{"gene":"ROPN1","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2000,"finding":"Ropporin (ROPN1) binds to the PDZ domain of rhophilin via its carboxy-terminal four amino acids, as shown by yeast two-hybrid assay and deletion analysis. Ropporin also co-precipitates with RhoV14 (GTP-bound RhoA) in the presence of rhophilin in vitro, placing it in a RhoA-rhophilin-ropporin complex.","method":"Yeast two-hybrid assay, deletion mutagenesis, in vitro co-precipitation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — yeast two-hybrid with deletion mutagenesis identifying essential residues, plus in vitro co-precipitation, all in a single rigorous study","pmids":["10591629"],"is_preprint":false},{"year":2000,"finding":"Ropporin (ROPN1) dimerizes through its amino-terminal ~40 amino acid domain, which is homologous to the RII dimerization domain of cAMP-dependent protein kinase regulatory subunits. Dimerization was demonstrated by yeast two-hybrid assay and gel filtration of recombinant protein.","method":"Yeast two-hybrid assay, gel filtration of recombinant protein","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — two orthogonal methods (yeast two-hybrid and gel filtration reconstitution) in a single rigorous study","pmids":["10591629"],"is_preprint":false},{"year":2000,"finding":"Ropporin localizes to the inner surface of the fibrous sheath in the principal piece and end piece of sperm flagella, while rhophilin is present on the outer surface of the outer dense fiber, suggesting they form a complex spanning these structures.","method":"Immunocytochemistry, electron microscopy","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct immunolocalization by light and electron microscopy, two complementary imaging methods in one study","pmids":["10591629"],"is_preprint":false},{"year":2007,"finding":"Ropporin (ROPN1) interacts with Sperm protein 17 (Sp17), identified as a binding partner by yeast two-hybrid screening of a testicular cDNA library. Ropporin also functions as an anchoring protein for AKAP110.","method":"Yeast two-hybrid screening of testicular cDNA library","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single yeast two-hybrid screen, no reciprocal confirmation reported in this abstract","pmids":["17551920"],"is_preprint":false},{"year":2010,"finding":"CABYR binds to ropporin (ROPN1) through regions outside its RII-like domain; deletion of the CABYR RII domain abolished CABYR–AKAP3/AKAP4 interaction but did not abolish CABYR–ropporin interaction, indicating distinct binding interfaces.","method":"Yeast two-hybrid assay, deletion analysis, immunoprecipitation","journal":"Reproductive biology and endocrinology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-immunoprecipitation plus yeast two-hybrid with deletion mutagenesis, two orthogonal methods in one study","pmids":["20731842"],"is_preprint":false},{"year":2011,"finding":"CABYR, AKAP3, and ropporin (ROPN1) form a complex in human sperm fibrous sheath. CABYR was co-immunoprecipitated with AKAP3 and with ropporin; reciprocal co-IPs and yeast two-hybrid assays confirmed these interactions. CABYR binds AKAP3 via its RII domain and binds ropporin through additional regions outside the RII-like domain.","method":"Co-immunoprecipitation (reciprocal), mass spectrometry, Western blot, yeast two-hybrid assay","journal":"Asian journal of andrology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP confirmed by mass spectrometry and yeast two-hybrid, replicated across two studies (PMID:20731842 and PMID:21240291)","pmids":["21240291","20731842"],"is_preprint":false},{"year":2013,"finding":"ROPN1 and its paralog ROPN1L both bind AKAP3 and are required for fibrous sheath integrity, sperm motility, and PKA-dependent signaling. Single knockout of ROPN1 causes moderate motility impairment and reduced FSCB levels with increased basal tyrosine phosphorylation; double knockout (ROPN1/ROPN1L) causes complete immotility, fibrous sheath structural defects (thinning/shredding of principal piece), and reduced AKAP3 levels, demonstrating that the two proteins compensate for each other in maintaining AKAP3 incorporation into the fibrous sheath.","method":"Genetic knockout (single and double KO mice), sperm motility assay, Western blot, electron microscopy","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined