelling identifies an AMP-binding interface between CFAP45 and AK8, and proteomic profiling of CFAP45-deficient cilia links CFAP45 to an axonemal module that includes AK8 and dynein ATPases. This positions AK8 as part of an axonemal adenine nucleotide homeostasis module regulating ciliary and flagellar beating.","method":"Proteomic profiling (mass spectrometry) of CFAP45-deficient cilia/flagella, structural modelling of AMP-binding interface between CFAP45 and AK8, in vitro AMP binding assay for CFAP45, microtubule sliding rescue assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic interaction mapping and structural modelling identify the AK8-CFAP45 interface; AK8-specific binding validated by modelling but not by direct AK8 mutagenesis; single study","pmids":["33139725"],"is_preprint":false},{"year":2024,"finding":"AK8 interacts with the radial spoke (RS) of the sperm axoneme, as revealed by immunoprecipitation combined with mass spectrometry. AK8 knockout mice are infertile with impaired sperm motility. AK8 and AK9 cooperatively regulate ATP transfer in the axoneme and are concentrated at sites of energy consumption in the flagellum, as shown by ATP probes and metabolomic analysis.","method":"Immunoprecipitation combined with mass spectrometry, knockout mouse model, ATP probe assay, metabolomic analysis, human infertile male cohort analysis","journal":"Science China. Life sciences","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reciprocal Co-IP/MS, knockout mouse model with defined fertility/motility phenotype, and metabolic flux measurement; multiple orthogonal methods in one study","pmids":["38761355"],"is_preprint":false},{"year":2025,"finding":"EFCAB10 interacts directly with AK8 and RSPH3B, anchoring AK8 to the radial spoke in motile cilia. Loss of EFCAB10 results in complete absence of AK8 from cilia, whereas loss of AK8 does not affect ciliary EFCAB10. Both Efcab10 and Ak8 knockout mice display impaired ciliary motility and PCD-related phenotypes (respiratory defects and infertility).","method":"Biochemical co-immunoprecipitation, knockout mouse models (Efcab10-/- and Ak8-/-), ciliary motility assays, immunofluorescence localization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP establishing EFCAB10-AK8-RSPH3B complex, dual knockout mouse models with defined phenotype, directional epistasis (EFCAB10 required for AK8 localization but not vice versa); multiple orthogonal methods","pmids":["41055978"],"is_preprint":false},{"year":2025,"finding":"AK8 is identified as a component of the stalk of radial spoke 1 (RS1) in human and mouse sperm flagella. In IQUB-deficient sperm, RS1 (but not RS2 or RS3) is lost, and AK8 is among the RS1 stalk proteins that are correspondingly absent or reduced, placing AK8 structurally within the RS1 stalk.","method":"Protein mass spectrometry of IQUB-deficient human and mouse sperm, western blotting, immunofluorescence, Iqub knockout mouse model","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based proteomic identification of AK8 as RS1 stalk component in KO model; single study, no direct AK8 mutagenesis","pmids":["39849482"],"is_preprint":false},{"year":2007,"finding":"AK8 (then designated hypothetical protein FLJ32704) is present as a sperm antigen recognized specifically by sera of immunoinfertile women but not fertile women, identified by 2D gel electrophoresis and MALDI-TOF-MS/LC-MS of human sperm proteins. An immunodominant peptide (amino acids 151–159) was identified that reacted with 90% of immunoinfertile sera.","method":"2D gel electrophoresis, MALDI-TOF-MS/LC-MS, ELISA with synthetic peptide","journal":"Molecular reproduction and development","confidence":"Low","confidence_rationale":"Tier 3 / Weak — identifies AK8 as a sperm surface antigen by proteomic identification; no direct mechanistic functional experiment on AK8 itself","pmids":["16998854"],"is_preprint":false}],"current_model":"AK8 is a dual-domain adenylate kinase (with two independently active AK domains) that localizes to the axoneme of motile cilia and sperm flagella, where it is anchored to radial spoke 1 (RS1) via EFCAB10 and RSPH3B, and functions as part of an adenine nucleotide homeostasis/ATP transfer module that is essential for proper ciliary beating, sperm motility, and male fertility; loss of AK8 in mice causes primary ciliary dyskinesia phenotypes including hydrocephalus and infertility."