morphological and biochemical phenotypes, multiple orthogonal readouts, clear epistatic relationship","pmids":["23303679"],"is_preprint":false},{"year":2011,"finding":"Loss of ROPN1 alone does not affect ciliary beat frequency in airway cilia, demonstrating that ROPN1 is dispensable for ciliary motility (as opposed to sperm flagellar motility).","method":"ROPN1 knockout mouse, ciliary beat frequency measurement","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined functional readout, single lab, negative result explicitly reported","pmids":["22021175"],"is_preprint":false},{"year":2016,"finding":"FSCB phosphorylation suppresses SUMOylation of ROPN1 and ROPN1L during sperm capacitation, and phosphorylated FSCB has significantly higher affinity for ROPN1/ROPN1L than non-phosphorylated FSCB. Suppression of ROPN1/ROPN1L SUMOylation mimicked the effects of FSCB phosphorylation on sperm motility.","method":"Immunoprecipitation assay, SUMOylation assay, sperm motility assay","journal":"American journal of translational research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — immunoprecipitation demonstrating phospho-FSCB/ROPN1 interaction with functional readout, single lab, single study","pmids":["27398160"],"is_preprint":false}],"current_model":"ROPN1 (ropporin) is a sperm flagellar fibrous sheath protein that homodimerizes via an N-terminal RII-like domain, localizes to the inner surface of the fibrous sheath, and forms a multiprotein complex with AKAP3, AKAP110, rhophilin (connecting it to RhoA signaling), CABYR, and FSCB; it is required—redundantly with its paralog ROPN1L—for fibrous sheath structural integrity, AKAP3 incorporation, PKA-dependent signaling, and sperm motility, and its SUMOylation is suppressed by phosphorylated FSCB to promote capacitation."},"narrative":{"mechanistic_narrative":"ROPN1 (ropporin) is a sperm flagellar fibrous sheath protein that functions as a structural and signaling scaffold required for fibrous sheath integrity and sperm motility [PMID:10591629, PMID:23303679]. It homodimerizes through an N-terminal ~40-residue domain homologous to the RII dimerization domain of cAMP-dependent protein kinase regulatory subunits, and localizes to the inner surface of the fibrous sheath in the principal and end pieces of the flagellum [PMID:10591629]. Through this RII-like architecture and its C-terminal residues, ROPN1 nucleates a multiprotein complex: its C-terminal four amino acids bind the PDZ domain of rhophilin, linking it to GTP-bound RhoA signaling [PMID:10591629], and it associates with AKAP110/Sp17, CABYR, and AKAP3 within the fibrous sheath [PMID:17551920, PMID:20731842, PMID:21240291]. Genetically, ROPN1 acts redundantly with its paralog ROPN1L to anchor AKAP3 into the fibrous sheath and sustain PKA-dependent signaling; single ROPN1 loss causes moderate motility impairment and reduced FSCB, whereas combined ROPN1/ROPN1L loss produces complete immotility, fibrous sheath thinning and shredding, and depletion of AKAP3 [PMID:23303679]. During capacitation, phosphorylated FSCB binds ROPN1 with high affinity and suppresses its SUMOylation to promote motility [PMID:27398160]. ROPN1 is dispensable for airway ciliary beating, indicating its motility role is specific to the sperm flagellum [PMID:22021175].","teleology":[{"year":2000,"claim":"Established ROPN1's molecular architecture and first binding partner, defining it as a dimerizing scaffold that couples to RhoA signaling and resides in the fibrous sheath.","evidence":"Yeast two-hybrid with deletion mutagenesis, gel filtration of recombinant protein, and immuno-EM in sperm","pmids":["10591629"],"confidence":"High","gaps":["Functional consequence of the rhophilin/RhoA linkage for sperm physiology not tested","RII-like dimerization not shown to bind a PKA regulatory subunit ligand"]},{"year":2007,"claim":"Extended the ROPN1 interactome by identifying Sp17 as a partner and positioning ROPN1 as an anchoring protein for AKAP110.","evidence":"Yeast two-hybrid screening of a testicular cDNA library","pmids":["17551920"],"confidence":"Medium","gaps":["Single yeast two-hybrid screen without reciprocal confirmation","Functional role of the AKAP110 anchoring not defined"]},{"year":2010,"claim":"Resolved that CABYR binds ROPN1 through interfaces distinct from those it uses for AKAP3/AKAP4, refining the topology of the fibrous sheath complex.","evidence":"Yeast two-hybrid, deletion analysis, and immunoprecipitation","pmids":["20731842"],"confidence":"High","gaps":["Precise ROPN1 residues binding CABYR not mapped","Stoichiometry of the multiprotein complex unknown"]},{"year":2011,"claim":"Demonstrated that CABYR, AKAP3, and ROPN1 co-assemble into one fibrous sheath complex in human sperm, integrating the scaffold components into a single module.","evidence":"Reciprocal co-immunoprecipitation, mass spectrometry, Western blot, and yeast two-hybrid","pmids":["21240291","20731842"],"confidence":"High","gaps":["Assembly order and dependence within the complex not established","Signaling output of the assembled complex not measured"]},{"year":2011,"claim":"Showed ROPN1 is dispensable for airway ciliary motility, distinguishing its motility function as flagellum-specific rather than general to motile cilia.","evidence":"ROPN1 knockout mouse with ciliary beat frequency measurement","pmids":["22021175"],"confidence":"Medium","gaps":["Single-lab negative result","Whether ROPN1L compensates in cilia not addressed"]},{"year":2013,"claim":"Established the in vivo requirement for ROPN1 in fibrous sheath integrity and motility and revealed functional redundancy with ROPN1L in anchoring AKAP3 and sustaining PKA signaling.","evidence":"Single and double knockout mice with motility assays, Western blot, and electron microscopy","pmids":["23303679"],"confidence":"High","gaps":["Mechanism by which ROPN1 loss elevates basal tyrosine phosphorylation not defined","Direct biochemical role of ROPN1 in AKAP3 docking versus indirect stabilization not separated"]},{"year":2016,"claim":"Connected ROPN1 to capacitation regulation by showing phospho-FSCB binds ROPN1 with high affinity and suppresses its SUMOylation to drive motility.","evidence":"Immunoprecipitation, SUMOylation assay, and sperm motility assay","pmids":["27398160"],"confidence":"Medium","gaps":["SUMOylation sites on ROPN1 not mapped","Single-lab, single-study finding","Downstream effect of ROPN1 SUMO status on the fibrous sheath complex unclear"]},{"year":null,"claim":"How the RhoA-rhophilin-ROPN1 signaling axis, the AKAP/PKA scaffold, and ROPN1 SUMOylation are integrated to control capacitation and motility remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the assembled fibrous sheath complex","Causal link between RhoA signaling and ROPN1 function untested in vivo","Whether ROPN1 binds a PKA regulatory subunit despite its RII-like domain is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3,4,5,6]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[2,6]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[2]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2,6]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0]}],"complexes":["sperm fibrous sheath AKAP3-CABYR-ROPN1 complex"],"partners":["RHPN1","RHOA","SPA17","AKAP4","CABYR","AKAP3","ROPN1L","FSCB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9HAT0","full_name":"Ropporin-1A","aliases":["Cancer/testis antigen 91","CT91","Rhophilin-associated protein 1A"],"length_aa":212,"mass_kda":23.9,"function":"Important for male fertility. With ROPN1L, involved in fibrous sheath integrity and sperm motility, plays a role in PKA-dependent signaling processes required for spermatozoa capacitation","subcellular_location":"Cell projection, cilium, flagellum","url":"https://www.uniprot.org/uniprotkb/Q9HAT0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ROPN1","classification":"Not Classified","n_dependent_lines":22,"n_total_lines":1047,"dependency_fraction":0.021012416427889206},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ROPN1","total_profiled":1310},"omim":[{"mim_id":"618304","title":"GLUTAMINE-RICH PROTEIN 2; QRICH2","url":"https://www.omim.org/entry/618304"},{"mim_id":"611757","title":"RHOPHILIN-ASSOCIATED TAIL PROTEIN 1; ROPN1","url":"https://www.omim.org/entry/611757"},{"mim_id":"611756","title":"RHOPHILIN ASSOCIATED TAIL PROTEIN 1-LIKE; ROPN1L","url":"https://www.omim.org/entry/611756"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Principal