},"narrative":{"mechanistic_narrative":"AK8 is a dual-domain adenylate kinase that supplies local adenine nucleotide homeostasis to motile cilia and sperm flagella, where it is required for proper ciliary beating and male fertility [PMID:21080915, PMID:38761355]. It carries two complete, independently active AK domains within a single polypeptide; full-length AK8 and each isolated domain phosphorylate AMP, CMP, dAMP, and dCMP using ATP (or GTP) as phosphate donor, with highest affinity for AMP-ATP [PMID:21080915]. Within the flagellum AK8 is concentrated in the axonemal compartment, distinct from the outer dense fibers and mitochondrial sheath, where nucleotide interconversion shapes the ADP-driven flagellar waveform [PMID:24740601]. AK8 is a structural component of the radial spoke 1 (RS1) stalk, anchored there through EFCAB10, which bridges AK8 to RSPH3B; loss of EFCAB10 abolishes ciliary AK8, whereas loss of AK8 leaves EFCAB10 intact, defining a directional assembly hierarchy [PMID:41055978, PMID:39849482]. AK8 acts together with AK9 to route ATP transfer to sites of high energy consumption in the flagellum, and it associates with an axonemal nucleotide-homeostasis module that includes CFAP45 and dynein ATPases [PMID:33139725, PMID:38761355]. Loss of AK8 in mice produces primary ciliary dyskinesia phenotypes including congenital hydrocephalus, respiratory defects, impaired sperm motility, and infertility [PMID:21746835, PMID:38761355, PMID:41055978].","teleology":[{"year":2007,"claim":"Before any functional role was known, AK8 was flagged as a sperm-associated antigen, providing the first link of the protein to reproductive biology.","evidence":"2D gel electrophoresis with MALDI-TOF/LC-MS of human sperm proteins and peptide ELISA against immunoinfertile sera","pmids":["16998854"],"confidence":"Low","gaps":["No mechanistic or enzymatic function demonstrated for AK8","Antigen identification does not establish localization or molecular activity","Association with infertility is correlative"]},{"year":2011,"claim":"Establishing the biochemistry first: AK8 was shown to be an adenylate kinase carrying two independently catalytically active AK domains in one chain, defining its core molecular activity and substrate preferences.","evidence":"In vitro enzyme and substrate-specificity assays with domain truncations plus GFP-fusion localization","pmids":["21080915"],"confidence":"High","gaps":["Cytosolic localization in overexpression did not reveal the axonemal targeting later observed","Physiological substrate flux in vivo not measured","No interacting partners identified"]},{"year":2011,"claim":"Genetic knockout established that AK8 is required in vivo, linking its loss to motile-cilia dysfunction and congenital hydrocephalus.","evidence":"Ak8 knockout mouse with neuropathological analysis","pmids":["21746835"],"confidence":"Medium","gaps":["Mechanism connecting AK8 loss to ciliary failure inferred from histopathology only","No molecular placement of AK8 within the axoneme","Sperm/fertility phenotype not assessed here"]},{"year":2014,"claim":"Subcellular resolution placed AK8 specifically in the axonemal compartment of the sperm flagellum and tied its enzymatic activity to flagellar mechanics.","evidence":"Immunolocalization in mouse sperm with detergent-modeled motility assays using defined nucleotide substrates","pmids":["24740601"],"confidence":"Medium","gaps":["Direct AK8-dependence of the AMP-driven waveform not tested by genetic ablation","Anchoring mechanism within the axoneme unknown","Single-lab functional inference"]},{"year":2020,"claim":"AK8 was embedded in a defined axonemal module by linking it to CFAP45 and dynein ATPases, framing it as part of a nucleotide-homeostasis system regulating beating.","evidence":"Proteomic profiling of CFAP45-deficient cilia, structural modelling of an AMP-binding CFAP45-AK8 interface, and microtubule sliding rescue","pmids":["33139725"],"confidence":"Medium","gaps":["AK8-CFAP45 interface validated by modelling, not by AK8 mutagenesis","Functional consequence of disrupting the interface untested","Stoichiometry of the module unresolved"]},{"year":2024,"claim":"AK8 was shown to physically associate with the radial spoke and to act with AK9 in directing ATP transfer to flagellar energy-consuming sites, connecting its enzymatic role to