piece","reliability":"Approved"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"testis","ntpm":151.3}],"url":"https://www.proteinatlas.org/search/ROPN1"},"hgnc":{"alias_symbol":["ODF6","ropporin","ROPN1A","CT91"],"prev_symbol":[]},"alphafold":{"accession":"Q9HAT0","domains":[{"cath_id":"-","chopping":"15-60","consensus_level":"medium","plddt":76.9091,"start":15,"end":60},{"cath_id":"-","chopping":"131-212","consensus_level":"medium","plddt":89.3352,"start":131,"end":212},{"cath_id":"1.10.8","chopping":"64-129","consensus_level":"medium","plddt":91.1562,"start":64,"end":129}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HAT0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HAT0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HAT0-F1-predicted_aligned_error_v6.png","plddt_mean":85.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ROPN1","jax_strain_url":"https://www.jax.org/strain/search?query=ROPN1"},"sequence":{"accession":"Q9HAT0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9HAT0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9HAT0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HAT0"}},"corpus_meta":[{"pmid":"10591629","id":"PMC_10591629","title":"Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella.","date":"2000","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/10591629","citation_count":118,"is_preprint":false},{"pmid":"23303679","id":"PMC_23303679","title":"Loss of R2D2 proteins ROPN1 and ROPN1L causes defects in murine sperm motility, phosphorylation, and fibrous sheath integrity.","date":"2013","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/23303679","citation_count":83,"is_preprint":false},{"pmid":"21240291","id":"PMC_21240291","title":"CABYR binds to AKAP3 and Ropporin in the human sperm fibrous sheath.","date":"2011","source":"Asian journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/21240291","citation_count":39,"is_preprint":false},{"pmid":"20731842","id":"PMC_20731842","title":"CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath.","date":"2010","source":"Reproductive biology and endocrinology : RB&E","url":"https://pubmed.ncbi.nlm.nih.gov/20731842","citation_count":27,"is_preprint":false},{"pmid":"20705794","id":"PMC_20705794","title":"Functional expression of ropporin in human testis and ejaculated spermatozoa.","date":"2010","source":"Journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/20705794","citation_count":26,"is_preprint":false},{"pmid":"17551920","id":"PMC_17551920","title":"A yeast two-hybrid system using Sp17 identified Ropporin as a novel cancer-testis antigen in hematologic malignancies.","date":"2007","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/17551920","citation_count":21,"is_preprint":false},{"pmid":"22021175","id":"PMC_22021175","title":"Loss of ASP but not ROPN1 reduces mammalian ciliary motility.","date":"2011","source":"Cytoskeleton (Hoboken, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/22021175","citation_count":19,"is_preprint":false},{"pmid":"21654522","id":"PMC_21654522","title":"Cancer testis antigen, ropporin, is a potential target for multiple myeloma immunotherapy.","date":"2011","source":"Journal of immunotherapy (Hagerstown, Md. : 1997)","url":"https://pubmed.ncbi.nlm.nih.gov/21654522","citation_count":18,"is_preprint":false},{"pmid":"29247344","id":"PMC_29247344","title":"Molecular study of human sperm RNA: Ropporin and CABYR in asthenozoospermia.","date":"2017","source":"Journal of endocrinological investigation","url":"https://pubmed.ncbi.nlm.nih.gov/29247344","citation_count":17,"is_preprint":false},{"pmid":"27398160","id":"PMC_27398160","title":"FSCB phosphorylation regulates mouse spermatozoa capacitation through suppressing SUMOylation of ROPN1/ROPN1L.","date":"2016","source":"American journal of translational research","url":"https://pubmed.ncbi.nlm.nih.gov/27398160","citation_count":16,"is_preprint":false},{"pmid":"39172012","id":"PMC_39172012","title":"TCR-Engineered T Cells Directed against Ropporin-1 Constitute a Safe and Effective Treatment for Triple-Negative Breast Cancer.","date":"2024","source":"Cancer discovery","url":"https://pubmed.ncbi.nlm.nih.gov/39172012","citation_count":10,"is_preprint":false},{"pmid":"21667248","id":"PMC_21667248","title":"Molecular cloning, sequence characterization, polymorphism and association analysis of porcine ROPN1 gene.","date":"2011","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/21667248","citation_count":8,"is_preprint":false},{"pmid":"33918976","id":"PMC_33918976","title":"Ropporin-1 and 1B Are Widely Expressed in Human Melanoma and Evoke Strong Humoral Immune Responses.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/33918976","citation_count":7,"is_preprint":false},{"pmid":"19947570","id":"PMC_19947570","title":"[Advances in the researches of spermatogenic protein, Ropporin].","date":"2009","source":"Zhonghua nan ke xue = National journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/19947570","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9152,"output_tokens":2204,"usd":0.030258,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9172,"output_tokens":2629,"usd":0.055793,"stage2_stop_reason":"end_turn"},"total_usd":0.086051,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"Ropporin (ROPN1) binds to the PDZ domain of rhophilin via its carboxy-terminal four amino acids, as shown by yeast two-hybrid assay and deletion analysis. Ropporin also co-precipitates with RhoV14 (GTP-bound RhoA) in the presence of rhophilin in vitro, placing it in a RhoA-rhophilin-ropporin complex.\",\n      \"method\": \"Yeast two-hybrid assay, deletion mutagenesis, in vitro co-precipitation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — yeast two-hybrid with deletion mutagenesis identifying essential residues, plus in vitro co-precipitation, all in a single rigorous study\",\n      \"pmids\": [\"10591629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Ropporin (ROPN1) dimerizes through its amino-terminal ~40 amino acid domain, which is homologous to the RII dimerization domain of cAMP-dependent protein kinase regulatory subunits. Dimerization was demonstrated by yeast two-hybrid assay and gel filtration of recombinant protein.\",\n      \"method\": \"Yeast two-hybrid assay, gel filtration of recombinant protein\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — two orthogonal methods (yeast two-hybrid and gel filtration reconstitution) in a single rigorous study\",\n      \"pmids\": [\"10591629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Ropporin localizes to the inner surface of the fibrous sheath in the principal piece and end piece of sperm flagella, while rhophilin is present on the outer surface of the outer dense fiber, suggesting they form a complex spanning these structures.\",\n      \"method\": \"Immunocytochemistry, electron microscopy\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct immunolocalization by light and electron microscopy, two complementary imaging methods in one study\",\n      \"pmids\": [\"10591629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Ropporin (ROPN1) interacts with Sperm protein 17 (Sp17), identified as a binding partner by yeast two-hybrid screening of a testicular cDNA library. Ropporin also functions as an anchoring protein for AKAP110.\",\n      \"method\": \"Yeast two-hybrid screening of testicular cDNA library\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single yeast two-hybrid screen, no reciprocal confirmation reported in this abstract\",\n      \"pmids\": [\"17551920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CABYR binds to ropporin (ROPN1) through regions outside its RII-like domain; deletion of the CABYR RII domain abolished CABYR–AKAP3/AKAP4 interaction but did not abolish CABYR–ropporin interaction, indicating distinct binding interfaces.\",\n      \"method\": \"Yeast two-hybrid assay, deletion analysis, immunoprecipitation\",\n      \"journal\": \"Reproductive biology and endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-immunoprecipitation plus yeast two-hybrid with deletion mutagenesis, two orthogonal methods in one study\",\n      \"pmids\": [\"20731842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CABYR, AKAP3, and ropporin (ROPN1) form a complex in human sperm fibrous sheath. CABYR was co-immunoprecipitated with AKAP3 and with ropporin; reciprocal co-IPs and yeast two-hybrid assays confirmed these interactions. CABYR binds AKAP3 via its RII domain and binds ropporin through additional regions outside the RII-like domain.