motility and fertility.","evidence":"Reciprocal Co-IP/MS, Ak8 knockout mice, ATP-probe imaging, metabolomics, and a human infertile-male cohort","pmids":["38761355"],"confidence":"High","gaps":["Precise division of labor between AK8 and AK9 not fully resolved","Direct radial-spoke binding partner not identified in this study","Causal AK8 variants in human infertility not functionally proven"]},{"year":2025,"claim":"The anchoring mechanism was resolved: EFCAB10 directly binds AK8 and RSPH3B to tether AK8 to the radial spoke, with directional epistasis showing EFCAB10 is upstream of AK8 ciliary localization.","evidence":"Co-IP, Efcab10-/- and Ak8-/- knockout mice, ciliary motility assays, and immunofluorescence","pmids":["41055978"],"confidence":"High","gaps":["Structural basis of the EFCAB10-AK8-RSPH3B interface unknown","Whether anchoring is required for AK8 catalytic function untested","Tissue specificity of the anchoring complex not fully mapped"]},{"year":2025,"claim":"AK8 was placed structurally within the RS1 stalk specifically, distinguishing its location among the three radial spokes.","evidence":"MS of IQUB-deficient human and mouse sperm with western blot, immunofluorescence, and Iqub knockout","pmids":["39849482"],"confidence":"Medium","gaps":["No direct AK8 mutagenesis to confirm RS1 incorporation","Relationship between IQUB and the EFCAB10 anchoring pathway unclear","RS1-specific functional role of AK8 not isolated"]},{"year":null,"claim":"It remains unresolved how AK8's catalytic activity, its RS1 structural role, and its anchoring by EFCAB10 are mechanistically coupled to dynein-driven beating, and whether specific human AK8 variants cause primary ciliary dyskinesia or infertility.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the AK8-containing RS1 assembly","No demonstration that anchoring is required for in-axoneme catalysis","Causal human disease variants not functionally validated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[2,5]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[4,5]}],"complexes":["radial spoke 1 (RS1) stalk","EFCAB10-AK8-RSPH3B complex","axonemal CFAP45 nucleotide-homeostasis module"],"partners":["EFCAB10","RSPH3B","CFAP45","AK9","IQUB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96MA6","full_name":"Adenylate kinase 8","aliases":["ATP-AMP transphosphorylase 8"],"length_aa":479,"mass_kda":54.9,"function":"Nucleoside monophosphate (NMP) kinase that catalyzes the reversible transfer of the terminal phosphate group between nucleoside triphosphates and monophosphates. Has highest activity toward AMP, and weaker activity toward dAMP, CMP and dCMP. Also displays broad nucleoside diphosphate kinase activity","subcellular_location":"Cytoplasm, cytosol; Cytoplasm, cytoskeleton, cilium axoneme","url":"https://www.uniprot.org/uniprotkb/Q96MA6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AK8","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/AK8","total_profiled":1310},"omim":[{"mim_id":"615365","title":"ADENYLATE KINASE 8; AK8","url":"https://www.omim.org/entry/615365"},{"mim_id":"615364","title":"ADENYLATE KINASE 7; AK7","url":"https://www.omim.org/entry/615364"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Flagellar centriole","reliability":"Approved"},{"location":"Mid piece","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"},{"location":"End piece","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"epididymis","ntpm":18.2},{"tissue":"fallopian tube","ntpm":24.3},{"tissue":"testis","ntpm":17.8}],"url":"https://www.proteinatlas.org/search/AK8"},"hgnc":{"alias_symbol":["FLJ32704"],"prev_symbol":["C9orf98"]},"alphafold":{"accession":"Q96MA6","domains":[{"cath_id":"3.40.50.300","chopping":"58-179_208-259","consensus_level":"high","plddt":90.4298,"start":58,"end":259},{"cath_id":"3.40.50.300","chopping":"271-299_343-472","consensus_level":"high","plddt":91.8262,"start":271,"end":472}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96MA6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96MA6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96MA6-F1-predicted_aligned_error_v6.png","plddt_mean":87.