\",\n      \"method\": \"Co-immunoprecipitation (reciprocal), mass spectrometry, Western blot, yeast two-hybrid assay\",\n      \"journal\": \"Asian journal of andrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP confirmed by mass spectrometry and yeast two-hybrid, replicated across two studies (PMID:20731842 and PMID:21240291)\",\n      \"pmids\": [\"21240291\", \"20731842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ROPN1 and its paralog ROPN1L both bind AKAP3 and are required for fibrous sheath integrity, sperm motility, and PKA-dependent signaling. Single knockout of ROPN1 causes moderate motility impairment and reduced FSCB levels with increased basal tyrosine phosphorylation; double knockout (ROPN1/ROPN1L) causes complete immotility, fibrous sheath structural defects (thinning/shredding of principal piece), and reduced AKAP3 levels, demonstrating that the two proteins compensate for each other in maintaining AKAP3 incorporation into the fibrous sheath.\",\n      \"method\": \"Genetic knockout (single and double KO mice), sperm motility assay, Western blot, electron microscopy\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined morphological and biochemical phenotypes, multiple orthogonal readouts, clear epistatic relationship\",\n      \"pmids\": [\"23303679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Loss of ROPN1 alone does not affect ciliary beat frequency in airway cilia, demonstrating that ROPN1 is dispensable for ciliary motility (as opposed to sperm flagellar motility).\",\n      \"method\": \"ROPN1 knockout mouse, ciliary beat frequency measurement\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined functional readout, single lab, negative result explicitly reported\",\n      \"pmids\": [\"22021175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FSCB phosphorylation suppresses SUMOylation of ROPN1 and ROPN1L during sperm capacitation, and phosphorylated FSCB has significantly higher affinity for ROPN1/ROPN1L than non-phosphorylated FSCB. Suppression of ROPN1/ROPN1L SUMOylation mimicked the effects of FSCB phosphorylation on sperm motility.\",\n      \"method\": \"Immunoprecipitation assay, SUMOylation assay, sperm motility assay\",\n      \"journal\": \"American journal of translational research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — immunoprecipitation demonstrating phospho-FSCB/ROPN1 interaction with functional readout, single lab, single study\",\n      \"pmids\": [\"27398160\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ROPN1 (ropporin) is a sperm flagellar fibrous sheath protein that homodimerizes via an N-terminal RII-like domain, localizes to the inner surface of the fibrous sheath, and forms a multiprotein complex with AKAP3, AKAP110, rhophilin (connecting it to RhoA signaling), CABYR, and FSCB; it is required—redundantly with its paralog ROPN1L—for fibrous sheath structural integrity, AKAP3 incorporation, PKA-dependent signaling, and sperm motility, and its SUMOylation is suppressed by phosphorylated FSCB to promote capacitation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ROPN1 (ropporin) is a sperm flagellar fibrous sheath protein that functions as a structural and signaling scaffold required for fibrous sheath integrity and sperm motility [#2, #6]. It homodimerizes through an N-terminal ~40-residue domain homologous to the RII dimerization domain of cAMP-dependent protein kinase regulatory subunits, and localizes to the inner surface of the fibrous sheath in the principal and end pieces of the flagellum [#1, #2]. Through this RII-like architecture and its C-terminal residues, ROPN1 nucleates a multiprotein complex: its C-terminal four amino acids bind the PDZ domain of rhophilin, linking it to GTP-bound RhoA signaling [#0], and it associates with AKAP110/Sp17, CABYR, and AKAP3 within the fibrous sheath [#3, #4, #5]. Genetically, ROPN1 acts redundantly with its paralog ROPN1L to anchor AKAP3 into the fibrous sheath and sustain PKA-dependent signaling; single ROPN1 loss causes moderate motility impairment and reduced FSCB, whereas combined ROPN1/ROPN1L loss produces complete immotility, fibrous sheath thinning and shredding, and depletion of AKAP3 [#6]. During capacitation, phosphorylated FSCB binds ROPN1 with high affinity and suppresses its SUMOylation to promote motility [#8]. ROPN1 is dispensable for airway ciliary beating, indicating its motility role is specific to the sperm flagellum [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established ROPN1's molecular architecture and first binding partner, defining it as a dimerizing scaffold that couples to RhoA signaling and resides in the fibrous sheath.\",\n      \"evidence\": \"Yeast two-hybrid with deletion mutagenesis, gel filtration of recombinant protein, and immuno-EM in sperm\",\n      \"pmids\": [\"10591629\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the rhophilin/RhoA linkage for sperm physiology not tested\", \"RII-like dimerization not shown to bind a PKA regulatory subunit ligand\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Extended the ROPN1 interactome by identifying Sp17 as a partner and positioning ROPN1 as an anchoring protein for AKAP110.\",\n      \"evidence\": \"Yeast two-hybrid screening of a testicular cDNA library\",\n      \"pmids\": [\"17551920\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single yeast two-hybrid screen without reciprocal confirmation\", \"Functional role of the AKAP110 anchoring not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved that CABYR binds ROPN1 through interfaces distinct from those it uses for AKAP3/AKAP4, refining the topology of the fibrous sheath complex.\",\n      \"evidence\": \"Yeast two-hybrid, deletion analysis, and immunoprecipitation\",\n      \"pmids\": [\"20731842\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise ROPN1 residues binding CABYR not mapped\", \"Stoichiometry of the multiprotein complex unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated that CABYR, AKAP3, and ROPN1 co-assemble into one fibrous sheath complex in human sperm, integrating the scaffold components into a single module.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, mass spectrometry, Western blot, and yeast two-hybrid\",\n      \"pmids\": [\"21240291\", \"20731842\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Assembly order and dependence within the complex not established\", \"Signaling output of the assembled complex not measured\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed ROPN1 is dispensable for airway ciliary motility, distinguishing its motility function as flagellum-specific rather than general to motile cilia.\",\n      \"evidence\": \"ROPN1 knockout mouse with ciliary beat frequency measurement\",\n      \"pmids\": [\"22021175\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab negative result\", \"Whether ROPN1L compensates in cilia not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established the in vivo requirement for ROPN1 in fibrous sheath integrity and motility and revealed functional redundancy with ROPN1L in anchoring AKAP3 and sustaining PKA signaling.\",\n      \"evidence\": \"Single and double knockout mice with motility assays, Western blot, and electron microscopy\",\n      \"pmids\": [\"23303679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which ROPN1 loss elevates basal tyrosine phosphorylation not defined\", \"Direct biochemical role of ROPN1 in AKAP3 docking versus indirect stabilization not separated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected ROPN1 to capacitation regulation by showing phospho-FSCB binds ROPN1 with high affinity and suppresses its SUMOylation to drive motility.\",\n      \"evidence\": \"Immunoprecipitation, SUMOylation assay, and sperm motility assay\",\n      \"pmids\": [\"27398160\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SUMOylation sites on ROPN1 not mapped\", \"Single-lab, single-study finding\", \"Downstream effect of ROPN1 SUMO status on the fibrous sheath complex unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the RhoA-rhophilin-ROPN1 signaling axis, the AKAP/PKA scaffold, and ROPN1 SUMOylation are integrated to control capacitation and motility remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the assembled fibrous sheath complex\", \"Causal link between RhoA signaling and ROPN1 function untested in vivo\", \"Whether ROPN1 binds a PKA regulatory subunit despite its RII-like domain is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 4, 5, 6]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"sperm fibrous sheath AKAP3-CABYR-ROPN1 complex\"],\n    \"partners\": [\"RHPN1\", \"RHOA\", \"SPA17\", \"AKAP4\", \"CABYR\", \"AKAP3\", \"ROPN1L\", \"FSCB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}