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AK8","jax_strain_url":"https://www.jax.org/strain/search?query=AK8"},"sequence":{"accession":"Q96MA6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96MA6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96MA6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96MA6"}},"corpus_meta":[{"pmid":"21746835","id":"PMC_21746835","title":"Congenital hydrocephalus in genetically engineered mice.","date":"2011","source":"Veterinary pathology","url":"https://pubmed.ncbi.nlm.nih.gov/21746835","citation_count":115,"is_preprint":false},{"pmid":"24127692","id":"PMC_24127692","title":"Next-generation sequencing, FISH mapping and synteny-based modeling reveal mechanisms of decreasing dysploidy in Cucumis.","date":"2013","source":"The Plant journal : for cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24127692","citation_count":59,"is_preprint":false},{"pmid":"33139725","id":"PMC_33139725","title":"CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33139725","citation_count":56,"is_preprint":false},{"pmid":"21080915","id":"PMC_21080915","title":"The characterization of human adenylate kinases 7 and 8 demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes.","date":"2011","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/21080915","citation_count":37,"is_preprint":false},{"pmid":"24740601","id":"PMC_24740601","title":"Adenine nucleotide metabolism and a role for AMP in modulating flagellar waveforms in mouse sperm.","date":"2014","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/24740601","citation_count":30,"is_preprint":false},{"pmid":"8056032","id":"PMC_8056032","title":"T cells which do not express membrane tumor necrosis factor-alpha activate macrophage effector function by cell contact-dependent signaling of macrophage tumor necrosis factor-alpha production.","date":"1994","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/8056032","citation_count":30,"is_preprint":false},{"pmid":"31134708","id":"PMC_31134708","title":"Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient.","date":"2019","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/31134708","citation_count":29,"is_preprint":false},{"pmid":"19597321","id":"PMC_19597321","title":"Glycosaminoglycan degradation-inhibitory lactic acid bacteria ameliorate 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice.","date":"2009","source":"Journal of microbiology and biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/19597321","citation_count":27,"is_preprint":false},{"pmid":"30868165","id":"PMC_30868165","title":"The story of promiscuous crucifers: origin and genome evolution of an invasive species, Cardamine occulta (Brassicaceae), and its relatives.","date":"2019","source":"Annals of botany","url":"https://pubmed.ncbi.nlm.nih.gov/30868165","citation_count":24,"is_preprint":false},{"pmid":"28487430","id":"PMC_28487430","title":"RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea.","date":"2017","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/28487430","citation_count":22,"is_preprint":false},{"pmid":"16998854","id":"PMC_16998854","title":"Molecular identities of human sperm proteins reactive with antibodies in sera of immunoinfertile women.","date":"2007","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/16998854","citation_count":21,"is_preprint":false},{"pmid":"2574679","id":"PMC_2574679","title":"Selective induction of growth factor production and growth factor receptor expression by different signals to a single T cell.","date":"1989","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/2574679","citation_count":21,"is_preprint":false},{"pmid":"38761355","id":"PMC_38761355","title":"Adenylate kinase phosphate energy shuttle underlies energetic communication in flagellar axonemes.","date":"2024","source":"Science China. 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Full-length AK8, as well as each domain separately (AK8p1 and AK8p2), all exhibit AK enzyme activity. AK8 phosphorylates AMP, CMP, dAMP, and dCMP with ATP as phosphate donor, and AMP, CMP, and dCMP with GTP as phosphate donor, showing highest affinity for AMP with ATP. AK8 localizes to the cytosol, demonstrated by GFP-fusion protein imaging.\",\n      \"method\": \"In vitro enzyme activity assays, substrate specificity assays, GFP-fusion protein subcellular localization imaging\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic characterization with multiple substrates, domain mutagenesis/truncation, and direct localization experiment; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21080915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"AK8 is present in the axoneme compartment of the mouse sperm flagellum (a third flagellar compartment distinct from outer dense fibers and mitochondrial sheath where AK1 and AK2 reside), as shown by immunolocalization. Addition of AMP to ATP-containing medium recapitulated the ADP-driven flagellar waveform, consistent with AK-mediated nucleotide interconversion in the axoneme.\",\n      \"method\": \"Immunolocalization/immunofluorescence in mouse sperm, detergent-modeled sperm motility assays with defined nucleotide substrates, digital image analysis of flagellar waveforms\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment placing AK8 in the axoneme with functional nucleotide experiments; single lab, two orthogonal approaches\",\n      \"pmids\": [\"24740601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Knockout of Ak8 in mice results in autosomal recessive congenital hydrocephalus, with dysfunctional motile cilia identified as the underlying pathogenetic mechanism, placing AK8 as required for proper motile cilia function in vivo.\",\n      \"method\": \"Knockout mouse model, neuropathological analysis\",\n      \"journal\": \"Veterinary pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout with defined phenotypic readout (hydrocephalus) linked to ciliary dysfunction; single study, histopathology-based mechanistic placement\",\n      \"pmids\": [\"21746835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CFAP45 structural modelling identifies an AMP-binding interface between CFAP45 and AK8, and proteomic profiling of CFAP45-deficient cilia links CFAP45 to an axonemal module that includes AK8 and dynein ATPases. This positions AK8 as part of an axonemal adenine nucleotide homeostasis module regulating ciliary and flagellar beating.\",\n      \"method\": \"Proteomic profiling (mass spectrometry) of CFAP45-deficient cilia/flagella, structural modelling of AMP-binding interface between CFAP45 and AK8, in vitro AMP binding assay for CFAP45, microtubule sliding rescue assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic interaction mapping and structural modelling identify the AK8-CFAP45 interface; AK8-specific binding validated by modelling but not by direct AK8 mutagenesis; single study\",\n      \"pmids\": [\"33139725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AK8 interacts with the radial spoke (RS) of the sperm axoneme, as revealed by immunoprecipitation combined with mass spectrometry. AK8 knockout mice are infertile with impaired sperm motility. AK8 and AK9 cooperatively regulate ATP transfer in the axoneme and are concentrated at sites of energy consumption in the flagellum, as shown by ATP probes and metabolomic analysis.\",\n      \"method\": \"Immunoprecipitation combined with mass spectrometry, knockout mouse model, ATP probe assay, metabolomic analysis, human infertile male cohort analysis\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reciprocal Co-IP/MS, knockout mouse model with defined fertility/motility phenotype, and metabolic flux measurement; multiple orthogonal methods in one study\",\n      \"pmids\": [\"38761355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"EFCAB10 interacts directly with AK8 and RSPH3B, anchoring AK8 to the radial spoke in motile cilia. Loss of EFCAB10 results in complete absence of AK8 from cilia, whereas loss of AK8 does not affect ciliary EFCAB10. Both Efcab10 and Ak8 knockout mice display impaired ciliary motility and PCD-related phenotypes (respiratory defects and infertility).\",\n      \"method\": \"Biochemical co-immunoprecipitation, knockout mouse models (Efcab10-/- and Ak8-/-), ciliary motility assays, immunofluorescence localization\",\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 — reciprocal Co-IP establishing EFCAB10-AK8-RSPH3B complex, dual knockout mouse models with defined phenotype, directional epistasis (EFCAB10 required for AK8 localization but not vice versa); multiple orthogonal methods\",\n      \"pmids\": [\"41055978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AK8 is identified as a component of the stalk of radial spoke 1 (RS1) in human and mouse sperm flagella. In IQUB-deficient sperm, RS1 (but not RS2 or RS3) is lost, and AK8 is among the RS1 stalk proteins that are correspondingly absent or reduced, placing AK8 structurally within the RS1 stalk.\",\n      \"method\": \"Protein mass spectrometry of IQUB-deficient human and mouse sperm, western blotting, immunofluorescence, Iqub knockout mouse model\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based proteomic identification of AK8 as RS1 stalk component in KO model; single study, no direct AK8 mutagenesis\",\n      \"pmids\": [\"39849482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"AK8 (then designated hypothetical protein FLJ32704) is present as a sperm antigen recognized specifically by sera of immunoinfertile women but not fertile women, identified by 2D gel electrophoresis and MALDI-TOF-MS/LC-MS of human sperm proteins. An immunodominant peptide (amino acids 151–159) was identified that reacted with 90% of immunoinfertile sera.\",\n      \"method\": \"2D gel electrophoresis, MALDI-TOF-MS/LC-MS, ELISA with synthetic peptide\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — identifies AK8 as a sperm surface antigen by proteomic identification; no direct mechanistic functional experiment on AK8 itself\",\n      \"pmids\": [\"16998854\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AK8 is a dual-domain adenylate kinase (with two independently active AK domains) that localizes to the axoneme of motile cilia and sperm flagella, where it is anchored to radial spoke 1 (RS1) via EFCAB10 and RSPH3B, and functions as part of an adenine nucleotide homeostasis/ATP transfer module that is essential for proper ciliary beating, sperm motility, and male fertility; loss of AK8 in mice causes primary ciliary dyskinesia phenotypes including hydrocephalus and infertility.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AK8 is a dual-domain adenylate kinase that supplies local adenine nucleotide homeostasis to motile cilia and sperm flagella, where it is required for proper ciliary beating and male fertility [#0, #4]. It carries two complete, independently active AK domains within a single polypeptide; full-length AK8 and each isolated domain phosphorylate AMP, CMP, dAMP, and dCMP using ATP (or GTP) as phosphate donor, with highest affinity for AMP-ATP [#0]. Within the flagellum AK8 is concentrated in the axonemal compartment, distinct from the outer dense fibers and mitochondrial sheath, where nucleotide interconversion shapes the ADP-driven flagellar waveform [#1]. AK8 is a structural component of the radial spoke 1 (RS1) stalk, anchored there through EFCAB10, which bridges AK8 to RSPH3B; loss of EFCAB10 abolishes ciliary AK8, whereas loss of AK8 leaves EFCAB10 intact, defining a directional assembly hierarchy [#5, #6]. AK8 acts together with AK9 to route ATP transfer to sites of high energy consumption in the flagellum, and it associates with an axonemal nucleotide-homeostasis module that includes CFAP45 and dynein ATPases [#3, #4]. Loss of AK8 in mice produces primary ciliary dyskinesia phenotypes including congenital hydrocephalus, respiratory defects, impaired sperm motility, and infertility [#2, #4, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Before any functional role was known, AK8 was flagged as a sperm-associated antigen, providing the first link of the protein to reproductive biology.\",\n      \"evidence\": \"2D gel electrophoresis with MALDI-TOF/LC-MS of human sperm proteins and peptide ELISA against immunoinfertile sera\",\n      \"pmids\": [\"16998854\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No mechanistic or enzymatic function demonstrated for AK8\", \"Antigen identification does not establish localization or molecular activity\", \"Association with infertility is correlative\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Establishing the biochemistry first: AK8 was shown to be an adenylate kinase carrying two independently catalytically active AK domains in one chain, defining its core molecular activity and substrate preferences.\",\n      \"evidence\": \"In vitro enzyme and substrate-specificity assays with domain truncations plus GFP-fusion localization\",\n      \"pmids\": [\"21080915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cytosolic localization in overexpression did not reveal the axonemal targeting later observed\", \"Physiological substrate flux in vivo not measured\", \"No interacting partners identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Genetic knockout established that AK8 is required in vivo, linking its loss to motile-cilia dysfunction and congenital hydrocephalus.\",\n      \"evidence\": \"Ak8 knockout mouse with neuropathological analysis\",\n      \"pmids\": [\"21746835\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting AK8 loss to ciliary failure inferred from histopathology only\", \"No molecular placement of AK8 within the axoneme\", \"Sperm/fertility phenotype not assessed here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Subcellular resolution placed AK8 specifically in the axonemal compartment of the sperm flagellum and tied its enzymatic activity to flagellar mechanics.\",\n      \"evidence\": \"Immunolocalization in mouse sperm with detergent-modeled motility assays using defined nucleotide substrates\",\n      \"pmids\": [\"24740601\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct AK8-dependence of the AMP-driven waveform not tested by genetic ablation\", \"Anchoring mechanism within the axoneme unknown\", \"Single-lab functional inference\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"AK8 was embedded in a defined axonemal module by linking it to CFAP45 and dynein ATPases, framing it as part of a nucleotide-homeostasis system regulating beating.\",\n      \"evidence\": \"Proteomic profiling of CFAP45-deficient cilia, structural modelling of an AMP-binding CFAP45-AK8 interface, and microtubule sliding rescue\",\n      \"pmids\": [\"33139725\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"AK8-CFAP45 interface validated by modelling, not by AK8 mutagenesis\", \"Functional consequence of disrupting the interface untested\", \"Stoichiometry of the module unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"AK8 was shown to physically associate with the radial spoke and to act with AK9 in directing ATP transfer to flagellar energy-consuming sites, connecting its enzymatic role to motility and fertility.\",\n      \"evidence\": \"Reciprocal Co-IP/MS, Ak8 knockout mice, ATP-probe imaging, metabolomics, and a human infertile-male cohort\",\n      \"pmids\": [\"38761355\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise division of labor between AK8 and AK9 not fully resolved\", \"Direct radial-spoke binding partner not identified in this study\", \"Causal AK8 variants in human infertility not functionally proven\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The anchoring mechanism was resolved: EFCAB10 directly binds AK8 and RSPH3B to tether AK8 to the radial spoke, with directional epistasis showing EFCAB10 is upstream of AK8 ciliary localization.\",\n      \"evidence\": \"Co-IP, Efcab10-/- and Ak8-/- knockout mice, ciliary motility assays, and immunofluorescence\",\n      \"pmids\": [\"41055978\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the EFCAB10-AK8-RSPH3B interface unknown\", \"Whether anchoring is required for AK8 catalytic function untested\", \"Tissue specificity of the anchoring complex not fully mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"AK8 was placed structurally within the RS1 stalk specifically, distinguishing its location among the three radial spokes.\",\n      \"evidence\": \"MS of IQUB-deficient human and mouse sperm with western blot, immunofluorescence, and Iqub knockout\",\n      \"pmids\": [\"39849482\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct AK8 mutagenesis to confirm RS1 incorporation\", \"Relationship between IQUB and the EFCAB10 anchoring pathway unclear\", \"RS1-specific functional role of AK8 not isolated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how AK8's catalytic activity, its RS1 structural role, and its anchoring by EFCAB10 are mechanistically coupled to dynein-driven beating, and whether specific human AK8 variants cause primary ciliary dyskinesia or infertility.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the AK8-containing RS1 assembly\", \"No demonstration that anchoring is required for in-axoneme catalysis\", \"Causal human disease variants not functionally validated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016301\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"complexes\": [\"radial spoke 1 (RS1) stalk\", \"EFCAB10-AK8-RSPH3B complex\", \"axonemal CFAP45 nucleotide-homeostasis module\"],\n    \"partners\": [\"EFCAB10\", \"RSPH3B\", \"CFAP45\", \"AK9\", \"IQUB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}